Embryology in Relation to Physiology and Genetics*

Embryology in Relation to Physiology and Genetics*

Embryology in Relation to Phygiology and Genetics* . .. P MAHESHWARI and N S RANGASWAMY Department of Botany. Univerdy of Delhi. Delhi. India . I...

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Embryology in Relation to Phygiology and Genetics*

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P MAHESHWARI and N S RANGASWAMY Department of Botany. Univerdy of Delhi. Delhi. India

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I Introduction ...........................................................

I1. Pollen

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................................................................. 221 A. Longevity of Pollen ...........!k ..................................... 221 B . Uermination of Pollen ............................................... 223 C. Pollen Tube ........................................................ 227 231 111. Control of Fertilization ................................................. A. Trentment of the Stigma .............................................232 B. Treatment of the Style ............................................... 232 C. Intraovarian and in Vitro Fertilization ................................. 234 237 IV. Embryo ............................................................... A. Urowth of Embryo in Relation to Sccd hvclopment .................... 237 B . Dependence of Embryo on Endosperm ................................ 239 C. Specificity in Nutrition of Embryo ..................................... 230 240 V . Endoaperm ............................................................ A . Constituents of Endosperm ........................................... 240 B. Role of Endosperm in Seed Development., ............................. 242 C. Culture of Endonperm ................................................ 843 248 VI. Embryo Culture ....................................................... A . Cultural Conditions. ................................................. 241 B. QrowthMedia ...................................................... 260 C. Applloatione of Embryo Culture ....................................... 256 D. Limitetiom of Embryo Culture ....................................... 262 263 VII. Culture of Ovules ...................................................... 266 VIII. Culture of Ovariee and Flowers .......................................... 273 IX. Parthenocarpy ......................................................... X . Polyembryony ......................................................... 280 A . Advontivo Embryony ................................................ 280 B. Embryonal Budding ................................................. 28f1 2M XI. Parthenogenes ie ........................................................ 300 XI1. Androgeneeis ...:...................................................... XI11. Anther Culture .......................................................... 301 304 XIV. Control of Sex Expression ............................................... xv . Concluaions............................................................ 309 Acknowledgements ...................................................... 310 References ............................................................ 310 \

1. INTRODUCTION During the years i8&1-1830 Alnici made some fundamental discoveries when he observed that pollen grains germinate on the stigma. and the pollen tubes grow into the style and ovary. and Snally enter the ovules Schleiden (1837) confirmed the observations of Amid but erroneously thought that the tip of the pollen tube ifself became transformed into the embryo . In 1849 Hofmeister reported the presence of germinal vesicles (i.e. constituents of the egg apparatus) in embryo saw and * The main eurvoy of the litcrkturc WHB complctcd by thc end of 1963.

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emphasized that it was ond of these germinal vesicles that gave rise to the embryo and not the tip of the pollen tube. He believed that the fluid discharged by the tube activated the germinal vesicle to develop. Strasburger (1884) observed the sperm nuclei in the pollen tube and their discharge into the embryo sac. He demonstrated the fusion of a sperm with the egg and showed that it is the product of the union of the gametes that gives rise to the embryo. Nawaachin (1898) and Guignard (1899) completed our knowledge of the basic facts of fertilization in flowering plants by showing that while one out of the two male nuclei brought in by the pollen tube unites with the egg to produce an embryo as already found by Strasburger (1884), the other fuses with the secondary nucleus to give rise to a tissue called the endosperm. Whereas the embryo is diploid and is the progenitor of the next generation, the endosperm is normally triploid and acts 88 a nurse, tissue for the embryo. With the year 1900 came the rediscovery of Mendel's laws followed by an unprecedented activity in the hybridization of varieties, species and genera in order to produce newer and more useful types. Plant breeders put the pollen upon the stigma and looked for results in the ovary, often succeeding but not always. Their failures to obtain the desired hybrids stimulated more intensive research into the morphology and physiology of the changes that follow pollination and ultimately lead to fertilization and then the formation of the seed and the f i t . These aspects have gradually been recognized aa constituting a new discipline, namely experimental plant embryology. The beginnings of experimental embryology may be said to have been laid when Hannig (1904) grew the young embryos of certain plants in an artificial medium. Several others followed him and in 1925 Laibach gave a valuable hint to plant breeders that if a cross does not result in a fertile hybrid and the sterility is due to the death of the resultant embryo it may be useful to excise the embryo at an early stage and grow it in a nutrient medium. This in Witro culture of embryos has now become a common practice in plant breeding and n more detailed account of it is given in a later section of this essay (see p. 246). Although an exckion of the young embryo and its culture in an artificial medium may sometimefl save it from premature death and thus prove helpful, there are other and still more primary barriers to successful hybridization. The chief among them are: (a) the relatively short life of the pollen, (6) the inability of the pollen to germinate on the stigma, (c) the slow growth of the pollen tubes 60 that they do not reach the embryo sac in time, (df the failure of the sperm to fuse with the egg, and (e) the diafunction of the zygote, or endooperm, or both.

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Some of these impediments are indicators of a sexual incompatibility which manifests itself as a physiological imbalanoe between the male gametophyte and #e carpellary tisaues or between the produots resulting from syngamy and triple fusion. We shall, therefore, disouae both the pre- and post-fertilization events. The former deal with the role of the pollen in relation to the pistil, and the latter with the endosperm, embryo, and other tissues of the seed.

11. POLLEN A. LONGEVITY OF POLLEN

In natture pollination is aohieved through wind, water, insects or other agenoies. Sometimes pollination occurs even in the bud when the anther and the stigma come in direct contact or lie so close to each other that any little movement of the flower bud causes the pollen to land on the stigma. Most pollens lose their viability soon after shedding from the anther. The pollen of grasses is especially short-lived. Bennett (1969) reported that in Pmpalum dilatatum it loses its capacity to germinate within 30 minutm after shedding. Only in a few plants does the pollen remain viable for more than 2 or 3 days, and a period of a few weeks or months is rather rare although it?has been observed in several fruit trees and gymnosperms. Methods of prolonging the vitality of pollen are, therefore, of considerablevalue for they would enable the transport of pollen in plants wbioh are geographioally isolated. They would also help in bridging over the undesirable interval, if any, between the blooming periods of the two parents. The longevity of the pollen iu governed by a number of factors such as temperature, relativo humidity, light, and the time of blooming. Of special significance are temperature and relative humidity and their effects are interdependent. Sub-freezing temperatures (-6 to -10°C) and 25-60% relative humidity (RH) are conduoive t o a satisfactory storage of most pollens. The pollen of certain species of Niwtiam shred for a year at -6°C and 60% RH germinated aa easily aa fresh pollen (Daniel, 1965). Similarly, the pollen of apple stored at -16OC for 9 months showed 95% germination (Visser, 1966). King (1969) h u reported a successful freeze-drying of the pollen of Pinus although that of P e e ~ & ~ w was u affected adversely (Livingston et al., 1962). Aocording to Whitehead (1963) freeze-drying greatly increased the longevity of the pollen of Coc0.3 nuciferu; pollen stored at room temperatures over sulphuric acid remained viable for 3 week, that at low temperature over silica gel generally retained its longevity for 2-3 months, and pollen stored over damp calcium ahloride remained alive

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even up to 18 months. Hansoii (1961) has demonstrated that subfreezing of the pollen of alfalfa can prolong its vitality from 8-15 to 183 days. Ordinarily the pollen of mango remains viable for 8 days only, but at a temperature of 4.5-9"C and at 10, 26, or 60% RH it retained its longevity for 5 month8 and at -23°C and 0% R H for 14 months (Siiigh, 1962). Temperatures as low as -190°C obtained by using liquid air or liquid oxygen, have proved still better in enhancing the longevity of pollen. For example, Bredemann et al. (1947) stored the pollen of Lupinus polyphyllus at -190°C for 3 months without loss of vitality, whereas storage at 0°C proved unsatisfactory. The pollen of apple stored at -190OC was as effective as fresh pollen even after 2 years (Visser, 1966). Like low temperatures, a low relative humidity also prolongs the life span of most pollens. The pollens of apple, apricot, peach, pear, plum, sour cherry (Nebel, 1939), grape (Olmo, 1942), and pistachio (Stone ei al., 1943) remained viable for one year or even longer (6.5 years in sour cherry) at 10-60% RH and a temperature of 0-10°C. The pollen of Datum remained viable for only 2 weeks when air-dried, but when refrigerated over calcium chloride it kept alive for almost a year (Blakeslee, 1945). The pollens of Arachig hypogaeu, BrassiCa; nigra, Solanum melongem and S. tuberosum showed the highest viability at 31-40% RH (Vasil, 1962). Singh (1962) reports that the pollen of Marqiferu indicu remained viable not only for a longer period (20 days) but also showed a higher percentage of germination when stored at 0% RH than at 10, 25 or 60% RH. On the other hand, a low relative humidity is harmful to the pollens of members of the Gramineae which can be stored somewhat better, although still only for a short period, at a relatively high R H (70-100%). For example, Sartoris (1942) observed that in Sacchrum and Zeu the pollen remained viable for 10 days at 90-1000/, R H and a temperature of 4°C. Daniel (1955) reported that temperatures lower than 7°C and a relative humidity below 50% were detrimental to the pollen of Zea mays. Pennisetum typhoideum is an exception as its pollen can be stored for 186 days at 16-35°C and 0% RH, but a R H of 60% or above limited the viability to 5 days (Vasil, 1962). To minimize deterioration during storage, particularly duo to desiccation, the pollen ix often mixed with certain pulverized, anhydrow materials called diluents. Out of over 30 mbstances, lycopodium powder, egg albumen, casein and talc are the most acceptable diluents. However, they are useful with granular pollen only; with sticky pollen they cause agglutination and a decrease in viability, Of interest is also the, report that hand-collected pollen of Prunw amygdalus stored in a home freezer at about -18°C remained alive

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for 801 days and bee-collected pollen for 1130 days (Griggs et al., 1953). The maximum longevity so far recorded is 9 years for the pollen of some rosaceous fruit trees (Ushirozawa and Shibukawa, 1961). Although no conclusive physiological interpretations can be offered, the practical value of prolonging the life of pollen is recognized by all . . plant breeders. B. GERMINATION OF POLLEN

Whereas the life span of the pollen of many species can be extended by artificial devices, this should not be taken to imply that stored pollen would always retain its capacity for germination and subsequent potency for fertilization to the same degree as f m h pollen. Some probable explanations for this are: loss of moisture, depletion of food reserves, and a decrease and/or inactivation of the enzymes or other key substances. The logical corollary is that stored pollen may require for its germination a higher relative humidity and a higher level of nutrient material than fresh pollen. This ha6 been confirmed by some investigators. Pfeiffer (1944) observed that on providing a favourable humidity and temperature the pollen of Cinchona showed some recovery of its capacity for germination. The pollen of Pinw freshly collected in June yielded optimum germination in 2% sucrose, while a sample stored until December required as much as 20% sucrose (Kiihlwein and Anhaeusser, 1961). Similarly, the comeeponding requirements of Aradiie hypogaea and of the varieties T.65 I.C.1472 and T.25 of Pennisetum typhoideum were 10, 12.5 and 26% sucrose for fresh pollen; and 12.5, I5 and 27.6% for stored pollen (Vasil, 1962). For tomato Gorobec (1968) observed that 4-day-old pollen stored in the laboratory yielded fruits of normal size while older pollen gave only small fruits. At the other extreme it is recorded that the pollen of apple gave some germination even 9 years after storage (Ushirozitwa and Shibukawa, 1951). Nielwn (1956) observed that the content of pantothenic acid (chief constituent of coenayme A) decreases Hubstantially in the pollenn of Pinw and Alnua after storage for one year. This imbalance in coenzyme A upsets the general metabolism which in turn shortens the life of the pollen. It is of interest to note that stored pollen which fails to germinate in vitro may nevertheless cause a satisfactory seed set. The investigations of Olmo (1942) on grape, of Stone et al. (1943) on pistachio, of Hagiaya (1949) on tobacco and of Visser (1966) on tomato seme as examples. The stored pollen of these plants showed only 6% gemina0

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tion in vitro but proved quite effective for field use. Similarly, the pollen of potato stored at -344OC for 7-13 months gave almost no germination in culture but effected R good fruit set when used on the stigma (King, 1988). The pollen of cacao stored over calcium chloride at -20 to -30°C for 1-4 weeks showed only a low germination on an agar-dextrose medium, but field pollinations gave 40 - 50% fruit set (Soria and Denys, 1961). This is readily understandable because the stigmatic and stylar tissues help to make up some of the deficiencies imminent to storage and provide a natural environment for the germination of the pollen and the growth of the pollen tube. Thus, the failure of germination of stored pollen in cultures does not necessarily mean that the pollen is dead or useless. Similarly, the mere germination of stored pollen in w h o is no assurance that it wiU effect fertilization. For example, the pollen of wheat showed &lo% gennination on an agar medium but proved unsatisfactory for pollination in nature (KovkEik and Holienka, 1962). This implies that there are other factors which also control the germination of pollen. The pollen requires an adequate supply of moisture, inorganic elements, and a source of energy-usually a sugar. Some of these requirements may be met from the reserves of the pollen itself, but very often one or more of these act as limiting factors. The ambient humidity is critical for the germination of pollen. As substrates the lower surfaoe of fresh leaves of aquatic plants and sometimes even moist parchment paper have proved adequate. I n some instances good germination has also been obtained by merely placing the pollen near a hanging drop of water in a microchamber. Excessive moisture is deleterious to the pollen, and it is a common experience of plant breeders that pollinations carried out soon after rain or dew are frequently infructuoua. Schmucker (1933)reported that the pollen of Nymphueu germinated in a solution of glucose only when mixed with the stigmatic extract of the plant. Later, he detected boron in the extract and, therefore, replaced it'by traces of boric acid. This proved succeissful and quantitative estimations revealed that the pollen required almost the same concentration of boron aa was present in the stigma. Following Schmucker, many 0th- have identified boron in the stigma and style and have confirmed its favourable effect on the germination of pollen. It was believed that boron is related, in some unknown way, to the incorporation of carbohydrates in the pollen tube membrane. However, some studies indicate that it is essential to the spthe8ia of peotine in the middle lamella of newly formed calls (Spurr, 1967) and in the pollen tubes (Raghavan and Baruah, 1969). Stanley (1964) and Stanley and Luewus (1964) suggested that myo-inositol is probably an inter-

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mediate in the conversion of hexose sugar to pectin. On using tritiated myo-inositol with two conmtrations of boron (0.75 and 7.5 pg/ml) in cultures of the pollen of Pyrua communis they found that (a)boron combined with a speoific enzyme enabling it to bind and react with inositol, (6) myo-inositol waa readily converted into pectin, and (c) pectin synthesis inoreaaed at higher levels of boron. Theee observations suggest that boron plays a significant role in peotin synthesis in germinating pollen. . Like boron, calcium has been shown to enhance the germination of pollen aa well as the growth of the pollen tubes, and especially the latter. Its role is d i s o u s ~under h t i o n I1 C. Low conoentrations (0~001-0~0001 M) of dicarboxyb acids, such aa succinic, fumaric and adipic, have also been reported to stiIn&b the germination of pollen (Petrochenko, 1962). The pollens of many members of the family Ma1vaoea.e germinate only with some difficulty, but Bronckern (1961) obtained 7 0 4 2 % germination of the pollen of cotton iti artificial media in the preaence of amnaphthene. It is well known that during germination the pollen grains exercise a m m effeot (Brink, 1924). The ancient Arabs did not discard their stocks of old pollen of date palm but mixed it with the fresh lot and uaed the mixture quite liberally. It is possible that the old pollen, although inviable, contributed some chemical subatanoes to the mixture. Savelli and Caruso (1940) also mentioned a “mutual stimulation effect” in Nicotianu. When a large sample of pollen of one species wm added to a small population of pollen of another species both pollens were stimulated and showed better growth. Several investigators have also reported that in culturea a denser sowing of the pollen grains results in better germination than when only a few grains are wed

Fro.

1. Qerminetion of pollen showing masa effect; note good &tion pollon is dense (after Iwensmi, 1959).

w h v e r the

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(Fig. 1). Tigin (1962) found that tho addition of pollen of Hibiscus, Malva neglecta and Qossypium arboreum to that of cotton 108F, promoted the germination of pollen and the development of bolls as compared to selfing and pollination with limited pollen. Similarly, kedrina (1962) observed that supplementary pollinations as well as mixtures of pollen from more than one variety of maize increased the frequency of fertilization from 71*8-97*4y0 and fertilization was accomplished in 24-28 hours. On the contrary, selfing and pollinations with a restricted number of pollen grains caused a delay in fertilization and a decrease in the amount of food reserves in the grain. Likewise, VoZda (1962) has reported that mixed pollinations in maize resulted in an increased weight of the ears but the other characters remained unaltered. According to Bardier (1960) in wheat the grain set was considerably improved if rye pollen was used either before or a few hours after pollination by wheat pollen. Such a “mentor” effect of foreign pollen has also been reported for sugar beet (Kovarskij and Guzan, 1960), rye (Sulima, 1960) and sorghum (Zajceva, 1961), but all these observations need confirmation on the basis of more critical data. The receptivity of the stigma is another link in the chain of events leading to fertilization, although it is often rather difficult to control. Generally the receptivity of the stigma is determined by the age of the flower, and the ambient humidity and temperature. Jones and Newel1 (1948) have reported that under favourable conditions the stigma remained receptive for 19 days in Ruchloe dactyloides and for 24 days in Zea mays. According to Eghiazarjan (1962) stigmas of Nicotiana, less than a day old after anthesicc, possessed greater receptivity than those of unopened or older flowers, and mature pistils preferred self pollen to foreign pollen. The effect of the stigma is well known in certain phiits in which the pollen fails to germinato after self-pollinations (see Brewbaker, 1957). Jost (1907) attributed this self-sterility to a retarded growth of the pollen tubes. On the bad, of hi6 work on Corydalis cava, Lilium bzilbiferum, Secale cereale and other plants, he propounded the concept of “Individualstoffe”, meaning that many plants have a characteristic substance in the pistil which inhibits the growth of their own pollen tubes. Correns (1912) noted, however, that while individuals of the same clone or pure line were cross-sterile, they could hybridize freely with individuals of other clones or pure lines. He therefore postulated the presence of “Linienstoffe” as the underlying cause of self-sterility. I n other words, each pure line is charncterized by a substance which is specific to all of its individuals and is inhibitory to their pollen. This thcory opened the genetical approach to wlf-sterility, and Compton

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(1913) attempted to explain it in Mendelian terms. He itssumed that substances are formed in the pistil which stimulate or retard the growth of the pollen tube. He also drew an analogy between selfsterility and the growth of fungous hyphae into the host tissue end compared the mechanism of self-sterility with that of immunity against a pathogen. In some self-sterile plants successful self-pollinations can be made in the bud stage of the flower. Bmsica oleracea (Attia, 1960), Trifo2ium hybridum (Williams, 1961) and Nicotianu a h (Pandey, 1963) are some examples. In Muau the frequency of fertilization is often discouragingly low, even if abundant pollen is supplied. However, several clones show an increased fertility if bud pollination8 are m*de (Shepherd, 1954, 1960). No conclusive explanation is available for this phenomenon but it is postulated that some factors, which would inhibit the germination of pollen soon after natural self-pollination, might be absent or ineffective in the immature stigma. At the same time it may be noted that bud pollination is not always satisfactory and may lead to the formation of weaklings, heteroploids and sterile individuals (Iizuka, 1960). Liko bud pollination, bee pollination has also been reported to be beneficial. In certain intravarietal crowma of cotton, bee pollination resulted in an increased number of bolls, seod set, and the quality and yield of the fibre (Arutjunova and Skrebcov, 1962).

In the Cruciferae the incompatibility reaction is localized in the stigma and after self-pollinations the percentage of germination of pollen is negligible, Sometimes, this can be improved by increasing the ambient humidity. El Murabaa (1987) has reported that high temperatures (26°C) proved more favourable in overcoming self-incompatibility in Ruphanua sutivzcs than lower temperatures (17°C) which promoted crose-pollination. Sexual incompatibility is common in interspecific and intergeneric crosses. Nevertheless, several examples can bo mentioned of successful interspecific and intergeneric crosses, especially among the orchids, where the stigma has little or no deleterious effect on the germination of foreign pollen. C. POLLEN TUBE

The germination of pollen may not be NO diffigult as sustenmce of the growth of the pollen tube. A knowledge of tho extent of growth of pollen tubes in the pistil is tliereforo a pre-requieite in devising techniques to circumvent the bai~iersto crowability. Dissections, whole mounts and phase contrast microscopy are usually employed for this. In Rome studies on interspecific hylwiclizations in Nicotiana, Swnmina-

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than and Murty (1967a) demonstrated that autoradiography serves as a rapid and reliable meane for estimating the degree of growth of pollen tubes. Recently, Dempaey (1962) suggested the application of ultraviolet fluorescence for assessing the growth of pollen tubea in styles. Little direct evidence is available of the nature of the factors influencing the growth of the pollen tubes in the pistil. Some correlations have been suggested between the structure of the style and the growth of pollen tubes in it. Haeckel (1961) reported that (a) solid styles (i.e. where a transmitting tissue is present) generally hrcve a low starch oontent and show a very high phosphatase activity during the growth

A

Stigma /

Style

FIG.2. Growth of pollen tubes in Lilium lonqiflorYm. A. Part of style from which a pime. waa removed end thbn rephoed in inveree position and glued with gelatin. When pollen waa sown at the top, all the pollen tubes grew downward in the etyle. B and C. Growth of pollen t u b in horizontally orienhtwl etylee. From the region marked X in B Bome of the styhr tiesue wm ecooped out. In C an incision waa made in the style. &me pollen tuber grew toward the ovary end and others in the oppoeite direation. D. Excised stigma plsoed on M agar medium along with pollen ehowing growth of pollen tubes along ita inner wall (after Iwenami, 1959).

of pollen tubes, and (b) hollow styles (i.e. those which possess a canal) are rich in starch and show a high amylase activity. According to Brewbaker (1957), in incompatible plants having a solid style the growth of the tubes becomes arrested in the style itself, but where the style is hollow (LiZium and Lotus) the inhibition occurs in the ovary. Iwanami (1959) investigated the growth of the pollen tubes in LiZium longiJEormnt. In one aet of experiments he cut off a part of the style, replaced it upside down and glued it with gelatin (Fig. 2A). When the

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pollen was sown at the top almost rtll the pollen tubes were observed to grow downward in the style. In another experiment the style was placed horizontally and a portion of it was scooped out or an incision waa made in it, On sowing the pollen into the operated region some of the pollen t u l m grew toward the stigma and others in the opposite direction (Fig. 2B and C). I n still other experiments the stigma was cut at its baae and placed on an agar medium (Fig.2D). If the pollen waa sown around it, the tubes grew toward and along the inner w d of the stigma. On the basis of these observations Iwanami (1969) concluded that an “inductive substance” is present in the pistil and it serves to draw the pollen tubes which are initiated at the surface of the stigma first into the stigmatic tissue and then into the style. Once the pollen tubes enter the style they grow of their own acoord in the direction of the ovary. Among the physical factors influencing the growth of the pollen tubes, temperature is the most important. With an increase in tempemture, the rate of growth is appreciably enhanced, a maximum being reached at between 20 and 30°C. However, in many illegitimate selfpollinations the optimal temperature favouring the growth of pollen tubes is lower than in the corresponding legitimate cross-pollinations. As the temperature rises the growth rate of the incompatible tubes diminishes. Lewis (1949) attributes this to an inhibitory reaction which proaeeda faster at higher temperatures. A good deal of information has accumulated on the iniluenoe of several kinds of chemicals i n d extracts of tissues from h o s t all parts of the plant including even the root. Among the substances which have been reported to have a stirnulatory effect on the growth of pollen tubes are alcohols, amino acids, auxins, carotenoids, colchicine, gibberellic acid, kinetin, metallic ions, purines, sugars, vitamins and yeast extract (see Chandler, 1957; Sawada, 1958; Raghavan and Baruah, 1969; Takami, 1969; John and Vasil, 1961; Singh and Randhawa, 1961; Prasad and Mehrotra, 1963). Further investigatiom are required to determine whether these substances, particularly the carotenoids, also possess any chemotropic effect (see Rosen, 1962). The physiology of sexual incompatibility is not fully understood and moet of the explanations are only speculative. East (1929) propounded the immunity theory suggesting an antigen-antibody mction between the pollen and the pistil. Preformed substances in the style are thought to react with antigens from the pollen, and the reaction products are believed to cause an inhibition of the pollen tubes, On the basis of his experiments with Petunia,Straub (1947) believed that the pollen tubee produce a substance necessary for their growth through the style, but in illegitimate pollinations this substance is rapidly used up and its

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shortage terminates the further growth of the tubes. Later, Straub (1964) explained that in illegitimate self-pollinations certain inhibitory substances are formed which prevent the normal growth of the pollen tubes. The recent studies of Tomkovti (1969), Linskens et al. (1960), and Brewbaker and Majumder (1961) support this hypothesis. Tomkov6 demonstrated the presence of an inhibitory principle in the Rtyles of self-pollinated flowers of Nicotianu a h h . On the other hand, in the styles of cross-pollinatedflowers it was detected only in negligible amounts. In a self-incompatible Rpecies of Petunia, Linnkens et al. (1960) X-irradiated mature st'yles and elf-pollinated them immediately. Nearly 60y0of the treated flowers set seed. When flowers were selferl 20 hr prior to or 24 hr after irradiation, there was no seed set. It is explained that once the incompatibility reaction has begun to operate in the style it cannot be destroyed by irradiation, although it can be arrested by X-irradiating the unpollinated style at a stipulated dose and time. However, such an inhibition is reversible because the irradiation merely causes some biochemical changes in the immunological mechanisni and not any mutation. As already mentioned a mass effect occurs in artificial cultures of pollen (see Fig. 1). Whereas small populations of pollen germinate but poorly, large populations show good germination and an excellent growth of pollen tubes. Brewbaker and Majumder (1961) demonstrated that the population effect is due to the action of a stable, water-soluble and highly diffusible factor which was designated as the pollen growth factor (PGF). They suggested that in cultures of small populations of pollen the quantity of the PGF is deficient. Recently, Brewbaker and Kwack (1963) adduced experimental evidence to show that in artificial cultures the PGF leaches into the external medium and therefore the germination and growth of pollen tubes are negligible. They added cell-free extracts of anthers to the medium on which small populations of pollen were cultured. With incretwing concentrations of the anther extract (number of anthers extracted in 2 ml medium) the percentage of germination also increased. The optimum lev& of germination were obtained with the use of 100 anthers. The population effect could be replaced by a growth factor obtained from aqueous extracts of many plant tissues, even including the root. UHing pollenash as the starting material they identified this factor as the calcium ion. In further experiments Brewbaker and Kwack cultured the pollen in sucroseboric acid medium supplemented with different cations and observed that satisfactory germination and growth of pollen tubes occurred on a medium which included calcium, boron, magnesium and potassium. When these elements were used individually no germ-

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ination occurred. By an elimination technique, they conclude that all the four ions (calcium, boron, magnesium and potassium) me essential for germination and growth of pollen tubes. In the Cruciferas the incompatibility reaction is manifested right on the surface of the stigmatic papillae which contain an inhibitory substance (Kroh, 1966). Heinen and Linskens (1961), and Linskens and Heinen (1962) observed that in Brassica nigra the pollen tubes formed after s e h g are unable to penetrate the cuticle of the papillae, but this barrier is easily overcome by treating the stigma with a cutinase obtained from moulds. On the basis of his work on apple and tobacco, Tupg (1959) found that in compatible crosses the pollen tubes grow rapidly and the available glucose is utilized to build the wall material with hardly any midue left in the form of callose. In incompatible matings, on the other hand, tho pollen tubes grow very slowly. The utilization of glucose is appreciably restricted; and large quantities of callose accumulate and hinder the further growth of the pollen tubes. These observations are supported by the cytochemical investigations of Schlasser (1961) on Petunia. On the basis of tracer studies, Tupj (1961a,b) has further concluded that in incompatible pollinations the arrest in the growth of pollen tubes is not due to a deficiency of the respiratory substrate but to the inhibition of respiration. This explanation is based on the following observations: (a) during the growth of the pollen tubes in the style there is an increased inflow of carbohydrates; (b) in contrast with compatible crosses, in incompatible pollinations the utilization of the carbohydrates is limited and there is a relative deficiency of y-aminobutyric acid and alanine; and (c) such a deficiency of these two amino acids is due to lack of a-ketoglutaric acid which is involved in their transamination.

111. CONTROLOF FERTILIZATION The basic facts about fertilization have already been outlined in the introduction. This section deals with some of the barriers to fertilization and their control. One or both of the participants in the act of fertilization may mmetimes pose problems which eventually lead to the lack of embryo formation. Sometimev the failure of fertiha'tion is merely due to an early abmission of the flower. I n such instances i t may be possible to prolong the life of the flower by the application, of hormones (see Crane and Marks, 1952; Brock, 1954).

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It is also feasible to apply to the stigma an artificial medium which is more suited for tho germination of the particular pollen than the secretions of the stigma. For some fruit trees boric acid sprays have been reported to increase the germination of the pollen (Batjer and Thompson, 1949). I n interspecific crosses of Trifolium Evans and Denward (1956) found that the application of NOA to the stigma resulted in a successful germination of pollen. I n some intervarietal crosses of potato a better germination of pollen and improved seed set have been obtained by treating the stigma with an extract of the anthers of a freely crossing variety of potato (Frimmel, 1966). I n self-sterile plants such a8 Petunia and Niwtiana the stigma has heen reported t o be inhibitory to Relf pollen. Takfishima (1964) demonstrated that in Petunia a washing of the mature stigma followed by smearing it with the secretion from a compatible strain removed the barrier to self-sterility. Such a disguised stigma did not exhibit any antagonism to self pollen and allowed it to germinate normally. In the self-incompatible Nicotianu ahta seed sot was increased more than 200-fold by an application of the stigmatic secretion of a mature stigma of the female parent to a dry stigma of the recipient flower bud (Pandey, 1963).

n. TREATMENT OF THE STYLE More often, however, the cause for the failure of fertilization lies in the style. Many experiments have, therefore, been directed to an understanding of the physiological role of the style in the control of fertilization. Some of these go back to the year 1907, when Jost demonstrated that pollen tubes can grow through the styles of two species placed end to end. Later investigators improved this technique and successfully overcame many instances of incompatibility (see Maheshwari, 1960). Their work has shown that the incompatibility response operates at any level either in the stigma or in the style. Employihg R new technique of stylar grafting Hecht (1960, 1963) demonstrated that in Oenothra the incompatibility reaction k stronger in the stigmatic region than in the style. Prom a oompatible strain of 0. organensis he excised the style 16 mm below the stigma and grafted it on to the lower part of a similarly cut style of an incompatible strain. The stock and the scion were superposed by a “splint” made of 6% lactose in 10% gelatin and a piece of lens paper. The “grafts” were maintained at 27°C for 15 hr in petri dishes containing moist filter paper. Under these conditions the self pollen tubes grew through the

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compatible scion into the incompatible stock up to an average length of 22.1 mm. I n another set of experiments on 0.w p i t o 8 a , 0.orgamm&9 and 0. rhombipdab Hecht (1963) made grafts (a)botween n compatible scion and a stigmtio lobe (stock) which WILH RIAO compntible, and (b) between a oompntible mion and an incompatible stigma lobe. I n the first type the pollen tubes grew readily from the cut end of the scion into the stock. I n the second where an incompatible stigma lobe formed the stock the pollen tubes were completely halted at the stigmatic surface. To determine the degree of the incompatibility reaction in the stigma, and style Bali (1963) made similar grafts in 0.rhmnbipetala. In the pontrol the pollen tubes grew through the scion, then through the stigmatic lobe of the stock and finally up to the baw of the stook style. In the incompatible grafts, on the other hand, the pollen tubes growing through the scion failed to penetrate the stigmatic lobe of the’ stock. These experiments suggest that the incompatibility reaction is complete a t the stigma and the self pollen tubes are debarred from entering into an incompatible stigma even if they had previously passed through a compatible region. Whether these techniques of stylar grafting can be applied to overcome incompatibility in fertilization is yet to be ascertained. Hybridization of a long-styled female parent with a short-styled male parent is usually unsuccessful whereas the reciprocal cross between a short-styled pistillate and a long-styled pollen parent presents no special diffculty. This is in accordance with the observation that the longer the style posses~edby a species, the greater is the growth potency of its pollen tubes. For example, the pollen tubes of Nicotiana rustica (10 mm long style) fail to reach the ovary of N. pniculatac (20-30 mm long style) while the reciprocal cross is eaaily achieved, Mention may also be made of the experiments of Gardella (1950) on Datura inrwxia, D. quercifolia and D . ferox whose styles me 130-170, 30-40 and 25-36 mm long respectively. When I ) . innoxia waN used as the female parent and the other two species as male parents she observed that the seed set was negligible. To improve it G a r d e b performed two kinds of experiments. Jn the fir&the upper portion of the style of the female parent was cut away and substituted by the stigma-bearing stylar portion of the male parent so that the total length of the “graft” was reduced to about 20 mm. This permitted the pollen tubes to grow as far as possible in thoir own stylar environment. In the second method, called RtyIe insertion, the Htyle of the female parent was completely replaced by that of the pollen parent. The situation in Lathyrus odoratus (10 mm long style) xL. hirsutug (4 mm long style) is similar. Davies (1967) amputated the style of L. odoratw and pollinated the cut end. Swaminathan (1956) mcom-

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mended the use of an artifioid stigma for some interspecific crosses in tlie Mexican solanuma. By applying an agar-sucrosegelatin medium 011 the stub and covering it with moist cotton wool after pollination, he obtained the otherwise incompatible croeses between Solanurn pinnutisecturn xS. bulbocastanum, and 8.pinnutisecturn x S . h n c i f m e . A similar procedure was tried with success in the cross Nicotiana tabacum x N . r?mticu (Swaminathan and Murty, 1957b). When trisomic plants of Datura stramonium are used as ,-3 parents, fertilization by sperms of the ( n + l ) complement is only a chance occurrence owing to tho slow growth of the pollen tubes. Buchholz et al. (1932) showed that at tho time when the pollen tubes of the n type had advanced to the base of the style, those of the ( n + l ) type had grown to only about lialf the length of the style. If the lower part of the style was now cut away to elirninatc the n pollen tubes and the upper portion containing the ( n + l ) pollen tubes affixed to the ovary, fertilization was achieved by the ( n + l ) sperms. When a diploid plant is treated with pollen from a tetraploid there is usually a high degree of incompatibility. Generally the pollen grains fail to germinate and even if they do tlie pollen tubes burst in the diploid styles. In Datura it is explained that the epidermal layer which lines the interior of the stigma and style in the diploid female parent exercises an antagonistic effect on the diploid pollen tubes derived from a tetraploid male parent. To enable such a cross Satina (see Avery et al., 1959)troated the female parent with colchicine and induced the formation of a periclinal chimaera which now had a tetraploid epidermal luycr. although it was otherwise diploid. The pollen tubes grew in such a style and fertilized the emlwyo sace.

C. INTRAOVARIAN AND I N VITRO FERTILIZATIO

A direct introduction of pollen into the ovary was thought of aa early as 1886 by Strasburger wlio tried to make such int'rwvarian pollinations in orchids. Kusano (1915) repeated Strasburger's experiments with another oschid, Gmtrodia ehta. For introducing the pollinium, he severed tlie upper portion of the ovary or made an opening on it. A satisfactory germination of the pollinium occurred followed by fruit formation. Several ovules received the pollen tubes and the seeds showed a normal development of the embryos. When polliriia of Bletia hyacinthinu were used, germination still occitrred and stimulated the growth of the ovitrios of Gastrodia but the seeds had no embryos. The experiments of Dahlgren (1926) on CorEonopsi~ ovata, of Cappelletti (1937) on Digitalis purpurea and of Bosio (1940)

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on species of He11ebortu arid Paeouici fiirther indicated the feasibility of this technique. In Yaeonia some viiLble seeds were also obtained. More recontly Mahediwari i ~ n dKttntci (1961, 1962), and Kanta and Maheshwari ( 1963a) have tfasrrihetl the results of their experiments with Payaver rhoaa8, P.BO?NniJorrr?ti, fitwh.ucholzl:acali,fornicu,ArcJemone mexirana anti A . ochruleiccu. A pollen suHpenrrion was prepriretl in (I

Fro. 3. Intruovlrrinn pollinntion in I’upuver ~ o ) ~ i i i ~ e rA. t ~ Ovary )a. at t h e time of injection of jwllen suspension in I00 ppni boric: twill aolution; wrow shown point of injection. 13. Vertical half of ovary shown in A . C. T.S. Iiortion of ovirry 3 iluyw nftrr injct:tion of pollon nunpcnHirm; showing 4-(!1dli!iJ iiriw~nt~ryt~; note gcrminntion of p11i.n. 1). Id.niicmpylar portion ol‘ WIIIC notc pmiatcnt p l l c n tubc. 14:. 47-l)ny-oltl fruit. ol~titinidttirouyh intrtwvclritin pollinrrtion itnci fcitilization. 10. Vt?rticd lirrlf of C.ILPNII~I! sliown in E; whitc: Imlion are tlic tiitlJle HOC~JH. C. Ncrdling ruiard from ficld-borne nt*rtl. FI Swtlling rc:;iretl from secd obtained thr~iugh int,mov:iriun pollination niid fcrtiliziit~ion(nfter Kantrb and Miiliunliwari, 1963a).

.

2 1111 solution of O.Ol‘ll, boric acid in sterile, double dilltilled water. The flowers were eninsculated iind the surfitue of the ovary wiped with cotton soaked in ethyl alcohol. T w o punctures were made 011 the ovnry--ime to dlow the air to escnpe mid the other to inject the

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suspension of pollen (Fig. 3-4and 13). After the injection the punctures were se;tled with petroleum jelly. T n the treated ovaries germination of t,he pollen, entry of pollen tubes into embryo sacs, and double fertilization occurred as in nature (Fig. 3C and n). The ovaries

PIG.4. In oilro fertilimtion in P ~ J ~ J ~oirin,ijertlm.. IIVP~ A. Culture of I d l e n wit1 ~ V U ~ Con H Nit,srh’s iigiir medium. 1%i i r i t l ( ’ . 7-lhiy-oldd t o r e u ; tlic white bodies are the ilcvcloping rrcedri. 1). Whole mount of ovule frnm 3-iIriydI cdtiire showing sever&] germinating pollen grains. E. 1)isnc.ction from n 5-day-nit1 culture slinwinp n 4-uc.lletl proembryo imheddod in the endoqicrrn. I: rind G. (:loliul;ir c1llIJr;vO i d fully hrmrd embryo excised from 9-day-old and 2!?-dny-oltlW Y I ~o l ~ t , ~ i nini ~viilturc: l (after l i t m t t t e6 (I!,., 1962).

grew nonniilly aiid developed into capsules bearing viable seeds (Pig. 3E t o H ) . The nl~ovemetiiod lins becn used to hybridize A . mexicana (an = 28) and A . ochroleucn (2n -- 56). ‘L‘hey rarely cross in nature. Stigmatic pollinations to raise 11yhrids have been discouraging especially when A . och:sobuca is used its the fomaIe parent. Kanta and Maheshwari

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(1963a) applied the technique of intruovarian pollination and found that it proved markedly efficacious in tho cross A . ochrole~%X A . mexicuna. In the reciprocal crow also intraovnrian pollinations oonsiderably improved the seed set and the seeds were fully viable. These results suggest that intraovarian pollinations may be specially uaeful in instances where the zone of incompatibility lies in the stigma and the style. A still more promising method is to excise the ovules and rear them together with the pollen in an artificial medium, thus eliminating the gynoecial tissues altogether. Of much interest in this connection are the recent papers by Kanta et al. (1962), Kanta and Maheshwari (1963b), and Maheshwari and Kanta (1964). They excised the unfertilized ovules o f P . eomniferum, A . mexiccana, E . alifornim and Nicotiana tabacum just after anthesis and cultured them on an agar nutrient medium along with pollen grains collected from ripe anthers. All the stages from the germination of pollen to double fertilization were observed and mature seeds containing viable embryos were obtained in culture (Fig. 4). On germination the seeds gave rise to normal seedlings. The technique of test-tube fertilization involves two major stepsovule culture and gernlination of pollen. Whereas it is not too difficult to achieve a satiefactory germination of pollen, the chief concern is the rearing of unfertilized ovuletj to victble seeds through fertilization. With further refinements thiv technique, like that of embryo culture, would prove a boon in wide hydridizntiori in that it is possibly the best approach to overthrow the various incompatibility barriers instituted by the gynoecial tissues. Even when fertilization can be accomplished with reasonable success, there are other problems like the abortion of the young embryo. The control of these post-fertilization barriers demands a n understanding of the development of the embryo and its relationships with the endosperm and the maternal tissues of the seed.

IV. EMBRYO A. GROWTH OB EMBRYO IN RELATION TO

SEED DEVELOPMENT

The embryo is the young fiporophyto of the new generation. For its normal growth and difforentiation, it is dependont on it8 immediate environment, namely the endosperm. The entloHperm in turn draws upon the somatic tissues of the plant via the placenta and funicle. This implies that the parent sporophyte exercises control, at least in part, over the development of the seed by the nature and quantity of the material which it supplies to tho seed. Conversely, the seed in

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turn affecta the growth of the fruit. Thus, fruits with many seeds are usually larger than those with fewer seeds (see Nitsch, 1952; Luckwill, 1969). Gustafson (1939) demonstrated that in immature fruits of the tomato the seeds, placentae, septa and pericarp contain decreasing concentrations of auxin. This suggests that auxin is synthesized in the me& and transported to the other parts of the fruit along a decreming gradient. That auxins play a role in the growth of the fruit is also confirmed by other data. One indicator is that an application of synthetic auxins may stimulate the development of the fruit even in the absence of fertilization. The spraying of auxins to improve fruit set has now become a standard prtwtice in many countries for certain varieties of tomato, apricots, figs, grapes and oranges. I n the blackberry and strawberry there is often partial sterility or inadequate pollination leading to tho formation of fewer Reeds and a subnormal growth of the fruits. In uuah instanceiJ applications of auxins have proved beneficial in increasing the size of the fruits. Dionne (1968) observed a negligible seed set in some interspecific crosses of Sohnum. He attributed it to a poor development of the fruit which in turn was correlated with the presence of too small n number of seeds to stimulate the optimum growth of the ovary. A treatment of the ovaries, 24 hr after pollination, with 2,4-D (1 drop of a 3-6 ppm solution) promoted the development of the pericarp which in turn stimulated the formation of seeds. Similarly, in the self-incompatbile Lilium longifEOrum Emsweller et al. (1962) obtained a good seed set through the use of naphthalene acetamide and potassium gibberellate. At the time of pollination a lanolin paste of 1% naphthalene acetamide was applied to the wound caused by tievering one of the petals. Potassium gibberellate was used as an aqueous spray (0.01-0.2% plus 0.1% Tween 20). Alternate flower8 on tho inflorescence were left untreated. Observations made from the twelfth to the twenty-sixth day after pollination revealed that naphthalene acetamide prevented the senescence of the ovaries and ovules, and favoured their development into normal fruits and seeds. In the untreated controls, on the other hand, the ovules showed a progressive degeneration and collapse of the ovaries. Potassium gibberellate proved less efficacious than naphthalene acetamide. For raising hybrids between Bramica oleracea var. m p h a l a and Raphnw Honma and Otto (1962) used Nrn-tolyphthalamic acid. In this case a piece of cotton soaked in a 100 ppm solution was applied to the pedicel at the time of pollination. Following the treatment, several pods were formed, although the number of viable seeds ww discouraging. These experiments suggest that the development of the fruit and the formation of the seeds proceed in harmony and that both the phenomena are influenced by growth regulators.

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B. DEPENDENCE OF EMBRYO ON ENDOSPERM

In general the endosperm is regarded as a nurse tissue for the embryo and there are several circumstantial observations which support this view. 1. At the time of fertilization the embryo R ~ has C very little nutritive material. As the endosperm advmces in development it stores enough food substances to insure an adequate supply for the developing embryo. 2. I n the majority of flowering plants the zygote segments only after the endosperm has reached a reasonable stage of development. Even in suoh instances where the division of the zygote precedes or occurs simultaneously with that of the endosperm, the latter soon surpasses the embryo in growth. 3. Generally, the embryo develop8 only when the endosperm is properly organized. If the endosperm aborts, as in many incompatible matings, the growth of the embryo is adversely affected. 4. In the absence of the endosperm (as in Podostemaceae, Trapaceae and Orchidaceae) special provisions exist to ensub the nutrition of the developing embryo (see Subramanyam, 1960; Johri, 1962). I n the Podostemaceae a pseudoembryo sac ig formed as a substitute for the endosperm. T r a p has a large suspensor haustorium. I n the orchids also, the suspensor often develops into a haustorial organ of considerable dimensions. The hauRtorial branches seem to function as absorptive structures which enable the embryo to grow even when it is not accompanied by the endosperm. Large and hamtorial suspensors are no doubt recorded in many other plants which also have a functional endosperm (for examples see Maheshwari, 1950), but here they probably serve as accessory structures which convey food materials to the main body of the endosperm which in turn relearres them for the embryo. 6. During its growth the embryo depletes the surrounding cells of the endosperm of their contents. In many plants, such as members of the Leguminosae, Cucurhitaceae and Compositae, the embryo consumes nearly the whole of the endosperm. I n the Graminem, Euphorbiaceae and Solanaceae, on the other hand, the endosperm stores food uuhstances which are utilized during the germination of the seed. ln wch plants only those emdosperm cells are digested during the development of the seed which lie in immediutc proximity to tho embryo. C. SPECIFTCITY IN NUTRITION O F EMBRYO

Two questions may arise regarding the function of the endoHperm aa a nurse tissue: (a) what is the nature of the material which t h e

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endosperm provides to tlio embryo, and (b) how specific is this substance. In other words, can the endosperm of a species serve to nurse the embryo of another species? For convenience the second question is discussed first. The available information on the specificity of the endosperm is based chiefly on experiments with mature or nearly mature endosperm whose food reserves are normally utilized during the germination of the seed and not during the earlier development of the embryo. This is particularly true of heterologous transplantations, where embryos of one species are exciwd and cultured on tho endosperm of another species. Sting1 (1907) observed that embryos of Triticum grown on the endosperm of Secale frequently gave better seedlings than when the embryo was reared on the endosperm of its own species. Similar experiments have been made by Grekoff (1940), Ciimara (1943), Yamasaki (1947, 1960), David and SOchet (1948), Hall (1964, 1966) and Strun (1966). Clmara made interspecific embryo-endosperm transplantations in Aqibp8, Avena, Hordezim, ISecale and Trilicum at the “milk” stage of the endosperm and noted differences in some features of the seed produced in Fl plant8. Also, the embryo of Triticum vdgare grew better on the endosperm of T.duruni than on its own endosperm or that of T.turgidurn. Several Russian investigators have claimed heritable changes in the plants raised by this method. However, Mathon (1952) has explained that such genetic diversities arise by a modification in the rate of growth and differentiation of the embryo due to the altered environmental conditions. More critical observations are necessary before these conclusions can be accepted. Studies on cultured embryos of related taxa suggest that in earlier stages also the embryo does not show an absolute spocificity for the endosperm. At the hcart-shaped stage the embryos differ only slightly in their requirements for inorganic elements, sugars and nitrogen compounds, arid the range of their tolerance to nutrient materials is fairly large (see Rappaport, 1954). Furthermore, even embryos of plants belonging to diverbe fanlilies can grow in almost identical nutrient media.

V. ENDOSPERM A. CONSTITUENTS OF ENDOSPERM

As already mentioned, the available information largely concerns the nutritional requirements of older embryos while the cature of nutrients supplied by the endosperm to the embryo during its early growth is less understood. A knowledge of the Binds and quantities of food materials and other substances which are stored and /or synthesized

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in the endosperm during its development is therefore fundamental to an understanding of its role in the growth of the embryo. Van Overbeek et a,?. (1941, 1942) found that the milk of young coconuts had a stirnulatory effect on isolated embryos of Datura stramonium. The “embryo factor’’ in coconut milk is now known to be a complex of potent substances. According to Steward (1963) this is probably related to the formation of proteins, and its effect stems from a complex of substances each of which is individually unable to control growth to the same extent as the entire complex (see also Steward and Mohan Ram, 1981). Growth-promoting substances also occur in the liquid endosperm of Zeu m y 8 (Steward and Caplin, 1952), Allanblackia parvi,fora (Nitsch, 1963), Aesculus hippocastmum (Shaiitz and Steward, 1955), in the banana, in the feniale gametophytc of Ginkgo biloba (Steward and Shatnz, 1969), and in the endosperm of Sechium edule, Camellia japonica and Thea sinensie (Tto, 1961). Extracts of these tissues have the property of inducing the multiplication and enlargement of cells in explants. Some of the substances which have already been isolated and identified are : 1,3-diphenyl urca; indoleacetie acid; xanthine, chlorogenic acid, hexitola, purine-type conipounds and leucoanthocyanins. Once it is known what subrrtsnces are provided by the endosperm for the growth of the embryo, thcir specific actions can be studied. At present, however, we have only a partial knowledge of these. Earlier, Haagen-Smit et al. (1946)had reported that in the immature endosperm of corn only 9% of the auxin is accounted for by indoleacetic acid, thereby suggesting the presence of other substances of similar activity. Evidence is accumulating for the rather widespread occurrence in seeds of substances with gibberellin-like properties and they have already been recognized in the endosperms of Aesculus californica, Echinocystis mcrocarp, Persea amerimna, Prunw amygdalw, P . armeniaca and P . domestica (Phinney et al., 1957),Pyrus m l w r (Nitsch, 1968), Cocos nucifera (Radley and Dear, 1958), and in the seeds of Phaseolwr, Pisum and Zea muys (Radley, 1958). The endosperm thus contains a variety of growth-regulating substances which, at least partly, control the growth and differontiation of the embryo. The investigations of Avery et al. (1942)on developing maize kernels suggest that at the time of pollination their free auxin content is extremely low, but immediately after pollination it rises very sharply and a peak is reached within 1-3 weeks. No correlation could be established between the vigour of hybrids or polyploids and the amount of auxin stored in their kernels. Kernels with B sugary endosperm wem consistently richer in total auxin than those with a starchy endosperm. It is also suggested that the amount of auxin in the seed is nearly P

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proportional to the quantity of cellular endosperm present. The concentration of auxin in the endosperm is considerably higher than that in the embryo (see Luckwill, 1957). Duviuk (1962) has analysed the endosperm of four varieties of maize at different periods of development and reported the presence of nine amino acids and two amides. He found that most of the proteins of the glutein complex (alkali-soluble) are synthesized during the first half, and most of those of the zein complex (alcohol-soluble) during the second half of maturation of the grain. For Datura stramonium it has been shown that 2-3 weeks aftor fertilization the concentrations of amides and free amino acidx reach a maximum, but when the see& are mature the concentrations drop off considerably, and only asparagine and aspartic acid show a steady increase (Rietsema and Blondel, 1959).

Chemical analyses of the ertdoaperm like the above have, however, so far yielded only meagre data on the role of the endosperm in the growth of the embryo. Tho main source of information on this mpect is the culturo of isolated embryos, whose requirements may be expected to correspond largely with the materials received by them from the endosperm. However, the favourable effect of a substance on the growth of excised embryos in aseptic culture does not necessarily imply that it is also an active factor in the seed. Thus, in the seeds of Datura stramonium the embryos have much oil but no starch, whereas excised embryos grown on a medium containing sucrose accumulate starch instead of oil. Further, the endosperm is not a homogeneous tissue; often the cells frQm its different regions vary not only in size, shape and staining capacity but also in function. For example, the cells adjacent to the embryo become “dissolved” while those away from it continue to store reserve foods. The findings of embryo culture must, therefore, be interpreted with caution in explaining the role of the endosperm for the growth of the embryo. B. ROLE OF ENDOSPERM IN SEED DEVELOPMENT

It is more probable that the function of the endouperm is related in some ways to the quantity and proportions of the growth substances present in it. Any disturbance in tbia equilibrium leads to the abortion of the seeds. For example, the content of free amino acids in tho ~eeds of Datura innoxia after selfing and after crosfiing with D. d i a w h (d) varies considerably and the cross with D . discolor is incompatible because the ovules abort after fertilization (Rietsema and Satina, 1959).

The presence of growth substances in the seed becomes manifest immediately after fertilization. Murneek and Wittwer (1942) and

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Wittwer (1943) found that fertilization has a stirnulatory effect on the metabolism of the whole plant. In corn, McLane and Murneek (1962) reported a substance which they termed as syngnmin. According to them its concentration reached the peak 5 days after fertilization and it greatly stimulated tho growth of excised embryos of corn. MEIT* and Murneek (1963) stated that the hormones produced in the seed have an important role in regulating the movement and accumulation of carbohydrates and nitrogenous substances towards the developing fruits. In naturally pollinated ears of corn as well as in unpollinated ears treated with NAA or the ethyl ester of IAA they observed (a) a distinct increase in the content of sbiI3rch,( b ) a decrease in the content of sucrose chiefly during the first three days after treatment, (c) an increasing Concentration of reducing sugars, and (d) a moderate but significant increase of hexose phosphates. On the contrary, no significant changes occurred in the carbohydrate content of unpollinated controls. h r t h e r , the similar respon,ses of both t h e kernel and the cob to treatment suggest that hormones influence the carbohydrate metabolism not merely in the fruit and the seed but may also have analogous effects on the neighbouring tissues. The above discussion has indicated the iniportanco of the endosperm in the development of the seed. Many exampla are known in which the aborting seeds show a breakdown of the endosperm after a certain number of cell divisions. In incompatible crossos there often develop empty seeds which are much s~rinllerthan tho seods formed &B the result of successful pollinations. In other words, a degenerating endosperm may lead to a poor development of the seed itself (Rietsema et al., 1966). The formation of viable seeds is therefore a function of the balance between the embryo and the endosperm (Shepherd, 1960). This necessitates an examination of the factors which govern the growth of the two partners independently of each other. C. CULTURE OF ENDOSPERM

It is well known that of the two sperms delivered by a poIlen tube, one fertilizes the egg and the other tho secondary nucleus. The zygote organizes itself into the embryo and the triple fusion nucleus produces the endoaperm. What directs this differential behaviour is not understood. That it is not the difference in the chromosome numbers of the two tissues which is significant is indicated by the condition in the Oenotheraceae where the embryo and the endosperm are both diploid. A logical approach to the problem is to grow the endosperm and the embryo of the same species in vitro and to experiment with the tissues obtained from them.

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La Rue (1947) obtained a successful culture of the endosperm of Zecc m y 8 on a medium contnining tomato juice, or yeast extract, or the extract of unripe kernels of maize itself. Subsequently, Straus and La Rue (1954) cultured the endosperms of the sugary, starchy ,and waxy varieties of Zaa. Only the sugary endosperm readily formed a callus tissue. Tomato juice brought about an erratic growth but yeast extract was quite satisfactory, the Seitz-filtered solution being twice &B effective as the autoclaved one. Sternheimer (1954), and Tamaoki and Ullstrup (1968), also succeeded in rearing the endosperms of the sugary, waxy and starchy varieties of 8. m y 8 on a medium supplementod with yeast extract. Coconut milk did not prove beneficial to the cultures of the endosperm. In tosting the effects of several carbohydrates Straus and Ln Rue found Rucrose to be the most effective in supporting tho growth of the endosperm while glucose was the least. Some other sugar8 (arabinoso,galactose, glycerol, lactose and rhamnose) did not enhance the growth of the tissue at all; instead they caused a decrease in its weight. The endosperm tissue grown in vitro often showed a number of chromosomal aberrations leading to the formation of polyploid cclls. Since the endosperm grew in cultures only when yeast extract waa supplied, Straus (1960) studied the effects of some amino acids and amides present in the extract. Aspartic acid, glutamic acid, aaparagine and glutamina all supported the formation of a callus, but only asparagine could suitably replace yeast extract. The endosperms which had a pigmentod aleurone layer produced callus tissues which were purple due to the presence of anthocyanins. Straus (1959) reported that aspartic acid and cywteine strongly promoted the synthesis of anthocyanins in endosperm tissue cultures. Norstog (1956) excimd the endosperm of Lolium perenne at variow stages of development and cultured it on White's medium eupplemented with yeast extract or coconut milk. It proliferated vigorously FIQ.6 (See facing p a g e ) .

FIQ.6. Culturc of cmbryo and endosperm of Santalum albwm (hyp, hypocotyl; ra, radicle). A. 3-week-old culture of a seed on a modified White's basal medium; the seed enlarged slightly but failed to germinate. B. Culture of liimilar age on the basal medium supplemented with coconut milk (20%)+ caacin hydrolysate (400 ppm): the primarg root and hypocotyl are well developed; plumule and cotyledons arc still partially enclosed in the degenerated endosperm. C. 6-Week-old culture as in B; a normal plantlet has formed. D. 3-Week-old culture of seed on the basal medium+yeast extract (0.25%)+ kinetin (6 ppm)+P,CD (2 ppm). The endosperm hea proliferated along the sides and the embryo has just emcrged from it. E. A similar culture BB in D. from which the embryo waa excised. The endosperm continued ite proliferation and in 3 weeks formed an extensivc mass of callus tissue. F. 2-Week-oldsubculture of a fiagmcnt of the endosperm callus (after Rangaswamy and Rao, 1983).

1’

*

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and mitie of the cells developed into starch storing cells. Many showed c:hroniosomnl aberrations similar t o those in maize endosperm. Ntiktijimu (1902) excisctd the endosperm of Cucumia sativw 10 days d t e r pollination and cultured it with considerable success on White’s medium supplemented with yeast extract. A mixture of IAA, 1,3diphenylurea and casein hydrolysate could effectively replace yeast extract. According to Nakajinia the successful growth of the endosperm depends on the presence of an auxin, a kinin and an organic substance rich in nitrogen. Recently, Rnngaswaniy and Rao (1963) have successfully grown the endoRperin of Santdwn album. When the seeds were cultured on Wliite’n mediuni, there WIW no npprecinbla response except for an ovordl enlargenient of tho Heed (Fig. 6A). If the ba~ttlmedium was supplemented with casein hydrolysate (400 ppm) and coconut milk (‘LOO/,)the embryos gerininated, conmiming the endosperm. I n about G weeks normal seedlings were formed (Fig. 6B and C). If the seede were cultured on a medium supplemented with yeast extract (0-25y0), kinetin (5 ppm) and 2,4-I)(2 pprn), the seeds enlarged many times their initial size in 2-3 weeks. In t;0-70% of the cultures they grew into a large mass of callus tissue (Fig. SD). Both microtomepreparations and dissections showed that only the endosperm proliferated while the embryo merely elongated slightly. If the embryo was excised from the proliferating endosperm nnd grown separately it made no growth. The endosperm continued its proliferation and formed an extensive mass of callus tissue from which small masses of tissue broke away (Fig. SE). Fragments of the endosperm callus were subcultured and the cultures have been maintained for more than 12 months through four passages (Fig. BF). These investigations suggest a similarity of the growth requirements of the endosperms ofwidely aeparated taxa like Zen, Lolium, Cucumit~ and Santalum. For a satisfactory proliferation readily lending to subculture the endospermfi probably require an auxin and kinin (2,4-D and kinetin for Santalum) in conjunction with yeast extract. The establishment in tissue cultures of endosperm is of significance in understanding the physiological implications concerning embryo growth . I

VI. EMBRYO CULTURE In 1904 Hannig grew the embryos of a few plants in an artificial medium. Several others followed him, but the most significant contribution was that of Laibach (1925). In crosses between Linum perenne x L. austriacum he found that the seeds and embryos were inferior to

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those of the parents in size as well as weight. Further, in the reciprocal cross the fruits shed prematurely, and the seeds were incapable of germination. Lttibach excised the embryos from the young seeds and reared them t o maturity in a solution of 10-1570 sucrose. Prompted by his succew in obtaining the hybrid seedlings, Laibach suggested that in all cases of difficulty in obtaining viable seeds of hybrids it niight be appropriate to try t o excise their embryos and grow them in an artificial nutrient medium. Laibach's work gave a treniendous impetus to the technique of embryo culture. In the growth of the embryos of angiosperms there are three periods : ( a ) it period of slow growth following fertilization, (6) a phase of rapid growth. and findly (c) a period of physiological niaturation of the cmhryo. 'L'hc Hucccssfril culture of embryos, thercfore, comprises two niiLjor phawc": prcgorminal ant1 postgerminal (Rijven, 1952). The first is the proniotiori of embryotittl growth during which differentiation continues ; tlio second is trhegermination of embryo and the formation of a nornird needling. A. ClILTURAL C?ONDITIONS

The younger the embryo, the more difficult it is to excise it under aseptic conditions. Secondly, young embryos are extremely susceptible t o osmotic shock. In cultures of embryos of Hordeurn wu2gare the addition of casein hydrolybcate increiised the osmotic value and thus favoured the pregerminal growth (Ziebur et al., 1950). Rietsema et al. ( 1963) also reported that osmotic concentration influenced the growth of embryos of Dalzwa. Using maniiitol to obtain the desired osmotic values, they demonstrated that the younger the embryo the greater was its affinity for a medium of high osniotic concentration. According t o Rijveii (1958) a high concentration of phosphate buffer also had a similar influencc on the embryos of Capella bursa-pastoris. Contrarily, Raghavan and Torrey (19634 have reported that the osmotic value of the culture medium is of relatively little importance in the morphogenesis of cultured embryos. Early heart-shaped embryos (81-406 L,A in length, Fig. 6A) of C. bursa-paetoris grew normally in an agar nutrient medium containing mineral salts, three H-vitamins and only 2yo sucrose. Three weeks after culture the root and shoot primordia had already formed and the cotyledonary lobes were well marked (Fig. OB and C). The primordia of the first pair of leaves developed in 4-6 weeks (Fig. (ID). Globular embryos (<80 p in length, Fig. 6E) cultured on the basal medium failed to develop even after a long time. An increase in the concentration of the vitamins five or ten times over the initial quantity, variations in the p H of the medium, and a high

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oxygen tension failed to improve the growth of the globular embryos in vitro. However, a successful development of the globular embryos (80-80p in length) wns obtained, if the bnsnl medium was supplemented with indoleacetic acid, kinetiti and /or ittlenine aii1phiLte. A combinntion of d l tho thrcs ctdjiivttrrta providoti tho host wrz'liau for growth. Ihiririg the first 7-10 ckya, the bnll of ernbr.yonrd cells continuoti to atltl to its t t u further bulk by cell divieions and cell enliirgement (Fig. el!'). development was siiniltlr to that of older embryos (Fig. 6G). Thus, in C . bur.~a-pastoristhe younger embryos did not show a n absolute requirement for n high osmotic: concentration although a high percentage (12 or 18) of sucrose or a high quantity of mineral salts (ten times that in the basal medium) partly replaced the effect of the three growth substances in inducing their development. On the basis of these observations Raghavan and Torrey (1963a) suggest that the growth and differentiation of excised embryos in cultures are controlled not so much by the osmotic value of the culture medium but by the availability of specific growth factors. For the embryos of Bo,~,~ypium Mauney (1961) used White's medium with all the ingredients a t five times the usual concentration, and supplemented it by 0.7% sodium chloride and certain growth substances. This had the useful effect of retarding the enlargement as well as elongation of cells, which ia typical of germination, and of favouring the division of cells which is otisentiel for differentiation. When the embryos grew older, they were tratiafctml to ;L medium oontuining only 0-3(% sodium chloride. l'he lowered osmotic pres~ureof the medium improved the postgcrminal tieveloptncnb of the embryo. A low osmotic concentration, which i H easily achioved by using decreasing amounts of sucrose in the medium, promotes the germination of embryos (Ziebur and Brink, 1951; Rijven, 1952; Honma, 1955; Iwanowskaya, 1962). Recently, Ryczkowski (1962b) Btudied the changes in the osmotic values of' the embryo and endosperm in the

Fro. 8 (see fnring pxrge). FIG.0. I n rilro rultliro of rmbryos of (!fip3t?lhbur8ri-pmlori8 (cot, cotyledon; hyp, hyporotyl: s, uuspenaor).A. Early heart-sliaptl rmbryo (81 in lengt,hexclusivc of the Buspensor). 13. 2-\Vwk-old rulture on nil agiw nutrient, medium incubnted in dark; note formation of rotylrctons. C. 3-U'oek-old culture grown in light ; tho cotylcdon8 and the plumule are well developed. 1). Ax in C'; 5-h'eok-okl emtxyo ~howitigthe formntion of the first pair of leaves. P. Globular embryo (51 p in loiigt,h inclunive of tho aimpensor). P. 10-Day-old culture of n globular ambryo, rxriwd nt Htiigc wliown in E. on tho 1)nHuI mcclium supplmnmtcxl with J A A (0.2 ppm)+ kinetin (0.001 ppm)+ ibclfminu nolpliittc: ( O W 1 ppm). Ct. fLWt!t!k-olfl culturt! ahowing the differentintion of mot, hypowt~yluntl i.ot.yk!iltni~(ufbr Httfihttvun nr~d'rorruy, 196311).

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clevelopirig seeds of soine representiitives of both monocotyledonous and dicotyledonus plants. In both, tho embryo generally showed a higher osmotic value than the endosperm. However, the osmotic value of the embryo increased with its growth only in monocotyledonous plants. Ryczkowski Iias, therefore, suggested that a knowledge of the osmotic values of t'lie e m h y o tliiring its growth ant1 development in the o v i h would be Iiclpful iii tlonigniiig a propor nirtricnt medium for its growth irb ailro. I,ike tho oxinotic virlu~,the cwnwntratioii of the oxygen in the rne~liunialso intluenccs t l w growth of the embryo in culture. White (1939) reported that a tlecrcnsc in the content of the oxygen in the surrounding nietlium is essentid for enabling the embryo to follow the normal course of differentiatioii. "hi8 can be achieved by lowering the enibryos into the nutrient medium. Embryos of some gymnosperms, like Ginkgo hiloba and I'inw lambwtiana, showed their best growth only when their cotyledons were imhedded in the medium (Bulard, 1952; Brown and (:ifford, 1958). Sinii1:irly. embryos of Dafuru (Van Overbeek et at., 1944) and Cydamm pwsicum ((hrter, 1955) grew best only when they were implanted below the surface of the agar medium. However, other reports are contrary to this. Submerged embryos of Pinus hrnbertiuna failed to grow normitlly hut developed tumorous growths whereas those of Horderrm vulgaw did not grow a t all (Norstog, 1961). U. UROW'I'H M RI)IA

Aliot,lier wninioii t?xl)crieticcis t l l i b t t i nutritive medium suitable for older er11l)ryos is oftcn quite iiiisiiit;d)le for youngcr ern bryow. Also, yoling embryos usually grow prwociously in culture and produao inalformed seedlings. Thiw is vci*y i~il(lc!sirihlespecially when tho aim is t o raise plants from otherwisp iLborth embryos. Mu& of tho investigations on embryo culture arc. therefore, concerned in formulating the conditions and media w1iic.h would promote the normal growth of t,he eiiibryo in its pregcrminal and postgcmiinal phases. Sugars not only alter the osmotic value of the medium but also play it nutritional role. High osmotic values and therefore high concentrations of carbohydrates (as inuch as 12-18';;> mcrose) favour the pregerminal phase ; low osmotic values achieved by lower concentrations of sugars (2';; and below) promote the postgerminal phase (see Rappaport, 1954; Norstog, 1961). Sucrose has been found to be by far the best source of carbohydrate for embryo culturea. Van Overbeek el al. (1941) showed that unautoclavecl coconut milk induced normal, pregerminal growth of young embryos of Uaturu. Since then coconut milk has h e n used with success on embryos of several plants including Cocoa ncccifera.

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Merry (1942) excised embryos of Hordeurn salivum 7-28 days after fertilization and cultured them on an agar nutrient medium containing 2yh sucrose. The youngest embryos (7- and 8-day-old) lay quiescent even up to 8 weeks, whereas 9- to Il-day-old embryos initially showed some indications of growth. Only the 12-day-old or still older embryos grew normally in culture. Recently, Norstog (1961) cultured embryos of H. ndgare, ranging from fio to 1500p in length, on White’s medium Rupplemented with none or one or more of the following subtitanceti: danine, nspnragine, glutnmine, rnixtureti of amiiio acids, and coconut milk. On White’s iiictliiitii t ~ l o i i oembryos lotin than 500 p in length failed t o survive!, wliilc t,tiow 500 14 in length tihowed only a limited growth (Fig. 7A-C). It iH only those twyontl 50Op that attained a size coniparnblo to that of rtnihryos iiL r h o . However, no differentiation W ~ observable H in any of them. Mere ninino acitln were of little henefit, but coconut milk (10, 20 or YO)(,) iiiducetl a marked growth even in embryos which were smaller than 500 p. I n 2 weeks after culture roots and additional leaf primordia developed in 500 p long embryos (Fig. 7D and E). I n combination with glutamine (400 ppm) or with a mixture containing 585 ppm of a total of 14 amino acids, coconut milk caused excellent growth and differentiation of embryos of all ages. I n a few instances even 6Op long embryos produced masses of tissue which developed leaf and shoot priniordin (sometimes more than one) (Fig. 7F and (2). Nevertheless, the embryos grown in culture did not develop scutelluni. (’hang (1963) illtio reported that embryos of barley grown in. iiitro allowed tieveral deviations in their morphology and failed t o reach the mature stage. T n an attempt to discover a suitnhle sutwtitute for coconut milk, Matsubara ( 1968) tested the effects of (:wein hytlrolysdn, dried brewer’s yeast, skimmed milk, diffiinnteti froin the endotipcrtns of Ginkgo and froin the neetln of t ~ t imaiiy an iiinc rmgiofqmrns on yoling embryos. He observed that the alcoholic difFutiltten from young Heedn of Lupinus lutezu rLnd from old seeds of 8rchium edde were as effective as coconut milk in promoting the growth of embryos of Datura tutula which were only 0.15-0-55 mm in lengbh. Embryo factor activity, similar to that of coconut milk, has also been found in extracts of almond inenl, banana, Datura ovules, nondormant apple buds, wheat germ (Van Overbeek, 1942; Van Overbeek et nl., 1942), malt extract (Solomon, 1950), and peat and yeast extracts (Gorter, 1955). Like coconut milk, casein hydrolysate (a complex of several amino acids) is yet another substance which has been widely used in embryo culture. I n their experinients on Hord~urnuirlgare, Kent and Brink (1947) tried casein hydrolysate, Noditiin nucleate, and tomato juice

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and discovored that these substances too can promote the growth of young embryos t o maturity. Sanders and Burkholder (1948) observed that, unlike casein hydrolysate, individual amino acids or mixtures of a few of them did not favour t,he growth of embryos of Da.tirm

C

E

D

-

F

White’s medium i 20% coconut milk t 4 0 0 p p m glutornine OW 130 doys) (C.80cells)

Shoot 1 Shoot2 Shoot 3-

.

Shoot 5

Root

G

FIQ.7. Culture of young embryos and procmbryos of ffordeum vulyare. A. Embryo (0.5 mm in length) a t thc time of culture. U. 14-lhy-old culturc: on Whit& medium. C. Embryo from B in longisection; on White’s medium the growth was negligible. D and E. Embryos (0.5 mm in length) grown for 14 days on Whito’u mcdiurn+coconut milk (20%);note primordie, of additional roots and leaves and comprrc with I3 and C. F. Proembryo (60 p in length). G . 30-Uay-old culture of p ~ ~ l l l l J ~in y OWhite’s medium+ coconut milk (20%)+ glutamine (400 ppm). Tho embryo prodticwl n muss of tistme from which five shoots differentiated (nfter Norstog, 1961).

innoxia and D. stramoniurn. However, a mixture of 20 amino acids, or of cysteine and tryptophan i n concentrationu proportional to their

quantity in casein hydrolyrrate, proved favourable.

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According to Ziebur et al. (1950) tho amino acid atid sodium chloride components of casein hydrolysitte prevent the precocious germination of embryos, ant1 that the iitnino ;ic*ids together with the phosphate complex serve as nutrients in p m o t i n g the normal growth of embryos. Amino ttcids by themselves ftdetl. to diiplicate the effeclt of orwein hydrolysate. Therefore, Ziel)ur et ul. (l!)50) concluded that tho growth of immature embryos of barley, ohtiiined i n vitro by using casein hydrolysate, “may he the result of ;In interplay of both nutritional and physical factors”. The works of Lofland (1950) and Mauney (1961) on Cossypium, of Rijven (1952) on C’apscEb hursa-pasforis, and of Nakajima (1958) on Cucurbita maxirna confirm that casein hydrolysate strongly supports the growth of young embryo8. Tn his work on Cucurbita maxima Nakajima investigated the effects of casein hydrolysate, 1,3-diphenylurea (a component of coconut milk), kinetin, IAA, malt extract, and a n extract of the endosperm of its own species on young torpedoshaped embryos. The most marked promotion of embryonic growth and formation of seedlings occurred when casein hydrolysate and 1,3diphenylurea were used in combination. For the growth of proembryos of Citrus microcarp aluo, casein hydrolysate proved beneficial (Rangaswamy, 1961). Very young globular proembryos (14-28 x 14-28 p ) cdlapaed within 3 days of culturing on n modified Whitbe’s mccliunt (Fig. 8A). An addition of 3, 5 or even 10‘,~{~siicrose did not promote the growth of proembryos to any appreciable degree but merely prolonged their survival t o 2CJ days. With 10f;G sucrose the embryos enltirged t o twice their original size. The proembryoe, cultured on White’s medium supplementetl with casein hydrolysate (400 ppni), faithfully continued their pregerminal development through d l the entbryological utageu. I n 3-4 weeks fully organized embryos were formed in 8V1,,of the cultures (Fig. 8B and C). This is one of the few instsnc+esof successful growth of very young proenibryov (<18 p in diameter) in vifro. The fostering of the normal growth of young embryos 1)v sii1)stiincex x u d i as coconut milk and casein hydrolysate is iniportmt for rearing non-aberrant seedlings from cultures of proeinbryos. In addition t o rarboliydmteu, untitio acids and plant tissue extracts, a large number of other organic. coinpounds belonging to the class of vitainins, plant hormones, kinins, xterols, steroids and steroidal sapotiins have been screened with R view to discovering substances beneficial for the growth of ewisetl embryos (see Sanders ant1 Ziebur, 1963). Among these. gibberellitis i ~ n dkinetin have reooived increaning recognition :ts clieniioals c.apiLl)le of rcgiilatitig the growth untl tlovclopmerit of embryos.

of dvena fnliiu (Ni~yIorntitl Sitiil)son, 1961). Later, Simpson and Naylor (1962) tlcmonstrtttcvl that w i twyo tlormancy in A . f d u a in due to a critictil t):ilnrtce in tlic ; a m o u n t of' rnrrltiaso i r i the grrtinn. I f tho xynttlenis of fllibIttlS(? is hlockctl Or i t s c:orltcrlt is low, the! hydIY~lynis of stnrdi in prevwtetl i m 1 the oni t)ryo p s w into u state of'rlormancy. Naturally, in ciiltiirvs of' embryos exrisetl from dormant caryopnen a high conccntration of' exogenous nngar, or ntaltase, or gihttorellic. acid (50 ppni) W R S Iiclpf'iil hi overcoining the dormancy. That gihberellic acid is effective i n 1)rwtking tl(~~tiliL11~y tmtl promoting germination

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is explained as due to its capacity to stimulate the activity of maltme. I n embryo cultures of Hor&*um z~tilgarealso Paleg et al. (1962) found that GA, activated the mobilization of food reserves from the endosperm. Veen (1963)reports that gibberellic acid did not improve the growth rate of globular and heart-shaped embryos of Capselk bursap t o r d s . However, in cultures of torpedo-shaped embryos GA, increased the growth rate considerably by stimulating the activity of the root meristem as well as the development of root hairs, and induced epinastic curvatures of the cotyledons. Since these responses normally occur during germination Veen considered gibberellic acid to be a promoter of this process. Nickell's (1958) dbservation that gibberellin-like substances appear in very old tissue cultures of the cotyledons of Phaseolw uuZgaris is of interest, and suggests that GA-like substances occur in the embryo. Corcoran and Phinney (19W2) and Ogawa (1963a,b)have demonstrated a oorrelation between the growth of the embryos and the quantity of gibberellin-like substances present in the soeds. Like gibberellic acid, kinetin has also been reported to affect the growth of the embryo. I n Veen's experiments it showed no significant effect on the torpedo-shaped embryou of Capsella bur8a-pastm*8,but in to 10-0 g/ml it increased the growth rate of concentrations of globular as well US heart-shaped embryos. Nevertheless, this kinetininduced growth resulted only in malformed embryos. C. APPLICATIONS OF EMBRYO CULTURE

I n addition to these fundamental investigations a large number of papers have appeared on the applications of the embryo culture method, Since the time of Laibach (1926) the technique has been extensively used in agriculture and horticulture where the failure to obtain viable seeds is due to one of the two causes: (a) the embryo of the first generation hybrid aborts in the seed, or (b) the F, hybrid produces seeds which are incapable of supporting the development of the embryo to the germinable stage. In several instances of intergeneric and interspecific matings, which had been declared ineffectual or discouraging because of only a low yield of adult progeny, embryo culture has proved a boon. Some such achievements with cereal, pulses, fruit and vegetable crops and ornamentals etc. are dincumxi below. Species of EZymw are characterized by (a) thc development of a deep-growing and strong root system which enctbles the plantn to resist drought, and (6) the production of a large number of ueeds per ear. When Tritimm was crossed with Elpmw no hybridrc were obtained

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unless the embryos were cultured in artificial media. Iwanowskaya (1946) crossed T. durum x E . arenariua, excised the hybrid embryos, and grew them in culture. Later, Iwanowskaya (1962) raised several other hybrids and observed that the viability of the embryos of T. d u r u m x E . arenarius was greater than that of the embryos of T. durum x E. giganteus and of T . uulqare x E . arenariua. Many of these hybrids are said to be perennials having a height of 1.5 m and bearing abundant foliage and large ears. Uttaman (1949a-0) used tho technique of embryo culture to test the viability of young embryos of Zea mays. Three-week-old cobs were refrigerated immediately rtftor harvont and ten kernels were removed from them daily. The embryos excised from 3-week-old cold-stored cobs retained viability for about 46 days. Embryos excised directly from 4- to 7-day-old kernels (which were not cold-stored) and grown at 31&1°C showed only a slight growth and even this ceased five days after culture. The addition of n few drops of cold-preserved coconut milk (expressed from mature nuts) to 3- or 4-day-old cultures had a depressing effect, particularly on the root. If coconut milk medium was used to culture embryos excised from 2-week-old kernels, germination was suppressed; but good growth resulted when it was added on the second day after culture. Only embryos which were at least 16-day-old could readily develop into plants. Hordeum vulgare is completely self-fertile but susceptible to cold and to the powdery mildew, Erysiphe graminis f. h.uraei, whereas H . bulbosum, although loss self-fertile, is appreciably winter hardy and resistant to mildew. Hybrid progenies between the two species have always been desired, but in practical hybridization the resulting caryopses are frequently devoid of an endosperm so that the embryo dies prematurely. Thvim (1960) applied the tochnique of embryo culture to raim interspecific hybrids between H . vulgare, H . bulbosum and €?.californicurn. He performed cross-pollinations in nine combinations, excised the embryos 14-28 days after pollination, and cultured them on a modified Randolph-Cox medium. Seedlings capable of successful transplantation were obtained from the cross H . vdqare ( 2 n ) x H . bulbosum (4n). Seedlings were also obtained in other combinations but the transplantations were more dificult. Unlike H . vulgare, the wild H. hexupodium is resistant to Hdminthosporium sativum. When the two were crossed the endosperm usually failed to develop in the hybrid caryopses and the embryos were rendered inviable. To circumvent this difficulty, Schboler (1962) made cultures of excised embryos and successfully raised the Fl and also some subsequent generations. Similarly, in crossing Hordeum jubatum and Secale cereale many of

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the hybrid seeds oollapsed in 6-13 days after fertilization. At maturity even the largest seeds were malformed and failed to germinate. Histological examination showed that the embryos made considerable growth but were arrested because of an incompatible endosperm. To overcome this Brink et al. (1944) excised tho embryos from the hybrid seeds and cultured them on White’s medium. Out of 81 cultures, the majority showed undifferentiated growth, but one embryo differentiated normally and developed into a seedling. In interspecific crosws in O y z a it is generally observed that although fertilization occurs the caryopses fail t o produce viable embryos. I n crosses between 0. minuta and 0.sativa Nakajima and Morishima (1958) noted the formation of only imperfect grains containing only malformed embryos. When 0. sativa (diploid) was crossed with 0. mint& (tetraploid) the caryopses produced were viable but those obtained in the cross 0. sativa (tetraploid) x 0 . minuta were inviable. By culturing them it was possible not only to raise seedlings of this otherwise unsuccessful cross but also to improve the performance of the hybrids of the diploid 0. saliva x 0 . minula. Bouharmont (1961) has reared hybrids between 0. saliva ( 9 ) and three other species (0.schweinfurthiana, 0. perennea and 0. latifolia) which were used &B pollen parents. He cultured 7-day-old embryos on a nutrient medium containing dextrose and obeerved that the endosperm adherent to the embryos had no inhibitory influence on the formation of seedlings. However, according to another report a n extract of the endosperm of 0. sativa retarded the initial stages of the germination of embryos in culture (Sircar and Lahiri, 1956). Sirice indole-3-aceticacid duplicated the retarding effect, Sircar and Luhiri concluded that the endosperm contains a substance similar to IAA. The legumes are yet another group of plants to which embryo culture has been extensively applied, and many new hybrids have been obtained which show a blending of several desirable features. Of special interest are the investigations on Melilotw. M. oficinalis is more drought resistant and better adapted to grow in the phhs (in the USA) than M.dba;but it htts a high content of coumarin (harmfd to cattle) unlike M.dentata and some lines of M. alba. Webster (1955) reported that efforts to introduce genes for low coumarin from ill. dentata into M. oficinalis were a failure. Reciprocal croxses were then attempted between M . oficinalis and the low coumarin lines of M. alba. In these, the embryos grew for some time but failed to mature becaum the ovules aborted 8-10 days after pollination. Only through embryo culture was it possible to rear two hybrid individuah from the cross bl. OfiCiW& x H.a&. Almost identical rwults were obtained by SchlosserSzigat (1962) who has also raised a hybrid of M.oflcinalio x M . alba.

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According to Webster (1966) “other species crosses, previously thought to be impossible, should now be reconsidered”. I n Medicago sutiva (alfalfa) an abortion of o d e s is quite frequent after self-pollinations and in interspecific crosses. I n self-pollinated plants Fridriksson and Bolton (1963) excised the embryos when they were 21-day-old and nurtured them into seedlings. A few interspecific hybrids of L a t h y w cornicktw, L. tenub and L. uliginoszls have also been made possible through embryo culture (Davies, 1961). It is known that only “non-viable” or (‘physiologically sterile” seeds result from tho cross Phmeolus wulgnris x P. acutifoliw. However, when embryos were excised 14-24 days after pollination and cultured on White’s medium satisfactory seedlings were obtained (Honma, 1966). I n this case the embryos were initially cultured in a liquid medium and then gradually transferred to media containing decreasing concentrations of sucrose ranging from 4 to 0%. Interspecific crosses in the genus Prunw have been &,tempted quite frequently. Tukey (1938) applied the embryo culture technique and reported success with embryos of apple, peach, pear and plum, which often abort before completing their full development. Skirm (1942) germinated 414 embryos derived from 16 different interspecific crosses of Prunw. Lammerts (1942) demonstrated that embryo culture helps to shorten the breeding cycle of deciduous fruit trees such as apricot, nectarine end peach, and to hasten the germination of hybrid seeds. Nectarine and double-flowering peach hybrids reared by this method grew large enough to be used as female parents within two years and proved superior to the plants raised from seeds subjected to stratification (treatment with moisture followed by low temperature). Gilmore (1960) studied the growth of embryos of peach excised from seeds treated with various disinfectants. He also investigated the influence of light, temperature, moisture, and the composition and age of the medium on the growth of embryos in witro, and etandardized a technique for culturing the embryos of peach. Many raaceous fruit trees are characterized by a long period between the ripening of the seeds and the maturation of the embryos. The requirements for germination of embryos excisod from after-ripened seeds and of the embryos isolated from non-after-ripened seeds am variable (Tukey, 1944). Lesley and Bonner (1962) reported that it is helpful to store the fruits of Prunus at 2°C for 7 weekR before excising the embryos from them. Hesse and Kester (1966) and Kester and Hesse (1966) cultured the embryos of almond, plum, peach and cherry. They observed certain correlations between the ability of excised embryos to germinate in vitro and their developmental status at harvest,

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the method and length of storage of seeds or fruits before the excision of embryos for culture, and the nature of the medium. Embryos of the early ripening s p i e s were favoured by the presence of sugar in the storage solutions as well as in the culture media, whereas those of the intermediate ripening varieties readily germinated in a non-sucrose medium irrespective of a previous storage in sugar or non-sugar solutions. Embryos of late maturing varieties responded best when grown in a sucrose-deficient culture medium. Zagaja (1962) studied the after-ripening requirements of immature embryos of apple, apricot, pear, plum, sour and sweet cherry. He raised embryo cultures and subjocted them to low ,temperatures. Cold treatments significantly stimulated the germination of embryos. Zagaja reported (a) that the after-ripening requirements of immature as well as mature embryos were somewhat similar, and (b) that in immature bmbryos the cold treatment stimulated a 'better translocation of materials from the ootyledons to the embryo axes and in turn increased the capacity for cell divisions. Some wild species of Lywpersicum-L. peruvianum and L.glanduloeum--are resistant to viruses. Several attempts have been made to transfer this character to the cultivated tomato. However, the cross L. peruvianum x L . emulenturn proved infructuous and even in the reciprocal cross only collapsed seeds were formed (Smith, 1944; Choudhury, 1966). From his enibryologicalstudies Smith found that the endosperm collapsed first and then the embryo within 30-40 days after pollination. He excised the embryos prior to their undergoing necrosis, oultured them for about 10 days and obtained hybrid seedlings. Similarly, Choudhury (1966)excised the embryos nearly a month after fertilization when they were still turgid, and cultured them on a medium supplemented with 50% coconut milk. A week later when the seedlings were formed they were transferred to a medium without coconut milk, These hybrid seedlings grew rigorously and also produced flowers. Alexander (1950) reported that nearly 9% of the fruits resulting from the cross L. esculentum x L . peruvianum bore seeds from which the embryos could be cultured into hybrid plants. The embryo culture method is also useful for overcoming dormancy in seeds. To give an example, the seeds of Iri.9 hihve a variable period of dormancy. To reduce the long interval between the formation of the seed and the flowering of the plants Randolph and Cox (1943) employed the embryo culture method and thereby shortened the life cycle from two or three years to less than a year, Like many other ornamental plants the rose normally takes a whole year to come into flowering. However, by embryo culture it has been possible to produce two generations a year resulting in a shortening of Q

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its breeding cycle (Lamnierts, 1946; Asen, 1948). Dormancy of seeds and a slow growth of the seedlings are undesirable features in many other horticultural plants. Thus, in the crab apple (MaZuesp.) 3-4 years are required to obtain the F, progeny. Nickell (1951) tried to ourtail this period. The excised embryos germinated within 24-48 hr of culturing and in 3-4 weeks transplantable seedlings were formed. In soil the seeds germinated only 9 months after Eowing. In recent years other reports have appeared. In hybridizations between diploid and tetraploid Chrysanthemum boreale (9) and C. paci$cum, Kaneko (1967) observed that although fertilization occurred the embryos collapsed early during the development. Hybride were obtained by excising the embryos and culturing them on a nutrient medium. The brtyding of lilies has been greatly facilitated by embryo culture. All the attempts to cross Lilium [email protected] with L,regak. were unsuccessful until the embryo culture technique wm adopted (Skirm, 1942). When the varieties “Album” and “Rubrum” of L. spe&mm were treated with pollen from L. auratum, the resultant seeds were largely non-viable and contained embryos at various stages of degeneration. With the embryo culture technique Emsweller et al. (1962) successfully raised over 1000 seedlings of L. speciomm album"^ L. auratum and 100 seedlings of L. speciosum “Rubrum” x L. auratum. The F, hybrids, named L. p r k n i i , bloomed normally but were df-sterile. Interpollinations among them and back-crosses with their parents have resulted in a wide range of cultivars such as “Allegra”, “Aurora” and “Advance”. Among other economic plants whose improvement has been sought through embryo culture technique are the fibre-yielding crops such as C o r c k s (jute) and Qossypium (cotton). Corchorus q s u l a r i a yields the “white” jute of commerce. T t is fairly resistant to drought and flood, and can be cultivated on low lands. However, it is susceptible to certain pests and diseases, and although the fibre is white it is less strong than that of C. olitoriw. This species generally grows on high lands and produces a red but strong fibre. Plant breeders have long wished to evolve an interspecific hybrid combining the desirable features of both species. However, the croes 0.olituriw xC. txpw.hri8 gives a very poor fruit set without any viable a d s , and the reciprocal cross gives entirely negative regults owing to the abscission of the flowers shortly after pollination. Ganesan et al. (1967) investigated the causes for the failure of seed formation in the cross C. olitoriwrx C. capsularis and found that the hybrid embryo usually aborts before reaching the heart-shaped stage. Sulbha and Swaminathan (1969) adopted such techniques as smearing the ovary with hormonea and using the reciprocally grafted plants as parents. The b a t mentioned

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technique considerably increased the percentage of fruit set not only in the cross C. olitoriua xC. mpsulam's but also in C. cupaulat$sx C. olitoriwr. Islam and Raehid (1961) improved the fruit set by applying IAA to the pedicels of flowers soon after pollination, and obtained a few viable F, seeds in the cross C. ol~tom'usxC.cupsularis. More recently, Islam (1964) coupled the application of hormone and the technique of embryo culture to rear hybrid seedlings of the reciprocal crosa, C. capeulariip xC. olikdwr. A medium supplemented with 0.1% yeast extract and 0.06 ppm each of kinetin and IAA proved best for the germination of the hybrid embryos which grew into tramplantable seedlings. In Gbesypiunz repeated attempts have been made to obtain hybrids between the species of the New World and those of the Old World. According to Beasley (1940) in the cross B. arhmeurn (2n = 26) XU. h~rsutum (2n = 62) the endosperm disorganized in 15 days after pollination without reaching the cellular stage and the embryo aborted. J. B. Weaver (1968) excised the embryos from capsules varying in age from 20 days after pollination to maturity and oultured them on White's medium. Those which were excised from 30-day-old fruita grew satisfactorily and one normal seedling was obtained. I n the reciprocal cross, B. hireutum XU. arboreurn, the hybrid embryos grew normally for about 10 days after pollination. Thereafter, the growth was retarded and within 15 days after pollination the fruits became abscissed. In the few capsules which remained on the mother plant one or more ovules showed a cellular endosperm but there were no embryos. Out of 600 pollinations only one embryo grew to a stage that waa advanced enough for excision and culturing. This waa reared in vitro (J.B. Weaver, 1957). Among other investigations on embryo culture may be mentioned those of McLean (1946), Wall (1964) and Nishi el al. (1969). McLoan raised interspecific hybrids of D.ceratocaula with nine other herbaceous species of Datura. Wall (1964) successfully hybridized Cucurbitu p e p with C . moechata, and Nishi et al. (1959) obtained a hybrid Chinose cabbage. The work on the parasitic Caaytha jitiforrnie (Rangaswamy and Rmgan, 1963) haa shown that normal germination and the subsequent growth of the seedling can be obtained in culture without the intervention of any host plant. This confirms the earlier observations on other pamites, namely Cwcutu rejiexa, Dendrophthoe falcala and 8aWlum album. These have been discussed elsewhere in this Damr.

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D. LIMITATIONS O F EMBRYO CULTURE

Despite mtiny years of experimentation and its application in practical biology and fundamental research the problem of the culturing of embryos cannot be regarded as solved. The embryo undergoes a gradual transition from a stage of dependence in the beginning t o that of relative autonomy. Consequently, its requirements for growth also change with age. Of interest in this connection is the work of Raghavan and Torrey (1963b) on the embryos of an intervarietal hybrid of Cattlep, They observed that with progressive ontogenetic development the embryo acquired a capacity for the activation of certain enzyme systems, especially those concerned with the utilization of nitrogen compounds. The ability to assimilate nitrate nitrogen corresponded with the appearance of the enzyme nitrate reductase in the embryo. In the sced the embryo is nourished by the tissue adjacent to it, namely the endosperm (the Podostemaceae, Orchidaceae and Trctpaceae are the only exceptions in which little or no endosperm is formed). It is, therefore, logical t o presume that as a rule the changes in the contents of the endosperm largely correspond to the requirements of the embryo. In their attempts to provide the embryos with natural nutrients, many investigators have added extracts of endosperms, ovules, or fruits of the same species to the culture medium. Van Overbeek et al. (1944) reported that an extract of the ovules of Datura favoured the growth of the young embryos. Likewise, Ziebur and Brink (1951) observed that the endosperm of Hordeum vulgare had a high stimulatory effect on the growth of its embryos. Endospenns and embryos of similar as well as of different stages were tested. The endosperms were excised and placed around the embryos either directly on the surface of the agar medium, or in a box made of filter paper or cellophane. It was found that the older endosperms wer0 more effective in improving the growth of young cmbryoa. Further, freshly excised, entire living endosperms were superior to (a) overnight-frozen endosperms, ( b ) a crude extract of' endospernis, and (c) riutoclavod endosperm. i to nurse t h o omtryo That the main function of the eticlosperm N is cleitrly demonstrated by the work of Pieczur (1962) on Zea may8. Previously established tiwue cultures of its endosperm served as beneficial substrata for the growth of excised embryos of the parent species. It has ttlso beon reported that the addition of the excised endosperm to the cultme medium c a w s an increase in the protein content of the embryos of corn (Oaks and Beevem, 1901). Similarly, the best growth of the embryos of Cocos nucifera was obtained in a

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medium containing filter-sterilized milk from tender coconuts. The older cellular endosperm, heat-sterilized milk, and milk from mature coconuts markedly. inhibited the growth of these embryos in dtro (Cutter and Wilson, 1964). It is curious that sometimes “foreign” endosperms support the growth of embryos better than the endosperm of the same species. This is amply elucidated by the stimulative effect whioh the endosperm of coaonut has on embryos of widely separated species. The picture which emerges from this observation and other investigations carried out during the last six decades is that underlying the development of the zygote into the embryo there is an interplay of many nutrients, growth stimulants and inhibitors, whose exact duplication in artificial cultures is by no means easy. Consequently, when freed from the confines of the ovule, the young embryo often shows an unrestrained behaviour but also manifests growth responses of a most varied nature.

VII. CULTUREOF OWLES In orchids the interval between fertilization and formation of mature seeds lasts from several days to a few weeks. This lag is rather annoying, especially in epecies of ornamental value. The embryo is too tiny to be dissected out, but the fertilized ovules can be cultured as such and seedlings raised from them. Working with the ovules of Epidendrum cochlmtum, E . tarnpense and of a hybrid of Cattleya OctavexC. nws&e, Withner (1942, 1943) was able to shorten the duration between the pollination of the ovary and the maturation of the seeds, and thus hasten the production of seedlings. Curtis (1947) raised normal seedlings of Van& tricolor through ovule culture. On adding barbiturates to the medium the embryos showed a copious proliferation. Later, Spoerl (1948) tested the usefulness of different sources of nitrogen, and found arginine to be most satisfactory for supporting the growth of the unripe seeds and aspartic acid for that of the mature seeds. Vacin and Went (1949) obtained a better differentiation and germination of embryos in cultures made on a medium fortified with tomato juice or prominogen (a protein hydrolysate). It0 (1961) etates that the germination of the seeds and the growth of the protocorm of orchids do not take place satisfactorily in the absence of peptone in the medium. However, Rao (1963) was able to germinate the seeds of an interspecific hybrid of Van& on an agar nutrient mediuni without any special supplement. He observed that some of the seeds directly produced seedlings wheresu others developed into a callus which later differentiated into new plants. Similarly, Raghavan and Torrey (19638) obtained ready germination of weds and plantlet8 of an intervarietal hybrid of Cuttlega on a medium containing

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ammonium nitrate as the sole source of nitrogen. Other ammonium salts such as sulphate, chloride, acetate and oxalate were as effective as ammonium nitrate, whereas the nitrites and nitrates of sodium, potassium and calcium were poor sources of nitrogen for the growth of the embryos. Neither kinetin nor ascorbic acid proved useful. Professional orchid growers are well acquainted with several media and growth supplements which enable the raising of plants through seed culture. Vacin and Went’s medium, Knudson’s medium, RandolphCox medium, Difco orchid agar, and fish emulsion are some of them (see Withner, 1969). One of the advantages of ovule culture is that it serves as a tool (a) to support the growth of such embryos that fail to develop after isolation, and (b) to study the behaviour of embryos which are difficult to excise aseptically, Of interest in this connection is the work of N. Maheshwari (1968) on Papaver emnifetmm. She raised ovules containing a zygote (or 2-celled proembryo) and a few endosperm nuclei into viable seeds on Nitsch’s medium supplemenfed with 0.4 ppm kinetin. Subsequently, Maheshwari and La1 (196lb) reported that kinetin accelerated the growth and differentiation of the proembryos in the ovulos. They observed that in kinetin-treated ovules, 10 days after culture, the embryos had grown to a length of 450 p exceeding that attained by embryos in wivo. The cotyledons and the stem tip were also well developed. This initial rate of growth was, however, not maintained, so that the final length of the mature embryos (640 p ) in cultured ovules was less than that of the embryos which grew in the field controls (660 p ) . Using a 10% solution of sucrose PoddubnayaArnoldi (1960) successfully grew the ovules from pollinated ovariea of several orchids (Calunthe veitchii, Cypripedium insigne, DencErobium nobile, and Phaluenopsis scldleriana). I n such material she was able to trace the events commencing from the entry of the pollen tube up to the development of the embryo and also described the histochemical changes accompanying these stages. Work on the culture of the ovules and seeds of angioepermic parwittw is comparatively recent. It is generally believed that in obligafe root parasites such as Striga and Orobanche the formation of seedlings is dependent on some stimulants from the host root. Experimental verification of this point can be best carried out only through seed cultures. In dtrigu wiaticu, kinetin, certain other 6-substituted aminopurinee, scopoletin and 4-hydroxycoumarin (Worsham et al., 1969, 1962) and gibberellic’ acid (Williams, 1961) are said to replace the germination stimulant excreted by host roots. However, a aupplemontary treatment with kinetin is needed for the development of the

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shoots. Woraham e.t ad. (1962) also consider that although scopoletin and 4-hydroxycoumarin promote the extension of the radicle, the enlargement of the cotyledons as well us the growth of the shoot may nevertheless depend on a eupply of kinetin-like substances. In some experiments at the University of Delhi seed8 of Strigct euphwioick.8 were sown on White's medium supplemented with kinetin (10 ppm). In a few days they ruptured and released the embryoR, but instead of germinating into seedlings these proliferated and produced a mass of callus from the cotyledons (T. S. Rangan, unpublished data). Privat (1960) hm demonstrated that in Orobunche hederae a contact with the root of the host (ivy) is indispensable'for the initiation of the shoot. However, recent work on 0. uqyptima (Rangaswamy, 1963) has demonstrated that the differentiation of shoots can be induced in the absence of any stimulus from the host root (Fig. 9). Chopra and Sabharwal (1963) have recorded a beneficial effect of the placental tissue on the growth and maturation of the seeds in Qymndropeis gynandra but not in Impatiens balsamina. I n Opuntia dillenii also Sachar and Iyer (1959) observed that the ovules showed no growth on any of the media even when they were cultured along with the placenta. However, on a medium containing both IM and kinetin, the placenta sometimes proliferated to form a callus of limited growth. According to Niimoto and Sagawa (1961) the formation of a zygote is a prerequisite for the further growth of the ovules in vitro. In Dendrobz'umphalaenopsie, for example, they observed that fertilization did not occur oven when the placenta was excised with its ovules and pollen tubes and then planted on the culture medium. In this connection the experiments of Kanta el al. (1962) on in vilro fertilization are quite significant (see p. 235). Kazimieraki (1963) has reported that unlike Lupinus luteus, L. rothrrutleri produces much smaller seeds and is characterized by several other desirable qualities. In attempts to transfer these features into L.lulew,he observed that no well-developed seedu were obtained in the croae L. luteus xL. rothmleri whereas in the reciprocal cross ~ome viable seeds were formed although their number was negligible. The hybrids produced only about 20y0 fertile pollen. The high sterility waa mainly due to the failure of the ovules to form functioiial embryo mca. Ovule culture may possibly help in solving such problems. It may be worth while t o excise the ovules or the entire placenta bearing the primordia of ovules and nurture them up to the stage of the mature embryo sac and then to bring about fertilization in uitro. Ovule culture may also prove useful in the artificiai induction of parthenogenesis. A direct handling of' the eggs of angiosperms is by

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no means easy because of the problems involved in removing them without injury. However, there is no such bar to the use of unfertilized ovules. While ovules excised at the zygote or 2- to C-Celled proembryo stage can be reared in zrilro (see p. 268), unpollinated and unfertilized ovules have not proved amenable. According to Ryczkowski (1962a), a knowledge of the changes in the osmotic value of the o d e during its growth and development may prove helpful in understanding the requirements of excised ovules in cultures. He found that while in early stages the osmotic concentration of the central vacuolar sap of the ovule showed an increase, with the further growth of the embryo and especially the elongation of the cotyledons the value decreased considerably.

VZTI. CULTURE OF OVARIES AND FLOWERS La Rue (1942) waR the first to initiate the technique of culture of ovaries and observed root formation from the pedioels. Jansen and Bonner (1949) grew the ovaries of Lycopersicum pimpinellifolium on a medium supplemented with casein hydrolysate, IAA and a mixture of some B-vitamins. Ovaries with an initial diameter of 1 mm enlarged to a diameter of 4-8 mm and developed the pigment lycopene. However, no viable seeds were obtained. Nitsch (1949, 1951) cultured the ovaries of Lycopereicum mulenturn, Cucumis anguria (gherkin), Phseolue piulgaris (bean), Fragaria sp. (strawberry), and Nicotiana tubacum, excised two or more days after pollination. On media supplemented with 2,4-D or 2,4,5-Tor NOA even unpollinated ovaries of Lycopersicum esculentum formed smrtll but seedless fruits. Nitsch, concluded that certain growth substances can replace the stimuli of pollination and seed development on the growth of ovaries into fruits. Since the development of fruits in dtro followed a normal pattern he suggested that the technique of ovary culture could be used a tool for studies on fruit growth. Fro. 9 (See facing page). FIG. 9. In oilro growth of emhryoR of Orobonck aeqyptiuca (0, callus; end, endosperm; p, proliferation; t , testa). A. Whole mount of embryo excised from a seed grown for 2 weeks on a modified White’s medium+ caeein hydrolysate (a00ppm.). The radicle end shows some proliferation and the remainii portion has taken a deep stain with cotton blue in lactophenol. B. L.S. seed from a 3-week-old culture showing further proliferation of radicle end of the embryo. Some of the cells along the margin8 and plumular end appear necrosed. C. L.S. shoot apex differentiated on callus. Note apical dome, scale leaves, leaf primordia and proveacular strand; there is no indication of a root. D. 2-Month-oldsubculture of embryo callns on White’s medium+ casein hydrolysate (400 ppm)+ coconut milk (16%). E.Microtome preparation of part of callus showing “internal” divisions (after Rangaswamy, 1963).

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With a view to studying the effect of chemicals on the pattern of development of the embryo and endosperm Rau (1956) excised the ovaries of Phlox drummondii one day after pollination and cultured them on Nitsch’s medium. Both embryo and endosperm showed a normal development. If the cultures were made on a medium containing colchicine (0.1576) the endosperm nuclei showed aberrant divisions. Nuclear fusions and aggregations were common and eventually the endosperm degenerated. The embryo also showed certain cytological abnokmalities, although the general pattern of its development was normal and its size was also larger than that of the embryos in ovaries grown on the basal medium, If the ovaries were retained on the colcliieine niedium for more than 12-14 days, the seeds became malformed and the embryos aborted. Since young proembryos are l e s ~amenable to excision and their nutritional requirement8 are also not well understood, RBdei and RBdei (1955) cultured whole ovaries, flowers and spikelets of Triticum aestimm and T.apelta, excised 4-43 days after pollination, on a nutrient niedium containing yeast extract. If ovaries deprived of all the floral envelopes were cultured, the embryo8 seldom continued to grow. When ovaries enveloped by two paleae were cultured the embryo furthered its normal development. The development of the embryo as well as the caryopsis was better when both rachillas and paleae were retained. After growing the ovaries for 8-12 days, R6dei and RBdei excised the etnbryos, which hnd sufficiently progressed in growth by now, and cultured them on a medium supplemented with casein hydrolysate (0.50/,). The embryos continued their development and eventually germinated to form nornial seedlings. The experiments of R6dei and RBdei suggest that the floral bracts and rachillae have an iniportnnt role in fruit development. That the floral envelopes are not unessential organN but play a significant role in fruit development is aLo borne out by several other investigations. Chopra (1968, 1962) studied the effects of IAA, IBA, kinetin and GA8 on both unpllinater! tLnd poliinated ovaries of AZthuecc rmea cultured on Nitsch’s medium. On a medium supplemented with IRA (20 ppm) the unpollinated ovaries developed into parthenocarpic fruits comparable in size with those which developed in nature although the seeds were devoid of any endosperm or embryo. The addition of kinetin (0.5 ppm) or IAA ( 5 ppm) had no pronounced effect. However, when used in conjunction kifietin and IAA had a synergistic effect in inducing parthenocarpy. It is important to note that even the growth of the pollinated ovaries W&B considerably affected by the calyx. If the calyx was severed before implanting the ovaries in culture, the fruits p w to a diameter of only 12 mm, the endosperm remained free nuclear, and

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the embryo reached only the heart-shaped stage. On the other hand, when the calyx was left intact the fruits enlarged to a diameter of 19 mm, the endosperm became cellular. and the embryo showed its full development. Maheshwari and La1 (1958, 1961a) cultured the flowers of lhericr a m r a on Nitsch’s medium supplementod with the B-vitaminll. Flowers cultured one day after pollination produced normal fruits and the embryos grew to maturity. However, this was possible only when the calyx was not removed before implanting the flower hi the medium. If the calyx was removed, it was necessary to add 5% sucrose in order to obtain a satisfactory growth of the ovary, but even then the embryo did not grow as well as when the calyx was left intact. However, when the flowers were collected more than 8 days after pollination, the removal of the calyx did not affect the growth of the ovaries in vitro (Fig. 10). La Croix et al. (1968) found that in barley the development of the proembryos proceeded normally in florets whose lemma and palea were kept intact. If these were detached the development was impaired. In cultures of excised spikes the growth of the proembryos was normal even when all the florets had been dehulled provided a single leaf was retained on the tlpike. La Croix et al. concluded that a “hull factor” is necessary for a proper development of’the embryo and it ig supplied by the tissues external to the ovary. I n the absence of the hull faotor the cells of the proembryo merely enlarged and even showed a duplication of the DNA (twice the level of the diploid prophase), but failed to undergo mitoses. Kinetin could not replace the hull factor, It may be concluded that ovary, flower and inflorescence cultures are additional means for tackling difficult situations in rearing the embryos to maturity . That developing seeds are rich in auxin and control the growth of the fruit has been confirmed by the observations on ovary cultures. Sachar and Kanta (1958) studied the effects of certain growth substanaes on the ovaries of Tropueolum majus. Ovaries excised two days after pollination were cultured on Nitsch’s medium supplemented with three B-vitamins and glycine. Although initially the growth rate of the cultured ovaries matched with that of others grown in the field, eventually the test-tube fruits were smaller even when the medium was supplemented with one or more of the following substances : biotin, colchicine, casein hydrolysate, kinetin, 2,4-D, CA,, IAA, TBA, tomato juice and yeast extract. Sachar and Kanta (1958) also studied the development of the embryo and endosperm in the ovules of ovaries grown on the basal medium. At the time of btarting the cultures the ovules contained a young pro-

Fro. 1 0 . Culture of ovwien of l b r r h ( m u r # . A. Ovsry grown an Nitnch’n I J I L H ~medium (9-wwk-oId). B. Similsr culture on Nitwcli’n medium+ vitamins+ IAA (5 pprn). C and I). OvHrieH grown on Nitwh’n rnetliuni+ vitiirninfi-t kinetin (0.5 ppm)+IAA (5 ppm). E and P. OvwieR grown on Nitnrli’s mctliurn+ kinetin (04 ppm)+ I A A (10 ppm). G. Culture showing growth of fruit in Nitwh’a medium 1 vitamins+ kinetin (0.R ppm)+IAA (5 ppm). The pedicel i# hypertropltietl; compare with fruit Hhown in E ;&ndF. H. Culture Rhowing growth of fruit in Nitach’s medium+ vitamins+ I,inctiii (0.5 ppm)+ titleniitr (10 ppm); only rut end of pedicel in hypcrtropliirtl (after Miihl.rliwiiri snd 1.1~1, 19fillt).

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embryo and a nuclear endosperm. Although both embryo and endosperm began to develop normally, signs of degeneration were evident by about the third week. The influence of growth substances has also been studied on fruit and seed development in Linaria ntaroccaru~(Sachar and Baldev, 1958). Pollinated ovaries cultured on Nitsch’s medium developed into fruits, but these were smaller than the field-grown controls. The addition of kinctin, or TAA, or IBA, or 2,4-D, or adenine improved the growth only slightly. However, if yeast extract was added to the medium the fruits not only attained the natural size but also reached maturity at a much earlier stage (15-17 days) than in nature (21-23 days). A few ovule8 also developed into seeds containing viable embryos. With a view to determining the nutritional requirements of developing fruits Guha (1962) cultured the flowers of AZZium c e p excised two days after pollination. On the basal medium there was only a 6 7 % seed set. The addition of IAA or GA, increased this to 20%, and if tryptophan was also supplied seed set increased to 30% (see also Johri and Guha, 1963).

Ovary culture has also been useful in inducing polyembryonic tendencies in Ranunculm sceleratua (Sachar and Guha, 1962), Anethum gravedens (Johri and Sehgttl, 196%) and Foeniculum vulgare (Sehgal, 1964) which otherwise bear monoembryonate seeds. A reference t o these is made in the section on polyembryony (p. 280). Dulieu (1963, 1965) grew unpollinated pistils of Nicotiana tabacum on a nutrient medium and 24 hr later applied pollen to the stigmas. It was found that the number of the ovules which were fertilized depended on the quantity of pollen applied. Under the best cultural conditions nearly 25% of the ovules were fertilized and 50 seeds developed per ovary. Such a low seed set was due to the degeneration of a large number of ovules both before and after fertilization. Some of the seeds germinated while still in the ovary and produced normal seedlings. To increase the percentage of seed set Dulieu excised the ovules 5 days after fertilization and cultured them on a fresh medium. While the endosperm grew normally, the embryo attained only the heart-shaped stage even after 45 days, It is well known that double fertilization stimulates not only the formation of the embryo and endoeperm but alxo the development of the fruit. Even in most apomicts pollination stimulafes the growth of the ovary and seed although there is no fertilization. The culture of the pistils of apomictic plants may, therefore, help to elucidate the nature of the stimulus provided by pollination. So far the only cultural experiments of this kind are on Aerva tomentom (family Amaranthaceae) which is an obligate apomict. Since there are no stctminate

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plants at Delhi, seed formation is independent of the stimulus of pollination. Puri (1963)studied the effects of IAA, casein hydrolysate, coconut milk and yeast extract on the growth of the embryos in cultures of ovaries, flowers, and portions of the spike. I n ovary cultures only 7% of the ovules developed into seeds and even these contained only poorly differentiated embryos. In flower cultures raised on the basal medium 15% of the ovules bore mature embryos. Yeast extract and casein hydrolysate increased the seed set to 20%’ and with coconut milk 26% of the ovules matured into seeds. The best response was elicited when portions of the spike were cultured instead of individual flowers. I n the presenoe of IAA and yeast extract the seed set was comparable to that in nature. Except for slight variations in size, the growth of the embryo and endosperm waH quite normal. In Zephyrantha which is also an apomict, Kapoor (1959) observed that ovaries, excised two days after pollination, developed into normal fruits on the basal medium itself. In the confinements of the culture vial ovaries often fail to grow into full-size fruits. To overcome this Ito (1961) devised a “partial sterile culture method” in which, instead of implanting the entire pistil, only the flower stalk is inserted into the aseptic nutrient medium through an opening in the stopper of the culture vial, and the ovary is left free to grow outside the vial. Using this technique Ito studied the growth requirements of the ovaries of Dendrobium nobile. The ovaries developed well on Nitsch’s medium containing only inorganic salts and a sugar. Generally, the disaccharides were superior to the monosaccharides, and among the former maltose and lactose were better than sucrose. Organic supplements were not indispensable, but vitamins B, and B, stimulated ovary growth, and tocopherol acetate (vitamin E) increased seed fertility. The seed set was best when all the three vitamins were used together. Coconut milk as well as peptone favoured fruit growth and also increased seed fertility. The partial sterile culture method is useful for studying the effects of variouR physical agencies on the growth and development of fruits, especially in plants bearing pedicellate flowers and in which the mature fruit is many times larger than the ovary. The floral meristem has been intorpretetl as a system of reactions which pass through several phases that correspond with the initiation of the floral organs (Wardlaw, 1957; Heslop-Hamison, 1969). To test this, Tepfer et al. (1963)used the culture technique. Flower buds of Aquilegia fwnwaa ranging from the pre-sepal t o the young carpel stage were excised and cultured on White’s medium supplemented with coconut milk, ten water-soluble vitamins, IAA, kinetin and GA,. When the buds were cultured a t the ‘%tar-carpel&age” the carpels

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surpaased both the stamens ailti the atamiiiodirt in growth (Fig. 11A and B). Nearly 26 days after culture the carpels had reached the maximum stage. The stamens and staminodia were poorly differentiated and later aborted. If the buds were cultured at “dimpled-carpel stage” the carpel8 were very well developed cbnd tho petals grew to about the normal oize for t h i H carpel stago, but the androeoium aborted (Fig. 11 C and U).When the buds were cultured at the “grooved-carpel stage” in 19 days after culture they reached the “erect-carpel stage” (Fig. 11 E and F). However, the stamens and staminodia aborted. In many cultures the carpel reached a length of 10-16 mm but showed no ovules. Fewer carpels differentiated in vitro than in buds on the control plants. The filaments and anthers differentiated but %hesporogenous tissue developed only up t o the pre-meiotic stage. The sepals developed in a manner similar to those in buds of intact plants. Tepfer d al. (1963)also observed that if the sepals enclosed the floral apex the inner floral whorls ceased to develop and when the sepals were removed the cther members resumed their growth.

TX. PARTHENOCARPY That fruits can develop without the act of fertilization (parthenocarpy) was first recognized as early as 1849 by Giirtner who obtained seedless fruits of certain cucurbits by “pollinating” the ovaries with the spores of Lywpodium. Massart (1902)placed dead pollinia on the stigma of some orchids and observed n r~wellingof the ovary. That some chemical substances might be involvud in the development of parthenocarpic fruits was inferred from the observations of Fitting (1909). He injeoted an aqueow extract of the pollinia into the ovaries of certain orchids and found that it not only prevented the abmission of the flowers but also caused a swelling of the ovaries. Kusano (1915) confirmed the observations of Massart and Fitting and also performed several other experiments 011 tho orchid Gastrodia ehta. Yasuda (1930, 1940) succeeded in producing pathernocarpic fruits of nearly normal size in several plants, especially those belonging to the Solanaceae and Cucurbitaceae, by treating the flowers with extracts of pollen. After Thimann’s (1934; see also Fukui el al., 1958) demonstration of the presence of indoleacetic acid in pollen, this and several cther growth substances belonging to the indole and the naphthalene group and the gibberellins have been utilized to induce parthenocarpy in a number of plants (see Guetafson, 1942; Maheshwari, 1960; Leopold, 1956). There are also reports of the natural o c c m n c e of auxins, gibberellins, and other substances of R similar nature in ovules, seeds, ovaries and fruits (Teubner, 1953; Luckwill, 1957; Von Bargen, 1960; Corcopn and Phinney, 1962).

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Many varieties of pear grown under low but not freezing temperatures produce an abundant blossom and yet fail to give a satisfactory fruit set. This is generally attributed to inadequate pollination and/or a failure of fertilization. Osborne and Wain (1961) reported parthenocarpic fruit formation after treatment with naphthoxypropionic acid. Ratjer and Uota (1951) and GriggA et al. (1961) considerably increaRed the fruit set in the variety “Bartlett” by treatments with 2,4,6-trichlorophenoxyacetic acid (2,4,S-T). However, similar attempts with other varieties were unsuccessful. Luckwill (1960) and Griggs and Iwakiri (1961) reported that GA, increased the fruit set in pears. The fruits resulting from gibberellin treatments had a more elongated form than the pollinated controls. Thompson (1963) used GA, and certain other growth substances on five varieties of pear. At full bloom, lanolin emulsions of GA,, IAA, IBA, and 2,4,5-T were injected, individually or in combinations, into the calycine cup. Applications of lanolin emulsions only (without any test chemical) resulted in an early nbscission of the flowera which could not be remedied by IBA. On the contrary, 2,4,5-T usually delayed abscission; whereas GA, promoted the retention of the flowers as well a8 the parthenocarpic development of the fruits. Crane (1963) investigated the effects of GA, on the development of fruits of peach. Aqueous solutions of a potassium salt of GA, (1000 ppm) were sprayed a t different stages after emasculating the flowers. Fruits which were obtained from treatments given after the petals had fallen matched very well with fruits developed in t h e openpollinated controls. With apples also the gibberellins have proved superior to other substances. Davison (1980) obtained seedless fruits in some four varieties by treating the flowers with gibberellic acid. After removing the petals, stamens and style a lanolin paste of 1% GA, was smeared on the cut surface as well as on the inner region of the receptacle. Thin induced the development of parthenocarpic fruits which compared well with naturally formed fruits in quality, colour and the time of ripening. FIG.11 (See facing page).

FIG.11. In vitro culture of flornl buds of Aquilegia fwmosn (cl-c5. carpels; st, staminode). A. C and E. Top view of buds from which sepals wcre removed before photography. B, D and F. Same buds after their growth on White’s medium+ IAA (O*6ppm)+QA, (2 ppm)+ kinetin (0.6 ppm) +coconut milk+ 10 vitamina. A. Bud at “star-carpelstage”; note the five carpels arrsnged in a pentarch manner; the crescent-shaped structures immediately next to the carpels arc the staminodes; and the remaining outcr whorls represent the stamens. B. Bud shown in A photographed 19 days after culture; all tho floral organ8 ere well developed. C. Bud at “dimpled-carpelstage’’ showing the dimpled carpels, stamene and etaminodis. D. Bud shown in C grown for 27 days in culture; only two petals and part of one sepal were retained for photography. E and P. ‘‘Croovc4cq-A RtUge’’ of bud beforo and 19 days afbr culture (after Tepfw el nl., 1988).

Fro. 11. Soe Icgcntl facing page.

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Dennis and Edgerton (1962) induced parthenocarpy in the McIntosh apple by applying a lanolin paste of potassium gibberellate to the emasculated flowers, Bukovac (1963) applied lanolin smeara of gibberellin A, to the cut surface of the style and the adjacent regions of the receptacle resulting in the development of parthenocarpic fruits. The gibberellins have elicited a similar response in several other rosaceous fruit crops belonging to the genus Pr?inzs.Crane et al. (1960) obtained a successful induction of prthenocarpy by spraying aqueous solutions of 50, 260 and 600 ppni of potassium gihberellate a t full I)loom or eight h y s after anthesis. With ttlmoncl arid npricot two applicatioiis of tho gibl)erellnte, ; L t ill1 intervid of eight tlitys, induced the fornitition of I)artlieiiocarl)i(.fruits. ‘l’lie peach rexp)ntletl best to all the colicelitrations of GA, irrespective of the nuiiiber of applications aiid the partlietiorarpic. fruits iiintc*lietl those formed naturally. However. the tretttnieiitq were ineficctive on cherry and plum. Rebeiz and (‘rane (19fil) sprayed tLqueous solutioiis of (;A3 alone or niethioniiie (2.4-DM) suppleniented with 2,S-dic~liloro~~Iienoxytloetyl on “Bing” cherry. However. the pnrtlienocnrpic fruits were smaller than the pollinated cwitrols. For strawberries Zielinski aiitl (hrren (19.38) reported that NAA (80 ppni) caused a 301:, increase in fruit set if sprayed two weeks after pollination when the production of auxin in the achenes reaches a peak. Lord t ~ n dWhite (1902) obtnined seedless strawberries by treating the emasculated flowers of five varieties with lanolin miears, solution smears, cqueous sprays. or eniulsions of naphthalene acetamide and IBA, iisetl indivictually or i n mixtures. However, the parthenocarpic fruits were not superior to the control berries. r i l h c genus Hosii c*oinprisesiii)otnictic*ILS well ILH noii-ai)otiiic:ticHpecieH. I’artheiioc,nri)y is i i iioriiinl feature in tlic fornicr hut not of the nonitponiicts. tJii(’kS0II tint1 1’1~sser( 1950) ctttcti1l)tetl to intlucc pnrthenociirpy iii the iio!i-iLl)oriiic‘tic.qx.icx : ii. uriarutiH, K.r?iVoeu nntl H.spinosiesima. Lanolin piistes of N A A , NAI) iLll(1 2,4,6-‘I’ were applied to emasculated flowers of field-grown plants. All treatments gave parthenocarpic fruits in H. ncqosu. However, 2. spinosiaeim was less responsive tliaii II. riigovo antl the best fruit set (W;:,)waH obtained with NAA-treatment. R . urvenvis wns the least responsive to any of the treatments. If’ the greenhouse plants of II. spinosiasima were treated with NAD, or UA,. or a mixture of both, parthenocarpic fruits resulted. Both NAD and (;A, gave W-70”0 fruit set, and a mixture of the two gave a fruit set of 9-j+:;,. Zatyk6 (19fi2a,b, 1963) induced parthenocarpy in GrossuZaria reclinata (cultivated gooseberry), in Hibes nibrum and R. niyrum (red and black currant) antl in some t w o interspecific hybrids of Ribee

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throiigh treatments with GA, and IAA. A pronounced synergistic effect of the two chemicals was observed in the gooseberry. Schroeder and Spector (1967) demonstrated a similar effect in the growth of pericarp tissues in vitro. Tn temperate countries serious lo~sesoccur in the glass-house pmduction of tomatoes during winter owing to a paucity of good pollen. To mitigate such losses tomato growers have resorted to the use of TAA, NAA and 4-chlorophenoxyctcetic acid all of which have been found very effective. Wain (1950) nhowod that a mixture of NAA and 4-chlorophenoxyacotic acid N i alro very useful. Pollination is not excluded in tomatoes ; the application of chemicals suppleme& the work of pollination and improves the fruit set. McGuire (1952) and later Dempsey (1962) showed that old pollen of tomato induced the formation of berries without participating in fertilization. Sell el aE. (1963) observed that rnethylated and ethylated esters and chlorinated derivatives of IAA wore more effective than IAA itself in inducing parthenocarpy in tomato. Some of the gibberellins have also been reported to be 500 times more potent than IAA in inducing parthenocarpy (Wittwer el al., 1957). Bukovac and Wittwer (1958) found that even very low concentration (3 x lo-%) of GA,, GA,, and GA, was inore effective than IAA. Weaver and Williams (1950, 1951, 1952) found that spraying of 4-chlorophenoxyacetic acid considerably improved the fruit set in the seedless varieties of Vitis viniferu. Stewart et al. (1958) reported that if GA, wns sprnyod eithcr at nnthesis or shortly thereafter it incremed the fruit set aB well as the size of the berrien. Similar results were alno obtained by R. J. Weaver (1968), Weaver nnd McCuno (1959), and Wittwer and Bukovac (1958). Pratt and ShaulL (1961) intlucecl parthenocarpy in grapes by tho application of gibberellic acid. In tho absence of pollination a B well as under conditions of inadoquato pollination GA, induced the dovelopment of oarly maturing, parthenocarpic berries. Among citrus fruitx the iiavol orange is naturally parthenocarpic, but Furusato and Suzuki (1955) were able to induce parthenocarpy in Citrus nataudaidai by a treatment with 2,4,5-T or NAA. Tn fig culture the quality of the fruits is dependent on caprification. Stowart and Condit (1949) found that spraying6 of 2,4-D and 2,4,5-T could induce the formation of seedless figs which were comparable to the caprified fruits in size and in the content of sugar. Blondeau and Crane (1950) induced parthenocarpy in the Calimyrns fig using aqueous or oil emulsion sprays of IBA, NAA, 2,4,5-T and para-chlorophenoxyacetic acid. IBA was effective at 200 ppm; NAA was increasingly so at 26-250 ppm; and 2,4-T (10 ppm) not only induced parthenocarpic

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developnmlt but also accelerated the maturation of fruits. The treatments with para-chlorophenoxyaceticacid (40, 60, 80 ppm) resulted in parthenocarpic fruits which matured normally and were comparable to the caprified controls in size, colour and taste. Balasubramanyam and Rangaswami (1959) observed that the artificially pollinated ovaries of some varieties of Psidium quajma developed into profusely seeded fruits whereas those of “Allahabad Round” yielded parthenocarpic and seedless fruits. They attributed the development of the parthenocarpic fruits t o the action of a “pollen hormone”. On applying an aqueous extract of the pollen to the emasculated flowers nearly all the ovarie6 developed into seedless fruits which ripened earlier than those formed through natural pollinations. On making chromatographic analyses of extracts of the pollen and the ovaries of “Allahabad Round”, Rangaswami and Kaliaqerumal (1960) detected traces of an indole-like compound in the extracts prepared immediately after antheah and much larger quantities of the name substanco in 5-day-old ovaries of selfed flowers. Tho cornpound could not be detected in 20-day-old ovaries a8 well as in the seeded varieties. However, exogenous applications of neither IAA nor JBA were effective in inducing parthenocarpy . Instead, parthenocarpic development was obtained by treatments with N U , NOA and 2,4-D. Teaotia et al. (1961) and Shanmugavelu (1962) were able to induce the formation of parthenocarpic fruits through the use of gibberellic acid. Such fruits showed a granular pulp and contained more ascorbic acid than the normal fruits. The induction of parthenocarpy is of considerable value in the production of greenhouse cucumbers, since it helps in bypassing tho laborious process of hand pollination. Wong (1941) obtained parthenocarpic fruits in several members of the Cucurbitaceae and Solanaceae through treatments with NAA, IBA, potassium naphthalene acetate, colchicine, acenaphthene, sulphanilamicle and trimothylamino. Dzevaltovsky (1962) reported that while IAA, gikhorellins, and tho sodium salt of 2,4,5-T had little or no effect 2,4-1) (0-002”/) readily induced prirthenocarpic development of the ovaries of Homo mom how of the Cucurhiticeae. Yakar-Olgun (1962) induced parthetioc:arpy in Viciafaba by the application of a 100 ppm aqueoun nolution of (;Aa. The parthenocarpic fruits were larger than those which developed from the pollinated controls. Sachar (1962) studied the eReect of gibberellin6 and certain indole compounds on the self-incompatible Pereskia aculeata (family Cactaceae). Sprays of IAA (50-500 ppm) delayed abscission of the ovaries, but. failed to induce partbenocarpy. IBA (50-500 ppm) induced 15% fruit set ; with GA, (100-500 ppm) it was loo(:/, .

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Britten (1947, 1950) sprayed the dehusked ovarieH of Zaa vliays with an emulsion of 0.1% NAA. Trentnients given 24 hr before pollination stimulated the ovaries to develop part1:enocar~)ically. If the ovaries were treated after pollination tho incidence of parthenocarpy was less pronounced. Sachar and Kapoor (1959) found that both GA, and IAA were effective in inducing parthenocarpy in Zephyranthes. The parthenocarpic fruita obtained by gibberellin treatment excelled the untreated controls in size. Kinetin, as well as mixtures of kinetin and IAA, were ineffective. According to Nitsch (1952, 1963) parthenocarpy may be of three types ; genetical, environmental, and chemical or induced. The navel orange, several varieties of cucumbers and the cultivated banana may be cited as examples of the first kind. Sometimes environmental factors such as temperature can also cause a parthenocarpic development of fruits, Lewis (1942) obtained parthenocarpic pears by exposing the flowers to freezing temperatures. Yasuda (1934) made similar observations on Solunum mehgenu and Nicotianu tabacum. Many instances of induced parthenocarpy have already been cited above. According to Gustafson (1939) plants which have a high content of auxin in the ovary show a tendency to be naturally parthenocarpic. Muir (1942) reported that soon after pollination the auxin content in the ovary increases considerably (see also Lund, 1966). For the successful development of the fruit a supply of auxin is essential. In naturally parthenocarpic fruits this is met partly by the pollen and partly by the seeds, whereas in induced parthenocarpy the auxin is supplied exogenously. Thus, pollen has two chief functions: (a) participation in fertilization and (b) an aid in fruit development. This is s u p p o h d by the experiments of Nitsch (1960) on strawberry. If the fertilized achenes were removed the growth of the receptacle ceased. A removal of the achenes as late as 21 days after pollination also elicited a similar response. If the achenes were removed and lanolin pastes of NOA or IBA were applied in their place, fruits of normal size and shape developed. The investigations carried out during the last decade emphasize the importance of the gibberellins in the development of parthenocarpic fruits. Recent reports of the occurrence of gibberellins A,, A,, A, and A, in the seeds of Phaseolus muEtiJorus (MacMillan and Suter, 1968; MacMillan et al., 1961) and gibberellin-like substances in the ondonperm of apple (Nitsch, 1958) indicate that theBe substances play an important role in the growth of fruits. Of interest also is the report of the occurrence of a kinetin-like substance in apple (Goldacre and Bottomley, 1969). However, further studies are needed on the upecific role of the gibberellins and kinins in the growth of the fruit.

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x. 1’OLYEMURYONY As early as 1719 Leeuweiihoelr noted that orange seeds contained more than one embryo. Since then many other plants have also been found t o produce polyembryonilto seeds. Stradmrgor (1878) Rhowed that the additional eiiibryos usually originate from the maternal tissues like the nucellus or the integument (adventive embryony) and later protrude into the embryo sac, Subsequently, other sources of accessory embryos were discovered. Among these are a cleaving or budding of the zygotic embryo (cleavage polyembryony), an3 a development of the synergids or antipodals either parthenogenetically or after fertilization by a male gamete. A. ADVENTIVE EMBRYONY

Hatierlandt (1921, 1922) assumed that the degenerating cells of the ovule secrete some substances (“necrohormone”) which stimulate the neighbouririg cells of the nucellus to develop into embryos. He attempted to induce tho formation of adventive ombryos in Oencl-thera by pricking the ovules or by syuceziilg the ovary HO as to cmse a Blight damage t o some cells. I n one of the treated ovulecl he observed two embryos and considered one of them to be of nucellar origin. Bedemaim (1931) followed Haberlandt’s technique and obtained D 2-celled embryo in Mirabilis unijlora, but whether it originated from a nucellar cell or the egg wa8 not ascertained. Similar efforts by NBmec (19351, Doak (1937) and Ivanov (1938) on other plants were unsuccessful. Despite his careful and intensive experiments Beth (1938) was unable to induce adventive embryony even in Oenotheru lurnarkiunu which was the object of Haberlandt’s studies. According to some workers adventive embryos are the products of a “somatic fertilization” of nucellar cells (see Glushtchenko, 1956). They believe that when two or more pollen tubes enter an ovule, the male gametes are sometimes discharged into the nucellar celh and also fertilize them. However, this observation remains unconfirmed (see Maheshwari end Rangaswamy, 1958). The adventive embryos are of considerable importance to the horticulturist as they are genetically uniform and roproduce the genotype of the seed parent without inheriting the variations kJrOUght about by the chromosomal segregations during q~orogcnminor by the gene recombinations in fertilization. Adventive emhryon are, therefore, used for propagating selected varieties of some horticultural CrOIJS and methods of artificial induction of adventive embryony have acquired special significance. Van Overbeek et al. (1941) injected several chemical suhstances

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into the ovaries of Datura stramonium. A treatment with O*l‘:/b NAA or IBA produced in some ovules a multicellular, tumoroid structure from the endothelium. Similar results were also obtained by Chopra and Sachar (1957) with D. fastuosa. However, the tumoroid structures did not differentiate into embryos. Fagerlind (1 946) reported that if II 1 (;(, lanolin paste of heteroauxin was applied to unpllinated pistils of Hosta they produced young adventive embryos. However, owing t o the lack of an endosperm the embryos failed to develop further. In many species of Citrus nucellar embryos are common. Furusato (1953) studied the possibility of controlling their number in C. natsudaidai and C . unshu. When a solution of maleic hydrazide was injected into young fruits the seeds showed a reduced number of embryos, sometimes only one per seed. More critical studies are, however, necessary to determine whether the treatments had any tjelective effect on the zygotic embryo or the embryos arising from the nucellus. In recent years the technique of tissue and organ culture has been used as a tool in studies on polyembryony. Rangaswamy (1961) excised the nucellar tissue from the fertilized ovules of C. microcarpa and cultured it on a modified White’s medium supplemented with casein hydrolysate. The nucelli proliferated and formed an exuberant callus. The callus differentiated into embryo-like regenerants (designated as “pseudobulbils”) which eventually developed into plants (Fig. 12). Similar observations have also been made on a few other species of Citrw (Sabharwal, 1963). These investigations demonstrate that if the nucellar tissue is freed from the confinements of the ovule and grown on an appropriate nutrient medium it can be activated t o unlimited growth and induced to yield a continuous supply of nucellar embryos. As yet, this has been found to be true of the nucelli of only such Citrus species that are naturally polyembryonic. The nurturing of the nucelli of monoembryonic species on the nucellar callu8 of a polyemhryonic Citrus is a line for future investigation. The zygote is a unique cell since it carries the genetic c:omplcment necessary to build a new individual. Recent rexearch ha8 s h o w n that the capacity to produce a new plant hody may well he manifcxtcci hy alniost any living cell of the organism. Somatic c c h rdc~s(!dfrorn differentiated tissues such as the vascular parenchyma of‘rootn,rncclullary parenchyma of stems, leaf parenchyma, and even thc ccllx c ~ fthe endosperm (unpublished observations on Sunhlurn ulhum) have heen observed to form enibryo-like structures. Steward et uE. ( I $ % ) and Steward (1963) showed that full-grown plants of BUUCUB wrnto can be raised from its phloem cells. Iliscs of phloem were excised from the root and cultured on a nutrient solution supplemented with coconut r\

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milk. I n 80 days the explants increased 80-fold in weight. On trcbnsfer to shake cultures the tissue i i i n ~ sdiusorittted to form t~ suspciision of single cells. The individual cells developed into cell aggregates which typically resembled the early stages of growth of the embryo. When transferred to an agar medium they established a root-shoot axis. On transplanting into the soil they gave rise to new and normal plants of carrot. Almost similar observations were made by Reinert (1959, 1963), Butenko and Yakovleva (1962), Kato and Takeuchi (1963), and Wetherell and Halperin (1963). Reinert (1959) found that on a medium containing coconut milk the callus tissue derived from cwrot root formed only roots. The differentiation of the root and the shoot could be controlled by changing the composition of the medium. In the presence of auxins (IAA or 2,4-D) root formation was inhibited, whereas in their absence the shoots and buds were inhibited. The roots generally arose as typical monopolar organs while the shoots originated from bipolar adventive embryos which differentiated in the callus. Butenko and Yakovleva (1962) observed that the growth and differentiation of the carrot root explants occurred in two phasee. On a modified White’s medium supplemented with the four RNA nucleosides, casein hydrolysate, coconut milk and 2,4-D, the explants proliferated vigorously and also showed the formation of buds. The second phase of development, namely the differentiation of roots and an active growth of the buds, occurred only when nucleosides and amino acids were both excluded from the medium. A transition to the second phase was stimulated by introducing into the medium antimetabolites of nucleic acid, of protein, as well as of auxin metabolism. Kato and Takeuchi (1963) reared callus tissues from excised root discs of carrot cultured on an agar medium containing yeast extract ( O . l * a ) and IAA. The callus showed two patterns of differentiation into embryos and their development into seedlings. Initially the callus was orange yellow. In one pattern a colourless tissue emerged along its periphery (40 days after culturing) and showed several nodule-like growth points (Fig. 13A and B). Some of them already exhibited a root primordium (Fig. 13C). In another 20 days a shoot primordium also originated in the nodules and completed the root-shoot axis (Fig. 131)). In the second pattern of differentiation the friahle callus rliseociatcd into single cells and small cell aggregates in about four months after culture. Through successive stages of differentiation resembling those of normal embryogenesis the cell colonies established ~~lantletrl (Fig. 13E-J). When a single cell was cultured in complete isolation

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on a fresh medium it failed to manifest the totipotency, but on a medium which had previously supported the growth of the callus tissue or when several of them were nurtured in a mass, differentiation occurred suggesting the normal pattern of embryogenesis (Fig. 13E-I),

Explant from tap root

Seedling

formation

\/A Production of roots

Torpedo- shoped stage

/

t

Heart-shaped sloge

Formotion of shoot; also isolation j f single cells

Globular stage

Late globular stage

FIG.13. Formation of seedling^ from root disc8 of Daucu.9 awolr~culturctl on Wltito'n agu medium flupplemcnted with yeaat cxtrtwt (0.20/,)mil JAA ( I or 1 0 pprn). A,I$,~;,IJ und J represent one pnttarn of devehpmcnt of Iihntlatn. 1G.J iridii:8ktf>tho nc:c:ortd ptterrt in which embryo8 are organixc!rl prior t o formation of pliintlotn (after K i i t ~ ,and 'Ikkr:ur+ti, 1 !)fcjI.

ThurJ, multicellular units (nodules) as well as siriglc cells U'CI'C h J t h capable of producing plantlets (see also Mackawa ct al., 1968). Wetherell and Halperin (1963) cultured the root tissue of' thc wild carrot on an agar medium of semi-solid consistency. 'J'ho tissou p r Y J -

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duced a callus capable of continued growth. Portions of this were transferred to liquid cultures which were put on a rotator. Ten days after transfer 50-100 embryo-like structures developed in some of the cultures. These ranged from globular embryos (0.2 nun in diameter) to torpedo-shaped embryos. Subsequently, they devclopcd typical roots and green cotyledons. On transfer to a moist agar substratum containing only the mineral components of the ciilture medium, the plantlets developed into adult plants. Bearing close relevance to the above reports of the totipotency of carrot root cells are the recent observations of Bergmann (1959, 1960)

FIG. 14. Formation of embryo-like Ntructurw olinervcd in cell culturoa tlerivcd from pith pnrenehyma of Nicofiana h h ~ c u m(hfter Ibrgmann, 1851)).

on Nicotiana tabaczim and of Wadhi and Mohan Ram (1964) on Kalanchoe pinnata. Bergmann raised callus tissues from portions of the stem of Nicotiana cultured on White’x medium xupplemented with 7% coconut milk and 0.5 ppm 2,4-D. When the callus wax grown in liquid shake cultures it dissociated into small clusters and xingle cells. Suspensions of these were plated on an agar nutrient medium in petri dishes. Several cells showed active growth and gave ri8e to Glamentous structures some of which were organized like proembryos (Fig. 14). The leaves of Kalanchoe pinnata bear the so-called “foliar embryos” in their marginal notches, With a view to ascertaining their morphogenetic behaviour, Wadhi and Mohan Ram excised the leaf notches and cultured them on a modified White’s medium containing coconut milk and 2,4-D. The explants produced a callus tiswe which

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Y . MAHESHW.4RI A N D N . 9. R A N G A S W A M Y

eventually differentiated into structures reminiscent of proembryos. Whether these can estnblish themselves into plnntlets st’illremains t o be investigated. A reference may also be made here to the investigations of Ito (1960) on the protonemata and prothalli of Pteris vittata, and of Schroeder et al. (1962) on the pericarp of Persea gratissima (avocado). By pricking with a fine glass needle Ito killed all but one cell in some mono-layered portions of the gametophytes of Pteris vittata and cultured them on Knop’s agar medium. Each cell isolated in this manner promptly regenerated into i t normal gametophyte. Schroeder et al. (1962) observed that in explants of the pericarp of avocado cultured on White’s medium the callus tissue developed typical roots. Undoubtedly “the apparently unlimited growth in culture of a tissue definitely limited in its natural growth” is of much interest t o morphogenesis. That an already organized tissue like the vascular parenchyma of roots, or the nucellus, or the leaf parenchyma, or the medullary parenchyma of stems, or the fruit parenchyma can be made t o proliferate and then again differentiate in culture completes the cycle of differentiation--,dedifferentiation=redifferentiation. This confers on the somatic tissue a capacity simulating that o f the zygote. Being a potential and continuous source of adventive embryos such tissue “banks” acquire a special interest for physiologists and geneticists (see Maheshwari and Rangaswamy, 1963). Whereas the artificial induction of adventive emhryos has undoubted advantages, at times i t is necessary to eliminate them. For example, there are obvious difficulties and uncertainties if the breeder has t o deal with a mixed population of seedlings, and hybridization experiments would indeed be rendered unfruitful if the nucellar emhryos suppress the zygotic embryos. If methods could he devised for a selective elimination of either one or the other kind of emhryos, it would be an important contribution to our techniques of plant improvement. B. EMBRYONAL BUDDINU

Like adventive embryos, identical twins and Nuper-twins resulting from a cleavage of the zygote are also of great interest to geneticists and plant breeders. Such cleavage polyembryony is common in gymnosperms but is much less frequent in angiosperms (see Maheshwari and Sachar, 1963). A plant which is of interest in this connection is Erardhis hiemlis (family Ranunculaceae). At the time of shedding (April-May) the seeds contain an undifferentiated embryo which is pear-shaped and possesses

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a long suspensor (Figs. 15 and 17). The radicle as well as the cotyledons differentiate several weeks later while the seed is in soil. Haccius (1955, 1957, 1963) investigated the effects of certain antimitotic substances on undifferentiated embryos. The seeds were soaked in solutions of the test chemicals for 24-96 hr imtnediately after shedding. In treatments with a O.loA, solution of 2,4-D, or 2,4,6-trichlorophenoxyacetic acid, or NAA, Haccius (1955) obtained many abnormalities such as syncotyly and pleiocotyly in nearly lOOY, of the embryos and twinning in 3-80,; of them (Fig. 16 A-C). Occasionally,the embryos

Fra. 16. Time schedule showing development of embryo of Emnlhis hipmulis after shedding of seeds. H+R, hj’pocotyl and radicle; L, lamina of cotyledons; S, cotyledonary sheath; and V, plumule. At the timo of shedding (May) the embryo is 0.1 mm. in length and undifferentiated. Further devaiopment takes place after shedding (after HacciuR, 1963).

showed a one-sided regeneration (Fig. 16D). It is explained that owing to the treatments ttie plumule uas damaged and the cells which were normally destined to produce the cotyledonn gave rise to ittldithrtl stem tips. In another set of experiments the frexhly harvested xeetls were treated with isopropyl-N-phenylcarhamate (0-2‘%,)and maleic hydrazide (O.l”/b). Four treatments administered within 24 hr caused a necrosis of the embryo yhich now appeared like a brown s p t . After a couple of months new regenerants originated from the suspenwr end of the necrosed embryd. If the same treatments were given 3 days after the shedding of the seeds, the development of the original ernhr.yo was arrested but there was no regeneration. Recently, Haccius (19639 has reported that the original embryo died if the test solutions were acidic. Xo effect was observed between

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5 and 7 , but 5004 of the embryos treated a t pH 3.5-4.5 showed a marked basipetal necrosis and after 2-3 weeks only the suspensor cells remained alive (Fig. 17A-C). Tn due course these were re-embryonized and gave rise to embryoids. Initially the suspensor enlarged and became club-shaped due to initotic activity in its uppermost cells, and its connection with the necrosed embryo became severed (Fig. 17D). Eventually, a new bipolar embryo was reconstituted which showed

pH

E

F

C

G

FIG.17 (see frrciny page). FIG. 17. Eranlhis hiemstlie (I), bud; dc, degenerating embryo; e, embryo proper; re. regenerating embryo; s, suspensor; v, plumule). A. L.S.part of secd at time of shedding showing undifferentiated embryo and elongated suspcnsor. B. L.S. seed 18 dayn after acid treatment; the embryo is necrosed but the cells of the suspensor immediately below i t have been activated. C. As in B, 22 days after treatment; the suspensor ha8 heCIJme club-ehaped owing t o the enlargement of the cells a t its upper end and the degenerated embryo has heen pushed aside. D. L.S. seed 6 weeks after acid-treatment showing prominent activity of the suspensor. E. As in D, 10 weeks after treatment. The new embryo is well developed and also shows a lateral bud (b). The remnants of the parent embryo have been completely pushed aside (after Hacrius, 1963).

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normal development (Fig. 17E). However, twins and nialformed embryos were also seen occasionally (Fig. 16E-H). Eunus (1955) X-irradiated the developing caryopses of Hordeurn vulgure and studied its effect on the growth anti development of the embryos. Generally, young embryos were more sensitive to the treatment than old embryos. When the caryopses were subjected to high doses of X-rays (400-800r) the embryo showed certain regions of deeply stained meristeniatic cells. These gave rise to numerous proliferations. Their frequency was highest in embryos irradiated with 800 r. Further, the proliferations developed in larger numbers near the haw of tho c:otylcdon r ~ n din regions directly oppowite or close to the plumulc. Sonictimes thc prolifcrtLtioiirr ronernhletl tho plrrmulc. itwlf. In studies on hybritlizations of U. ccratowulu with nine ottior herbaceous species of Dulura McLean (1946) observed thiht the hyhritl embryos became arrested during their growth. They varied from a completely undifferentiated mass to a differentiated but often malformed structure. With a view to raising F, plants McLean (1946) excised the undifferentiated embryos obtained in the crosses between the male parent D . ceratocuulu and three female parents D.discolor, D. innoxia and D. leichhrdtii, and in the cross D. cerutocaula (9)x D . metel. The excised embryos were cultured on an agar nutrient medium containing malt extract (0.5%). They frequently formed an undifferentiated mass and produced several buds (“multiple growth”). In one instance (D. cerutocuulu xD. metel) as many as 106 buds were produced. Eventually the buds severed from the parent mass and developed into normal seedlings. The orchids have proved specially favourable for studies on embryonal budding. Curtis (1947) cultured the seeds of Vundu tricolor. On the basal medium they prcdocctl normal Heetllinp, tjut if the medium was supplemented with harhituric acid or its sodiurn sdtn the embryos proliferated into a large numher of c.hkmq)hyllous tistme masses which readily lent themselvew to su t)culturing. Enihryon o f n

FIQ.18 (see facing p a g e ) . FIG.18. Culture ofembryos of Czrsculu rejnezn on a modified White’s medium (AE, accessory embryo; C, callus; NS. normal shoot). A-C. Stagm in germination of mature embryo. In home cultures the radiculsr end of the embryo callused and accessory embryos differentiated from it. D. Culture showing further growth of callus; arrow indicates an accessory emhryo. E. T.S. portion of cultured embryo shoring the development of accessory emtJryoH. F. Culture of young embryo ahowing formation of callus and its differentiation into awcsnory rmbryos. G. A few accessory embryos; the one on the extreme right show8 the formation of hhoot. H. 10-Week-old culture of accemory embryo; note repetition of cyc le of formation CJf Hiipernumerary embryos (after Maheshwari and Baldev, 1962).

292

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R f A H E S H W A R l AN17 N . S. I
hybrid Cymbidium also developed rdlus on a medirun supplemented wit11 0-l0i, peptone. Curtis and Nichol (1948) studied the further growth of the original explants of both the orchids. TWOpatterns of growth were observed in Van&. Explants of Cymbidium gave rise to five patterns which differed in the size of the individual lobes in the callus and the degree of development of the rhizoids. I n their morphology and anatomy the proliferating structures resembled the protocorms produced during normal germination of seeds. Further, they showed a capacity for. continued growth, and germination was generally inhibited. Occasionally, however, true seedlings originated in some cultures. Recently, Rao (1963) reported embryo proliferation and differentiation in an interspecific hybrid of Vanda. It may be noted that in some orchids the embryo possesses a natural capacity to cleave into additional embryos and this can be augmented under artificial conditions. As in orchids, in many angiospermic parasites also the embryo shows a remarkable capacity for proliferation and Hubsequent differenrqflvza tiation. Work done a t the IJniversity of Delhi on L'W~CU~U (Maheshwnri and Snltlcv, I M a ) , llendrophthoc. Ji~Zcatu (,Johri uiid Bnjaj, 1963) and Orohurbche uell.l/r,tiacu (Rangaswemy, 1963) are some examples. ~ were When young embryos (0.5-1-0 mm in length) of C U S C Ureflem cultured on an agar nutrient medium containing casein hydrolysate and IAA,they became greatly swollen and ultimately produced a mass of callus from the radical end. Within six weeks after culturing the resultant callus differentiated into small whitish or yellowish-green bodies resembling normal embryos (Fig. 18F-H). If larger embryos (1.0-2.5 mm in length) were cultured, they developed into wedlings in about a month (Fig. 18A-C). However, some of them formed a maus of tissue a t the radical end which produced supernumerary e m h r p similar to those originating on the callus from younger embryOS (Fig. 18D and E). On transfer to fresh medium the accessory embryos formed normal shoots, but if they were retained on the ' ' ~ l d "medium FIG.19 ( S e e facing page). FIQ. 19. C'ultllre O f Cmbrj'OS (If Ilerrrlrophlhos j f 4 h i 1 l l ( I L O , l&l!l'l:HHi)ry(!Ill IJQ'~J; ill, III'I'I~UMJ~~ leaf; c, d u n ; h, holdfant; pl, pluinular J i d ; r, rudir:ulrrr ond; N, nunrx!riHiw). A. 31atuw crritirycl. B. 10-Week-oldcmbryo grown on N'hitc'H rnetliurn+ I A A (1 ppm)+ ymxt extrnr+ (500 pprn); the cotylcdons and the rudiculiir cnd hrrw prolifcrutetl. C. 20-Wcck-old ~:ILIJIIH c*ultiv:rted on White's medium t IAA (0-5 ppm)+ casein hydrolysatu (500 ppm) Nhowirig w w r a l pupillate structures. D. 20-Week-old seedling showing one plurnular and mverul ncccxaory leaven, and a massive holdfast. E. Globular proembryo. F mid G . 3- and 6-week-old cultures nhowing the differentiation of accessory embryos from the tLalll:s.H. 10-Week-old pdyernhryona1 muss (after Johri and Bajaj, 1903).

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they gave rise to a tissue which was capable of repeating the cycle of polyembryony. Somewhat similar results have been obtained with Dendrophlhoe falmtu (Johri and Bajaj, 1962, 1963). Differentiated embryos (4-8 mm in length) usually formed normal seedlings, but occasionally the radical end or the cotyledons produced a callus or buds of limited growth (Fig. 19A-D). If globular embryos (0.8-1*5 mm in length )were cultured on a medium supplemented with casein hydrolysate, they proliferated abundantly and the callus gave rise to several accessory embryos (Fig, 19E-H). On subculturing, these produced normal seedlings. Like the embryos of Cuscuta and Dendrophthoe (stem parasites), those of Orobanche aegyptiaca (a root parasite) have also shown a considerable capacity for proliferation (Rangaswamy, 1983). Tn seed cultures the ovoid, untiifferentjatetl embryos pro(lucct1 a massive ~t~llris ay)able of continuou8 growth. Several shoot tips arose from this, but there was no evidence of root formation (see Fig. 9). These investigations suggest that under cultural conditions the embryos of orchids and parasitic angiosperniu have a remarkable capacity to produce tissue masses of unlimited growth which can, however, be made to yield embryonal buds and seedlings. Recently, Steward et al. (1963, 1964) have reported that the cells of immature embryos of a wild carrot (“Queen Anne’s Lace”) express their totipotency more readily than do the mature phloem cells of the root. The excised embryos were grown on a liquid medium containing coconut niilk. Here they proliferated and formed a dense suspension of cells. When this was spread on an agar medium in petri dishes the resulting growth comprised some large, vacuolated undifferentiated cells and a larger number of embryo-like forma termed as “emhryoirls”. These passed through the globular, heart-shaped, torpedo and cotyledonary stages of’ embryogeny and eventually produced mature plants (Fig. 20). In some instances polyembryony has been induced in cultures of ovaries. In Ranunculus scelerutus the ovaries were cultured to study the growth of embryos. The embryo usually developed r~ormallyo r 1 the basal medium (Sachar and Guha, 1962), but on a medium containing casein hydrolysate it showed a tendency to produce additional emhryolike structures from the hypocotyl (Fig. 21). More striking results have been obtained with Anethum pi:eolene (Johri and Sehgal, 1963a,b). When the ovaries were cultured thrce rlayH after pollination on a medium supplemented with casein hydrolysate, IAA and yeast extract, in 7-21?;, of the cultures the zygote underwent cleavage and budding to form 15-52 embryo-like regenerants which showed varying degrees of cotyledonary abnormalities. The Hwollen

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polyembryonal mass projected outside the niericarp and produced multiple shoots. If the regenerants were isolated and cultured they grew into normal plants. Recently, Sehgal (1964) has also obtained similar responses from cultures of‘ ovclriee of Foeniculwn uulgarp. Whereas the production of a callus has been reported from cultured embryos of many other plants, only those instances where differentiation has been achieved are of interest here in that they recall the cycle of differentiation, dedifferentiation and redifferentiation. They may

,cultured in medium DIUS

Storage root

/2 md phloem

@ /

explants

Transverse section of the root

in suspension

t 2 \

\

\

Embryoid from cultured

\

cells from embrvo

[email protected]

\

FIG.30. Prom individual cells of carrot to mature plant: diagrammatic representation of the cycle of differentiation in culture. Cells from tho phloem explants of the root as well 88 those from immature embryos pass through a basici~llysimilar course of development to form embryoida, plantlets and mature plants (after Steward et al., 1984).

also serve as a possible means of abundant clonal propagation and may turn out to be of special value in such inetance8 where ordinarily vegetative multiplication is not possible. XI. PARTHEPI’OOENESIN In angiosperms the first cell of theembryo, i.c. the zygote, i~ a product of fertilization. However, t h i g in not true of nucellar emhryon ]lor ~f the haploid embryos. Sometimes the haploidn arc a consequenr:c of fertilization of the secondary nucleus without an accompenying fertilization of the egg. Or, the zygote and one of the synergide mny both develop resulting in diploid-haploid twins. Since the discovery of the first haploid in Datura etramonium in 1921 (see Avery et al., 1959) Huch occurrences have been reported in more than 80 species belonging to

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as ninny AS 18 families (see Kiinber i w i d Rilcy, 1963; Milgooil tin([ Khanna, 1963). In ciicumber Aalders (1958) noted that embryos measuring 4-6 mm in length were likely to be haploids; these were reared in witro for further observations. Haploid sporophytes are of special interest to the geneticist because by a mere doubling of the chromosomes a haploid organism can be made to yield a homozygous diploid. In this way, plants homozygous for self-incompatibility alleles have been obtained without resorting to a lengthy and difficult programme of inbreeding. Owing to the great theoretical and applied value of haploids, many methods have been tried to induce their occurrence. Although considerable success has been achieved in this direction in animals, particularly the sea urchins and some amphibians, in angiosperms the occurrence of haploid sporophytes is still a rare and uncontrolled phenomenon.* Various techniques, such as treatment with abortive or irradiated pollen, delayed pollination, distant hybridization, and physical and chemical treatments, have been employed to raise haploids. Although abortive pollen is useless for fertilization, it may sometimes stimulate the egg to develop into a parthenogenetic embryo and might also induce an autonomous formation of the endosperm. Honic examples of this kind are Pharbitis nil (U, 1932), Nicotianu ylutinosa (Webher, 1933), and Oryza spp. (Nakamura, 1933). X-Ray treatment has been often reported to result in the forniation of haploid embryos. Ehrensberger (1948) pollinated normal plants of Antirrhinum nmjm with irradiated pollen and found some haploids in the progeny. Natarajan and Swaminathan (1958) treated the seeds, seedlings and inflorescences of Triticum aestivum var. N.P. 809 with Among algae Hiroe and Inoh (1954) obtaincd chemical intliiction of parthc*nr)gc*ncninin

8arga5r~tnpilulijwum. La1 (1963) induced the formation of haploid (apogamouq) rrImroljhytcw

in cultures of callus tissues raised from the protonema of the mom Phy8wm~triumcwqetzse. The young prothalli of Pterie cillnla grown in cultures in dark proliferated into callus tiwirs which showed a capacity t o produce apogamous aporophyteb (Kato, 1963).

FIQ.21 (see facing p z g e ) . FIQ.21. Rearing of ovarie8 of h h u n e u l u s acckralus. A. Owrion excisctl ti (hysafter r ~ t ~ I l i i ~ ; i tion and grown for 14 weeks on Sitsch’s medium+ c:nsi-iri hytlmlynntc (I000 prim); notc: formation of aeedhgs. !A mirropylar portion of :r~~honc! ro:ircsl for 2 wc!i!kn oil Sitwli’s ~ ; medium+ casein hydrolysatc ( M H J ppm); ttjc IJrcJf!mbryrJ drown txginniny of ;I ( ~ I V I I wljili? the endosperm has aborted. C. L.S. micropylar portion of achcmc! rwmd fijr 2 w w k H fJflSitnr+i’a medium+kinetin (0.5.ppm)+ 1A.a (5 prim); thc: niinpmsor is tiy~x:rt.r~~ptiicsl icrirl thr. c.nclrj. sperm has degenerated. b and E. \Vhole mountn of cmtJryon oxcisc:ri from l l ~ ! h f ~ f rcarctl l~H on ;L medium rontaining casein hydrolysatc ( 3 J f J ppm); noto the fiwiittionn. 1’. I,.S. (mi bryo dissected from achene reared on Sitsrh’s medium +casein hydrolysate (5fJO ppm) stmwirlg formation of supernumerary rotyledons and budding (after Sachnr and Gutla, I!&?).

u.

Fio. 91. See legend facing mxe.

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different doses of X-rays and obtained a few haploid embryos in such cases where the inflorescences had been subjected to 5200r for 2 or 3 days prior to anthesis. Like X-rays, radioisotopes have also been utilized in producing haploid sporophytes. Using P32Arnason et al. (1952) discovered a few haploid individuals in Trilicum wdgure. Natartjajtjan and Swaminathan (1958) reported that was effective in inducing haploidy in wheat when the treatment was given just before the onset of microsporogenesis. Pai and Swaminathan ( 1 959) observed a nulli-haploid with 20 chromosomes in the progeny of an S36-treatedplant of T . aestiwum (2n = 42). Delayed pollination has also been observed to enhance the frequency of haploids in certain crop plants. Kihara (1940) states that none of the 41 individuals of T. monococcum which were pollinated 3-5 days after anthesis produced any haploids, but three haploids were obtained from 8 individuals pollinated 9 days after emasculation. Pollinations made in the intervening periods gave a frequency ranging from 0-37". Smith (1946) confirmed Kihara's findingH ; in his experiments the frequency of the haploids increased from 0.1-20% by delaying pollination up to about 12 days after emasculation. In some distant hybridizations, although the pollen may not effect fertilization, it can often stimulate the parthenogenetic development of the egg into an embryo. The investigations of Chase (1952) on Zea mays indicated that the freqnency of haploids increased 20-fold by the use of certain male parents. Coe (1959) isolated a stock of maize which produced about 3% haploids and showed that the pollen of this stock was effective in inducing haploids in other lines also. Tiihara and Tsunewaki (1962) described the production of haploids in TriticaZe and in the cross Aegilops mudata x Triticum aestivum. Yasuda (1940) reported that by injecting an aqueous solution of belvitan (probably indoleacetic acid) into ovaries of Petunia violacea, the egg cells were stimulated to a few cell divisions resulting in small proembryos. A treatment with colchicine was reported to prwluce haploids in Nicotiuna langsdqfii (Smith, 1943) and I j o h v t ~ l y a r i x (Levan, 1945). Deanon (1957, see Magoon and Ktianrin, 1 Y M ) tre;Lf(:
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only. Huskins (1948) found that in roots of Allium cepa, which developed from bulbs grown in a solution of sodium nucleate (14%) for a period of 3-36 hr, reduction divisions and haploid nuclei occurred frequently. Similar investigations were made by Wilson and Cheng (1949) on Trillium erecturn, T. ovaturn and T. sessile, and by Patau and Patil (1961) on R b e o discolor. Haploid c e b have also been reported to occur occcisionally in root meristems of seedlings of Hordeurn vulgave and Triticum mnmoccurn raised fmm X-irradiated seeds (Swaminathan and Singh, 1968). During their studies on the effect of irradiation on sex expression in Citrullw vulgaria (2n = 22) Swaminathan and Singh (1958) found that a whole branch of a plant raised from an irradiated seed (48,000r) showed only the haploid number of chromosomes (2n = 11). This branch produced only one pistillate but many staminate flowers. Swaminathan and Singh presumed that the X-irradiation probably caused somatic reduction in the meristem from which the haploid branch originated. Somatic reduction has also been reported to occur in callus tissue cultures of certain plants such as H q l o p p p w (Mitrtr, et at., 1960). Since somatic reduction occurs in cultured c e b and because single cells are known to exhibit totipotency, there is reason to hope that this may be a way of obtaining haploid plants and then a homozygous diploid progeny. Little is understood of the causal factors responsible for the development of the unfertilized egg. That agencies like X-rays, colchicine* and foreign pollen are effective only in certain but not in all instances suggests that the role of the genotype is also significant. Further, the frequency of haploidy is variable in different species and sometimes even within clones of the same species. This increws the importance of selection of desirable genotypes suitable for a high frequency of haploids. However, the paucity of a technique for easily producing gametic chromosome constitutions a t will has been the greatest limitation t o the wide use of haploids. Even granting that a parthenogenetic development of the egg can be induced by artificial methods, there is still a serious handicap if the caretaker of the embryo, namely the endosperm, does not develop in the embryo sac. I n other words, a successful induction of parthenogenesis would involve two steps: (a) normal cell divisions in the egg, and (6) formation of a nutritive tissue, whether it is endosperm or an aawptable substitute. A recent report on the culture of the unfertilized eggs of HorcEeum aativum (Walker and Dietrich, 1963) is of much interest. The eggs were excised and cultured in micro-chambers by the *~Cblobioinehas been generally effective in inducing chromoeomal duplication, but the reporta of Smith (1943) and Levsn (1945) that its application resultad in the formation of heploidn are intemting. S

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hanging drop technique, and the effects of kinetin and adenosine triphosphate (ATP) studied under the phase contrast microscope. ATP caused a rapid vacuolization whereas kinetin induced cell divisions. XII. ANDROGENESIS The origin of a haploid embryo is generally traced to the parthenogenetic development of the egg or some other haploid component of the embryo sac. However, a few examples are known of haploid sporophytes which reproduce the characters of the male parent only. I n such instances the embryo is believed to be formed by the male gamete and the sporophyte is described as androgenic.*? Among earlier records of androgenic haploids are those by Clausen and Lammerts (1929) for Nicotiana digluta (2n = 7 2 )x N . tabacum (2n = 48), by Kostoff (1929) for N . tabucum (3n = 7 2 ) x N . langadbrJii (2n = 18), by Kostoff (1934) for N . tabacum aylvestria hybrid x N . sylve8tria, and by Gerassimova (1936) for Crepia tectorum. In all these instances the paternal haploids were the result of interspecific crosses and /or experimental treatments. Androgenic haploidy has also been induced in Antiwhinum mju.8 by pollinating irradiated flowers with normal pollen (Ehrensberger, 1948), and in N . glutinosa (2n = 24) crossed with N . repanda (2n = 48) (Kehr, 1951). From crosses between the natural tetraploid Hordeum bulbosum (2n = 28) and the artificially induced tetraploid H . mdgare (2% = 28) three F, plants resembled the male parent, H. VuZqare, in several features suggesting an androgenic origin (Davies, 1958). Haustein (1961)has reported an androgen of Oenotkra ambra. Campos and Morgan (1968) obtained an androgenic haploid in Capaicum frutemena in an attempt to cross two varieties differing in the charactem of their foliage’and fruit. Since the Fl showed feature8 of the male parent only it was concluded that a sperm had given riw to the sporophyte. It is well known’ that during syngamy the male and the female gametes unite, but only the female gamete transmits its cytoplasm to the progeny. In many instances of androgenic haploidH it is presumed that the male gamete continues to develop in the milieu of the cytoplasm of the egg cell. This has considerable appJication in plant breeding. For example, Goodsell (1961) reported that androgenesis in maize is useful in transferring the genotypes of inbred lines into cytoplasm that causes male sterility. For example, “Texas-sterile” is a male sterile strain and “Nebraska 6 (N,)” is an inbred line of Zm

* For haploid spomphytas originating from sperms, several term8 have been used in the literature : androgens, androgenic haploids, androgenetic haploida and paternal haploids. t Haploid sporophytea are not known among gymnosperms. In CepJialOtanu, drupaaa, however, Favre-Duchartre (1966. 1967) observed supernumerary divisions of one of the nperms inside the archegonium and interpreted it a8 the initiation of an androgenic haploid.

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m a p When Texas-sterile was used as the cytoplasmic donor (female parent) for the homozygous N,, it resulted in the production of a few androgenic haploids. Reoently, Chase (1963) demonstrated that an appreciable proportion of androgenic diploids originated when the cytoplaemic donor or female partner was a tetraploid. A method of producing androgenic haploids is very much to be desired but so far no specific technique is known. Of interest in this connection &rethe haploid tissues derived from cultures of pollens of Ginkgo biZoba, T m (see Tulecke, 1961),Torreya nucifera (Tulecke and Sehgal, 1963; 688 also Tulecke, 1964) and EpWra fo2icbtQ: (Konar, 1963). In E . fo2iata about-to-dehisce microsporangia bearing 4-Or &nucleate pollen grains were cultured on a modified Reinert’s medium supplemented with coconut milk (15%) and 2,4-dichlorophenoxyacetic acid (1-5 ppm). In 10-12 days tiny mames of tissue were obtained in 57% of the cultures. Histological observations revealed two patterns of growth: (a) the pollen grains first underwent transverse diviRions to form uniseriate filaments which later assumed a multiseriate form ; or (b) the pollen grains showed an overall enlargement followed by cell divisions leading to the formation of discoid multicellular masses. I n about 90 days after culture the pollen tissue increased nearly 614 times its original mass. This pollen tissue is haploid and undifferentiated and haa shown considerable capacity for continued growth. If haploid pollen tissues could be made to differentiate into embryos, it would open a new area of research. No angiosperm pollen has, however, been known to yield a tissue. Yamada e l al. (1963)cultured stamens of Tradescartl&&rejieza, excised from very young buds, on White’s medium supplemented with a-naphthaleneacetic acid (0.8 ppm). A callus capable of subculture was formed and cytological obaervations showed it to be haploid (n = 12). Several cells of the callus also showed pollen tube-like protmions. According to Yamada et al. thia haploid callus tissue probably originated from the pollen mother cells.

XIII. ANTHER CULTURE During the last few decades many attempts have been made to culture excised sporogenous tissues and anthers. Shimakura (1934) grew the microspore mother cells of Trudemzntia at metaphase I in a solution of sucrose and found that 2 6 4 0 % of them formed tetrah. Gregory (1940) excised the anthers of Datura stramonium, Lywpereicurn emdenturn and LiZium longi-rn at various stagee of development and cultured them in nutrient solutions. Those of Datum and Lywper&um failed to continue their growth. The young anthers of LiZiurn excised

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at the archesporial or sporogenous tissue stage at fist showed similar growth rates both in culture and in vivo. However, the growth rate in vitro rapidly decreased at the time when meioses were to be initiated; the sporogenous ceUs lost their characteristic appearance and became elongated and vacuolated. If the anthers were cultured when the diplotene stage had been already initiated in the microspore mother cells the meiotic divisions were completed. Nevertheless, the microspore tetrads degenerated, and Gregory concluded that the initiation of meiosis is not vested in the mother cells themselves but is probably governed by an interplay of one or more materials transported to the anther. Taylor (1 950) studied the differentiation of sporogenous tissue and the formation of microspores in Tradescantia paludo8a. Anthers were excised at stages ranging from early sporogenous tissue (5-8 days prior to leptotene) to the completion of meiosis in the microspore mother cells. The nutrient media were supplemented with B-vitamins, coconut milk and IAA. He observed that younger anthers required a longer period to undergo differentiation in culture. Those excised during the formation of the sporogenous tissues showed a gradational development but failed to initiate meiosis. Anthers excised at preleptotene and leptotene grew only to a limited extent whereas those cultured at the zygotene-pachytene stage regularly completed meiosis. However, the development did not continue beyond the tetrad stage. When the anthers were excised at the tetrad stage the microspore nucleus divided, but even so the pollen grains failed to mature. In Trillium erectum Sparrow et al. (1955) excised the anthers at stages ranging from pachytene to diakinesis and cultured them on Taylor’s medium supplemented with coconut milk. An average of 76% of the anthers attained the stage of division of the microspore nucleus. Media supplemented with casein hydrolysate, yeast extract and glutamic acid were less satisfactory and in these an average of only 27% of the anthers progressed to the division of the microspore nucleus. An average of 22% of the anthers cultured at the pachytenc stage survived through microsporogenesis. If excined a t n Rlightly later stage, the anthers showed a much higher degree of Hurvival; at diplotenediakinesis it was 670/,. Vasil (1967, 1959) excised the anthers of Allium wpu and Rhow discoEo?.at the leptotene-zygotene and diplotendinkinenis ntagen. In Allium the davelopment progressed up to the one-celled microRpore stage on media supplemented with GA, and kinetin. In rndith Hupplemented with RNA almost all the microspore mother cells formed tetrads in Allium cepa as well as in R h e o discolor. However, in either case the development did not proceed beyond the one-celled microspore

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stage. Only when the medium was supplemented with the four nucleotides of RNA did the microspores develop into two-celled pollen grains (Vmil, 1963). Walker (1957) studied the effect of colchicine on microsporogenesis in the excised anthers of Tradescantia paludosa. In anthers cultured a t the pachytene-diplotene stage colohicine (0.01”/o) promoted the formation of dyads and tetrads. If the anthers were cultured at metaphase I in a medium containing colchicine (0.1%) this resulted in the formation of monads. Beatty and Beatty (1953) reported that a period of 7 days elapses between the initiation and completion of meiosis in T . plu&osa. Walker and Dietrich (1961a) showed that this period could be curtailed to less than 2 days if the anthers were excised at mid-diplotene and cultured in a sucrose medium supplemented with kinetin (0.26ppm). I n it sucrose-deficient medium there was a cessation of kinetin activity and a “meiotic stasis”, but this could be overcome by the addition of citrate. In a test involving a comparison between the two lobeA of the anther, Walker and Dietrich observed that the lobe treated with kinetin markedly surpaesed the untreated lobe in an acceleration of the prophase beyond pachytene. Walker and Dietrich (1961b) also cultured the anthers on a lactose medium. If cultured at the mid-diplotene stage for a period of 48 hours, the anthers went through the meiotic divisions but cytokinesis did not occur. An addition of kinetin did not improve the response; instead it arrested meiosis 11. When sucrose was tlubstituted for lactose the kinetin-induced stasis was overcome and wall formation occurred normally. Citric acid, uronic acids and amino-sugars also proved helpful. There is another question that arises from the experiments on anther culture. It is known that at least on some occasions the egg can develop pathenogenetically into an embryo. Is it possible to obtain something similar from pollen grains? Of special interest are the pollen-embryo Bacs sometimes observed in Hyacinthus orientalis (Stow, 1934) and Ornithogalum nulam (Geitler, 1941). Stow (1934) found that when the pollen-embryo sacs of Hyacinthus were placed on an agar medium together with some normal pollen grains, the pollen tubes formed by the latter coiled around the former. I n one instance a male gamete was observed entering the pollen-embryo sac and in another the pollen-embryo sac showed 10 nuclei, prefiumahly the products of diviaion of a triple fusion nucleus. Stow concluded that all pollen grains are potentially capable of antluming either the male or the female form and that whereas normally the “male potency” i R dominant, sometimes the “female potency” get8 the upper hand owing to A release of necrohormones. Naithani (1937) also ntudied plant8 of 8*

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H. orientalis which were treated for early flowering and confirmed that some of the microspores developed into pollen-embryosacs. He believed that temperature is the decisive factor in the formation of such structures. It would be of interest to repeak the experiments of Stow and Naithani by exposing aseptically cultured anthers to various physical and chemical agencies to see if in other plants also pollen grains can be made to form embryo sacs which can later be fertilized by male gametes from normal pollen grains. A reference may be made here to the observntions of Ram (1959) on Leptomeria billardierii. No normal pollen grains were observed in the anthers; instcad some of the sporogenous cells enlarged, became vacuolated and underwent three nuclear divisions to produce eight nuclei. These organized into embryo sac-like structures (Fig. 2'2). Such embryo sac-like structures originating directly from the sporogenous cells have not been reported in any other plant. Whether they can be induced artificially still remains to be investigated. Yet another problem which may be approached through anther culture concerns the Cyperaceae. TTnlike the majority of angiosperms in the Cyperaceae three of the four microspore nuclei of the tetrad degenerate and only one functions. Whether all the microspores of a tetrad can be made to develop into pollen grains in vitro remains to be investigated.

XIV. CONTROLOF SEX EXPRESSION One of the less understood facctn in plant biology is the expression of sex in flowers. From time to time several suggestiorm have been put forth to explain it, and recently Borne investigationn have been directod to assess the role of growth substances in sex expression. A few of these are discussed here. Mehndrium dioicum is a dioecious member of the family Caryophyllaceae. When the male flowers are infected with Uslilugo violacea, the anthers become filled with the spores of the smut and the formation of pollen is prevented. However, if the female plant is attacked, the flowers produce functional stamens. Love and Lave (1945) were able to duplicate the effects of the smut by treatments with animal hormones. When lanolin pastes of oestrone and oestradiole (female hormones) were applied to leaf axils of staminate plants, the stamens were partially suppressed and a pistil was formed in the flowers. Similarly, in the pistillate plants the male hormone testosterone suppressed the expression of femaleness and promoted the formation of stamens. That plant hormones control sex determination in flowers i8 a relatively new concept. Laibach and Kribben (19BO), Laibach (1952),

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and Nitsch et aZ. (1962) believe that the sex of the flower may be determined by the relative quantity of native auxin present at the time of the initiation of the flower. They made the interesting observation that in Cwurbita pep0 a treatment with auxin increased the number of pistillate flowers and decreased that of the staminate flowers. A pwte or an aqueous spray of IAA, NAA and 2,4-D was applied to the cut ends of the stalks of the first two leave8 of 16- to 18-day-old plants. There was a diminution in the total number of flowers but a definite

FIU.22. “Pollen-embryoeace” of LspsOrnerM billardierii. A. L.S. anther lobe showing two binucleata pollen-embryo eats developed from microepore mother oells. B and C. Squash preparation from anther ehowing organized pollen-embryoa c e (after Ram, lB59).

increase in the production of female floworu. Nitsch et al. (1952) also showed that if the plants were spraycd at the two-leaf stage with a 100 ppm solution of N U the first female flower buds were initiated even at the ninth node, whereas in the controh the Ihtillato flowcrn did not originate until 20 or mom nodes developed. Wittwer and Hillyer (1954) investigated the oxpreuHion of HCX in Cucumis sativus and Cucurbitu p ~ p oWhen . a solution of 100 ppm NAA, or 25 ppm 2,3,5-TIBA was sprayed at the two- or three-leaf utagc: the ratio of male to female flowers went down from 23: 1 to 8 : 1, and from 14: 1 to 2 : 1. In Cucurbitupep0 an application of NAA (100 ppm) af‘ter the unfolding of the first leaf resulted in a decrease in the ratio of male : female flowers from 1.47: 1 to 0.4: 1; and sometimes no staminate flowers were produced a t all. If N M was applied after the formation of four or five leaves, the treated plants bore only pistillate flowers for 8 days. Ito m d Saito (1957) also induced sex reversal in cucumber. The apical bud was removed and the cut end treated with 2,3,6-TIBA. Pistillate flowers arose at the lower nodes in place of the usual staminate flowers. Heslop-Harrison (1956) subjected plants of ~‘unnabissativa (dioec-

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ious) to a period of minimal photoperiodic induction (ten short days) and then studied the influence of NAA on sexuality. Immediately after the short day treatment, a lanolin paste of NAA (0.5%) was applied to the lower surface of the central lobe of one of the trifoliate leaves at the third node. Five days later similar quantities of NAA were smeared on both leaves at the fourth node. For interpreting the results all the NAA-treated plants were induced to flower 70 days after the beginning of the experiment. It was observed that in plants which were genetically male only female flowers had developed in sites which should have been occupied by male flowers. According to HeslopHarrison (1957) the differentiation of sex is controlled by the endogenous level of auxin in the regions adjacent to the floral primordium. The formation of the pistil may be favoured by high level of auxin in the vicinity of the differentiating primordium, and that of the stamens hy lower levels. Setyanarayana arid Rengab:wttmi ( 1959) inventigated the effect of 2,4-D, NAA and p-chlorophenoxyacetic acid (CLPA) on Luffa acutangula. The plants were sprayed at regular intervals from thc time of germination of the seed to the appearance of the first flower. CLPA induced an S070 reduction in the number of male flowers with a corresponding increase in the percentage of the female flowers. Yet another chemical which has been extensively used in studies on sex expression is gibberellic acid. Laibach and Kribben (1950) demonstrated that in the monoecious inbreds of Cucurnis sativus an application of NAA increased the female tendency whereas GA, decreased it. Wittwer and Bukovac (1957, 1968) reported that in cucumber gibberellins delay the formation of pistillate flowers but speed up that of the staminate flowers. Similarly, Peterson and Anhder (1960) demonstrated that GA, is effective in inducing staminate flowers on plants which otherwise bear mostly female flowers. Bukovac and Wittwer (1961) found gibberellin A, to be more effective than A,, A, and A, in inducing the formation of staminate flowers. Wittwer and Bukovac (1962) tested the effect of gibberellin8 A, to A, on exclwively pistillate plants.of cucumber. The chemicals were applied to tho fist plumular leaf at its emergencc and once again after a week. Tho number of plants that produced staminate flowers, the number of male flowers per plant, and the number of nodeb: bearing male flowern were taken as criteria for judging the effect of the test chemical. Gibbcrellin A, was the most active and gibberellin A, the lead in inducing rna1enosr;l. Gibberellins A,, A,, A, and A, had an intermediate effect in decreasing order. Since gibberellins A,, A,, A, and A, are structurally similar, Wittwer and Bukovac suggest that there is some relationship between the structure and degree of the biological activity of the gibberellins.

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In recent years the technique of tissue and organ culture has also been employed in investigations on sex expression. Petrt and htovsk? (1957) germinated the caryopses of some varieties of Zea mays on an agar medium. Prom 7-day-old seedlings the stem tips were excised and cultured on an,agar medium. They regenerated into new plants which were transferred to pots and grown in glasshouses, Tlicy produced only female infloreocences. In Cucumis sativuo gibberellins favoured the formation of staminate flowers (Galun, 1959). To investigate whether the growth substances have any direct control on sex expression Galun et al. (1962) made in vitro cultures of young floral buds (0-7 mm in diameter) of C. m t i m . Buds of potentially staminate flowers (borne on the eighth node) were excised from 20-day-old seedlings of a monoecious line and cultured on a modfied White’s medium. When these were grown for 20 days on a medium supplemented with IAA (0.1 ppm) the stamens showed early degeneration but there was a reasonably good development of the ovary. The effect of IAA was, however, reduced when GA, (0.33.0 ppm) was also added to the medium and in buds cultured on a medium containing both JAA and GA, the stamens continued their development. These results suggest that IAA and CIA, do have a direct effect on the differentiation of the ovaries and stamens and they can exert their influence on floral buds without any obligatory interference h m the leaves or other organs of the plant. It remains to be seen whether such female flowers could be made to grow into fruits through pollination and fertilization in vitro. In recent years various chemicals called “gametocides” have been reported to induce male sterility without seriously affecting female fertility. Maleic hydrazide (MH), 2,3-dichloroisobutyricacid (FW-460), end 2,2-dichloropropionic acid (dalapon) are some of them. Moore (1950) and Naylor and Davies (1960) reported that maleic hydrazide (MH)is effective in inducing male sterility in maize. Wittwer and HilIyer (1964) also obtained a similar response in some members of the Cucurbitaceae. A repeated dipping or spraying of the plants at intervals of 6-7 days with a solution of 100 ppm MH suppressed the development of male flower buds whereas the female flowers developed normally and were fertile. Rehm (1962) described similar results in Citrullus vulgaris and Lycopersicum ecrcukntum. Recently, much attention has been given to FW-450 and dalapon. Eaton (1957)induced male sterility in “Empire cotton” by thc applicntion of FW-450. Moore (1959) obtained total male sterility in tomato by spraying a 0.3% solution of a sodium salt of FW-460 at anthoeis. Similar results were obtained by Hensz and Mohr (1969) in Citrullw, vulgaris by using dalapon.

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Recently, Starnes and Hadley (196%)applied FW-450 (0-2000 ppm) as a foliar spray on soybeans. This resulted in the inviability of pollen and a failure of dehiscence of the anthers. At 750 ppm FW-450 effected maximum male sterility, the highest incidence (93%) of pollen abortion being observed in the variety “Dunfield”. Kho and De Bruyn (1962) tested the gametocidal activity of FW-450 on Antirrhinum wjw.Aqueous sprays of its sodium salt (0*6y0)soon after the appearance of first flower buds caused male sterility. More recently Nasrallah and Hopp (1963) reported induction of pollen sterility in Solanum rpelongena by the application of a sodium salt of FW-450. Aqueous sprayings of a 0.2% solution 2-3 weeks before anthesi8 or when the buds were 60-80 mm in diameter proved effective. Among other subetances carbon monoxido, NAA, 2,4-D, and GA, have been reported t o have a gametocidal action in some instances. Genetically monoecious plants of Mercurialis ambigwt produce exclusively male flowers a t the first one or two nodes, but at subsequent nodes they bear both kinds of flowers. Heslop-Harrison (1957) inveutigated the effect of carbon monoxide in inducing male sterility h.such plants, A treatment with carbon monoxide reduced the ratio of male to female flowers from 27: 1 in the controls to 6: 1 in the treated plants. The output of pollen waR relatively low in the treated plants but the female flowers were unaffected. Heslop-Harrison and HeslopHarrison (1958) obtained auxin-induced male-sterility in Silene pendukz (family Caryophyllaceae). In the male-sterile flowers the gynoecium showed a remarkably precocious development. The styles were frequently exserted and the stigmas became receptive even before the unfolding of the corolla. In a few instances a treatment of the leaves with auxins resulted in parthenocarpic fruits without pollination. Hensz and Mohr (1959) reported that a treatment with GA, (2000 ppm) induced male Sterility in Cucumia vulgari8. Choudliury and George (1962) induced male uterility in Solanurn rnelongenu by applications of MH (600 ppin), NAA (50 p~mi),and 2,4-L) (20 ppm), the percentage of pollen sterility ranging from 90 to 100. The application of NAA also caused sex reversal by converting the &amen8 into miniature pistils. Although the intricate phenomenon of floral organogeneuis is only imperfectly understood, there is some evidence t o show that sex expression in flowering plants can be modified through the application of growth substances notably auxins, gibberellins and gametocides. In this the technique of tissue and organ culture, as employed by Galun et at!. (1962), offers a promising field for further investigations. While the induction of inale sterility has an obvious value in plant

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breeding, much research is necessary on the effects these chemical6 produce on the ovules.

XV. CONCLUSIONS Ever since the discovery of double fertilization in 1898 tho etudy of the embryology of angiosperms has made rapid progress. Apart from its role in taxonomy (Maheshwari, 1904) embryology 1iaR a close relation to physiology and genetics. The in &ro oulture of embryos, endosperm, ovules, ovaries, flowers and anthers has considerably advanced our understanding of the physiology of these organs. The test-tube fertilization of ovules followed by their full development into seeds is another notable achievement. Nevertheless, thore in still a great paucity of dotailed information on many aspects of fertilization. Although one can pinpoint the site of incompatibility in the reproductive system of flowering plants, a knowledge of the factors whioh cause it remains a matter of theories and conjecture. The most rewarding approach to the study of control of fertilization would be through co-ordinated embryological, physiological and biochemical investigations on the pollen tube, stigma, style and ovary, coupled with genetical research on sexual incompatibilities. The artificial culture of embryos has an obvious application in plant breeding and in overcoming dormancy. From the viewpoint of physiology and morphogenesis a study of the embryos of the orchids, insectivorous plants and certain phanerogamic parafiites and naprophytos is likely to prove very rewarding. For the horticulturist tho artificial induction of polycmhryony is of great value. Its induction in those plant8 in which it doen not occur in nature and its control in thofie in which it exifitn an a normal fcuturo will both need much experimental work. The recent HtudieH on tho cultivation of single c e b have an important implication in t h i n context. These have clearly demonstrated the totipotency of vegctative cclk, in that they too, like the zygote, can build up a new organism (Steward, 1963).

Although the zygote and the endosperm are both products of fertilization, their morphogenetic destinies are so divergent that the former develops into an embryonic sporophyte while the latter remains merely as an attendant on it. Plants of the family Oenotheraceae, where both embryo and endosperm have the same chromosomal complement, are worthy objects for studying the nature of the factors which bring about such a functional diversity. Although the pollen grain and thc egg arc both haploid ntructuree, they behave quite differently. On some occctrjions the unfertilized egg

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may develop directly into a well-organized group of cells, namely the embryo, but the obtaining of a tissue from the pollen is far more difficult. In fact, even the initiation of meiosis and the maturation of the pollen gl‘ains in escised anthers have not yet provcii feasible. A parthenogenetic development of the ovum has becn achieved in many animals and a few cryptogams, but the angiosperms have so far defied all attempts in this direction. Haploids do occur in nature sometimes but all methods to induce the development of the unfertilized eggs of angiosperms have proved virtually infructuous. It is possible that excised ovules cultured in vitro may be more easily amenable to physical and chemical stimulations than those borne on the plant in nature. The control of sex expression has far-reaching applications and there is increasing evidence of the role of growth substances in this. Further research has to be directed towards an understanding of its physiological basis and for finding methods for a selective control of the two kinds of gametes. These problems raise several fundamental questions the solution of which c a b for a further strengthening of the bonds between embryology, physiology and genetics.

ACKNOWLEDGEMENTS We are grateful to our pupils, Messrs. P. S. Rao, T. S. Rangan, and K. R. Shivanna, and Miss S. V. Usha, for their diligent help and co-

operation in the preparation of the manuscript.

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