Intersubgeneric cross in Nymphaea spp. L. to develop a blue hardy waterlily

Intersubgeneric cross in Nymphaea spp. L. to develop a blue hardy waterlily

Scientia Horticulturae 124 (2010) 475–481 Contents lists available at ScienceDirect Scientia Horticulturae journal homepage: www.elsevier.com/locate...

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Scientia Horticulturae 124 (2010) 475–481

Contents lists available at ScienceDirect

Scientia Horticulturae journal homepage: www.elsevier.com/locate/scihorti

Intersubgeneric cross in Nymphaea spp. L. to develop a blue hardy waterlily Pairat Songpanich a, Vipa Hongtrakul b,* a b

129/106 Perfect Place, Rattanatibet Road, Mueang District, Nonthaburi Province 11000, Thailand Department of Genetics, Faculty of Science, Kasetsart University, Chatuchak, Bangkok 10900, Thailand

A R T I C L E I N F O

A B S T R A C T

Article history: Received 7 July 2009 Received in revised form 22 January 2010 Accepted 24 January 2010

Five waterlily cultivars, with good pod setting and completely formed flowers, from the subgenus Nymphaea (hardy waterlily) were used as pod (female) parent. Waterlilies from subgenera Anecphya (2 entities) and Brachyceras (20 entities), specifically the blue-flowered ones, were used as pollen (male) parent for transferring blue-flowered characteristic to the hardy waterlily. Hybridization of subgenus Nymphaea with subgenus Anecphya was unsuccessful. A successful crossing was obtained between the subgenera Nymphaea and Brachyceras, yielding one pod with 244 seeds, from which only 39 seeds germinated and yielded 20 good intersubgeneric hybrid plants. The characteristics of the hybrids were categorized by their flower colors into two groups, a pink-flowered group with 17 hybrids and a blueflowered group with 3 hybrids. In the blue-flowered group, there was a prominent one with beautiful blue-purple flower, which was determined by PCR-RFLP markers to be a hybrid between Nymphaea and Brachyceras subgenera, as well as inherited some plant characteristics, for example, ovary carpel, leaf (pad), rootstock, position of flower at flowering from the parent. The blue-purple flowered hybrid had important characteristics inherited from the hardy waterlily (subgenus Nymphaea); therefore, the hybrid has been named Nymphaea ‘Siam Blue Hardy’ to honor the birth place and its distinct flower color. ß 2010 Elsevier B.V. All rights reserved.

Keywords: Waterlily Blue hardy Pollination Intersubgeneric Hybrid Nymphaea Brachyceras

1. Introduction Waterlilies are in the Nymphaeaceae family, consisting of six genera: Barclaya, Euryale, Nuphar, Nymphaea, Ondinea and Victoria (Anon, 2008). Plants in the genus Nymphaea can be divided into two groups: Apocarpiae and Syncarpiae. The Apocarpiae group consists of three subgenera: Anecphya (an Australian tropical waterlily), Confluentes (an Australian tropical waterlily) and Brachyceras (day blooming tropical waterlily). The Syncarpiae group also consists of three subgenera: Hydrocallis (night blooming tropical waterlily), Lotos (night blooming tropical waterlily) and Nymphaea (hardy waterlily). Nymphaea waterlilies usually bloom for 3 days, blooming during the day and flower closing during the night with different blooming times for each subgenus. The Anecphya, Brachyceras, and Nymphaea are day bloomers (6 a.m.–2 p.m.), while Hydrocallis and Lotos are night bloomers (7 p.m.–10 a.m.) (Slocum, 2005; Jacobs and Porter, 2007). Waterlilies naturally have solitary-perfect flowers that cannot be self-pollinated within a single flower (Vandaveer, 2003). The protogynous phenomenon in the genus Nymphaea is the event in which the stigma is receptive for accepting the pollen from a different flower before the pollen in the same flower is ready to

* Corresponding author. Tel.: +66 2 562 54444206; fax: +66 2 579 5528. E-mail address: [email protected] (V. Hongtrakul). 0304-4238/$ – see front matter ß 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2010.01.024

shed. The stigma of the genus Nymphaea becomes ready to be pollinated during the second and third day of blooming. However, exceptions exist for some species where the pollen is ready to shed for fertilization on the first day at time of the stigma receptivity, enabling fertilization to occur within a same single flower. In order to create new desired traits of interest, breeders of new generation have tried to make intraspecific, interspecific and intergeneric hybridizations. In Nymphaea waterlilies, cross-pollination has been carried out between different species in the same subgenus to obtain unique waterlily hybrids not seen before, for example, pollinating hardy waterlilies to achieve the ones with blue flower, pollinating night blooming waterlilies to develop yellow-flowered varieties, and even pollinating perpetual bloomers with N. ‘Jongkolnee’ to generate more variety of flowers. None of the waterlilies in the Syncarpiae group provides blue flower. The Apocarpiae group, however, possesses sundry colors including blue in subgenera Anecphya, Confluentes and Brachyceras. In the past, intersubgeneric hybridization in Nymphaea had been performed for the hope of creating a ‘‘Blue Hardy Waterlily’’ hybrid. This has been a dream for hybridizers all over the world. Latour-Marliac (1893, 1899) hybridized hardy waterlilies with tropical waterlilies, but was unsuccessful in creating a blue flower hybrid. Reditt (1989) reported on the continuing attempt of Andrea Protopapas to create the blue hardy waterlily. Nash (1996) also mentioned Perry D. Slocum of the United States who has been trying to produce the blue hardy waterlily. The biggest obstacle for

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these enthusiasts is probably due to failure in developing mature intersubgeneric zygotes, due to the difference in chromosome numbers between subgenus of tropical waterlily and hardy waterlily. Variation in chromosome numbers in a single species are most apparent in a wide ranging species both in aneuploid and polyploid series based on x = 14, such as N. alba: 48, 56, 64, 84, 96, 105, 112, 160, N. stellata: 28, 56, 84 and N. lotus: 28, 56, 84 (Gupta, 1980; Pagels, 2000; Hossain et al., 2007). Speichert and Speichert (2004) mentioned the only colors still not found in hardy waterlily are blue and purple. From the efforts of many hybridizers and a period of more than 100 years of hybridization, new-generation hybridizers still dream and endeavor to create the blue flower hardy waterlily. This study reports the success of cross-pollination of hardy waterlily with blue-purple flowered tropical waterlily to become the first ‘‘Blue Hardy Waterlily’’ in the world. 2. Materials and methods Five cultivars of hardy waterlily with a history of successful fruit setting, completely formed and numerous flowers from the subgenus Nymphaea, including N. ‘Supranee Pink’, N. ‘Mayla’, N. ‘Perry’s Fire Opal’, N. ‘Splendida’ and N. ‘Madame Wilfron Gonne`re’,

were grown to the flowering stage (10 plants each cultivar) and used as pod (female) parents. The blue-flowered waterlilies subgenera Anecphya and Brachyceras were grown, two plants each species/ cultivar, and used as pollen (male) parents. Two species including N. gigantea and N. violacea were from subgenus Anecphya and 20 cultivars including 9 cultivars already named (N. ‘Director George T. Moore’, N. ‘Tina’, N. ‘Madame Ganna Walska’, N. ‘Pamela’, N. ‘Midnight’, N. ‘Muang Thummanoon’, N. ‘Panama Pacific’, N. ‘Royal Purple’, N. ‘King of the Blues’) and 11 un-named open pollinated hybrids (O.P. hybrid #1–11) were from subgenus Brachyceras. Pollination was achieved through direct crossing by brushing the pollen from flower blooming for 2 or 3 days onto the stigma disc of the pod parent. Pollinations were performed between 8:00 and 10:00 a.m. About 5 days after pollination, the cross-pollinated flowers began to sink beneath the water. The sepals and petals of the flowers rotted after about 2 weeks, the ovary enlarged into a pod containing seeds 3–4 weeks after pollination. The seeds were normally be covered by gelatin that helped the seeds travel along the water to disperse the seedling. The seeds were placed in a cloth bag and soaked for 2 days, then the gelatin was washed off and the well-formed seeds were sorted out. Seed dormancy was broken by stratification by maintaining seed at 4–5 8C for at least 2 months before planting.

Fig. 1. Research work flow chart.

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Table 1 Number of pollinations in each combination between subgenus Nymphaea and subgenus Anecphya/Brachyceras in 2003–2005. Pollen parent

Pod parent

Total

Subgenus Nymphaea N. ‘Supranee Pink’a

N. ‘Mayla’

N. ‘Perry’s Fire Opal’

N. ‘Splendida’

N. ‘Madame Wilfron Gonne`re’

Subgenus Anecphya N. gigantea N. violacea Total

15 8 23

19 9 28

5 3 8

19 10 29

10 13 23

68 43 111

Subgenus Brachyceras N. ‘Director George T. Moore’ N. ‘Tina’ N. ‘Madame Ganna Walska’ N. ‘Pamela’ N. ‘Midnight’ N. ‘Muang Thummanoon’ N. ‘Panama Pacific’ N. ‘Royal Purple’ N. ‘King of the Blues’ O.P. hybrid #1 O.P. hybrid #2 O.P. hybrid #3 O.P. hybrid #4 O.P. hybrid #5 O.P. hybrid #6 O.P. hybrid #7 O.P. hybrid #8 O.P. hybrid #9 O.P. hybrid #10 O.P. hybrid #11a

23 13 16 11 14 15 9 18 12 15 14 16 10 14 12 14 8 10 14 13

15 16 12 12 16 11 12 15 10 12 11 13 9 12 10 13 7 10 12 12

6 5 4 3 5 6 4 7 5 3 5 6 2 4 3 6 2 3 4 3

14 17 13 15 17 12 9 17 14 13 12 11 12 13 15 19 10 13 10 11

19 12 14 16 9 16 7 14 9 17 11 13 11 13 12 18 9 12 15 8

77 63 59 57 61 60 41 71 50 60 53 59 44 56 52 70 36 48 55 47

271 294

240 268

86 94

267 296

255 278

1119 1230

Total Grand total

O.P. = open pollinated. a Successful cross-pollination was obtained from crossing between N. ‘Supranee Pink’ and O.P. hybrid #11. Only one pod was achieved from 13 cross-pollinations of this combination.

Research was performed over a period of 5 years from 2003 to 2007 at Nonthaburi, Phathumtani and Bangkok provinces, Thailand (Fig. 1). For the verification of the hybrids, molecular markers were used, as well as morphological characteristics to compare the parents and their putative hybrids. Genomic DNA samples were extracted from the petals of the parents and the hybrids derived from the intersubgeneric crossing, following a modified CTAB extraction method (Doyle and Doyle, 1987). DNA fingerprints were generated based on a co-dominant PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) marker specific to ITS (Internally Transcribed Spacer) of nrRNA (nuclear ribosomal RNA) gene. The PCR reactions were performed in 12.5 ml of a mixture containing 20 ng DNA, 10 mM Tris–HCl pH 8.8, 50 mM KCl, 0.08% Nonidet P40, 0.2 mM of each primer, 2.0 mM MgCl2, 0.1 mM of each dNTP and 1 U of Taq DNA polymerase (Fermentas, Canada). The primers (F: 50 -TCGCTCCTACCGATTGAATG-30 and R: 50 -TCCTCCGCTTATTGATATGC-30 ) were at the conserved regions of 18S and 26S genes. The amplification condition was 3 min at 94 8C, followed by 35 cycles of 30 s at 94 8C, 1 min at 59 8C and 2 min at 72 8C, and finally for 5 min at 72 8C to complete extension. The amplified products of about 870 bp were cut with three restriction enzymes (AluI, RsaI, MseI), then electrophoresed in 1% agarose gel, followed by ethidium bromide staining and visualization under UV light. The characteristics of flower, leaf margin (pad), ovary carpel and rootstock of the putative hybrids were recorded and compared to those of their parent.

subgenus Brachyceras (N. ‘Director George T. Moore’, N. ‘Tina’, N. ‘Madame Ganna Walska’, N. ‘Pamela’, N. ‘Midnight’, N. ‘Muang Thummanoon’, N. ‘Panama Pacific’, N. ‘Royal Purple’, N. ‘King of the Blues’ and O.P. hybrid #1–11) were completed (Table 1). The cross-pollination (111 pollinations) of the subgenus Nymphaea with subgenus Anecphya did not yield any success, probably due to the difference in chromosome numbers and genetic incompatibility, resulting in abortive zygotes. The subgenus Nymphaea has the variation of chromosome numbers in different species and in a single species both in aneuploid and polyploid series based on x = 14. N. alba has chromosome numbers of 48 aneuploid, 56 (4x), 64 aneuploid, 84 (6x), 96 aneuploid, 105 aneuploid, 112 (8x), 160 aneuploid; whereas N. tuberosa and N. odorata carry chromosome numbers of 84 (6x). Subgenus Anecphya: N. gigantea has 2n = 16x = 224, while subgenus Brachyceras: N. capensis has 2n = 2x = 28 and N. nouchali (syn. N. stellata) has 2n = 28 (2x), 56 (4x), 84 (6x) (Pagels, 2000). The cross-pollination (1119 pollinations) of subgenus Nymphaea with subgenus Brachyceras were mostly unsuccessful. Only a successful cross between the subgenera Nymphaea: N. ‘Supranee Pink’ and Brachyceras: O.P. hybrid #11 was obtained, yielding only one pod with 244 seeds, from which 39 seeds germinated (16%). Twenty hybrid seedlings were grown to maturity and produced flowers (Fig. 2).

3. Results

N. ‘Supranee Pink’, our early hybrid (N. ‘Mayla’  N. ‘Perry’s Fire Opal’) as pod parent, had pink flowers and the subgenus Brachyceras: O.P. hybrid #11, our open pollinated hybrid as pollen parent, had blue-purple flowers. The flower colors of hybrids are displayed in Fig. 2, which could be characterized into two groups as follows:

A total of 1230 cross-pollinations of subgenus Nymphaea (N. ‘Supranee Pink’, N. ‘Mayla’, N. ‘Perry’s Fire Opal’, N. ‘Splendida’ and N. ‘Madame Wilfron Gonne`re’) with the blue-flowered waterlilies subgenus Anecphya (N. gigantea and N. violacea) and

3.1. Flower

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Fig. 2. Flowers of parent and their intersubgeneric F1 hybrids of subgenus Nymphaea (N. ‘Supranee Pink’)  subgenus Brachyceras (O.P. hybrid #11).

Pink-flowered Group contained 17 hybrids with the color ranging from light pink to dark pink. There were two interesting observed characteristics in this group. The first contained flowers with light pink color on the first day of blooming that tended to fade to whitish color in the inner petals, but the tips of the petals became more dark pink.

Examples can be seen in flowers of hybrids number 4, 8, 12 and 14. The second had flowers with dark pink that tended to have a lighter tone at the lower part of the inner petals. Examples are shown in flowers of hybrids number 1, 2, 9, 10, 15, 19 and 20. Both of these observed characteristics are rare in subgenus Nymphaea, but rather common in subgenus Brachyceras. The

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Fig. 3. The characteristics of the parent and their hybrids. Subgenus Nymphaea (N. ‘Supranee Pink’)  subgenus Brachyceras (O.P. hybrid #11).

remaining six hybrids in this group had constant pink-coloring flowers. Blue-flowered Group contained three hybrids (number 3, 16 and 18) with flower color similar to that of the pollen parent (bluepurple). The color at the petal base was lighter than other parts. The color on the anther tip of all hybrids was similar to that of the pollen parent. Most of hybrids had numerous flowers like the pollen parent. Only a few hybrids (numbers 16 and 18) had difficulty in producing flowers. The flowers of all hybrids and their parent were cup-shaped (Fig. 3).

3.4. Rootstock All hybrids generated had rootstock that tended to grow horizontally from and along a fleshy rhizotomous rootstock like the pod parent. ‘‘Eyes’’ at points along the rhizome produced new crowns. The underground rootstock for the pollen parent grew vertically from a single central crown and all leaves and flowers radiated from this point (Fig. 3). Most hybrids had numerous roots, the characteristic from the pollen parent, which was useful for nutrient uptake and root penetration into the ground. 3.5. Verifying hybridity

3.2. Ovary carpel Cross-sections of the flowers revealed that ovary contained many carpels, which either fused to form a compound pistil, the ‘‘syncarpous’’ or separate in single individual pistils, the ‘‘apocarpous’’. The carpel is an imporant characteristic for botanists to separate the Nymphaea plants into two groups. The Apocarpiae group contains three subgenera: Anecphya, Confluentes, Brachyceras and the Syncarpiae group contains three subgenera: Hydrocallis, Lotos, and Nymphaea (Conard, 1905; Jacobs and Porter, 2007). All our 20 hybrids were categorized to be syncarpous (Fig. 3), which indicated that they recieved this characteristic from the pod parent (Nymphaea). 3.3. Pad The pad or leaf margin characteristic of the hardy waterlily (Nymphaea) is entire (smooth edge) while that of the tropical waterlily (Brachyceras) is dentate (toothy). All hybrids generated had entire pad margins (Fig. 3). Leaves of all hybrids were rather thick and leathery like the pod parent, while the pollen parent’ leaves were rather thin. All hybrids were pubescent at the petiole and peduncle, like the pod parent. Thirteen hybrids had flecked pads like the pollen parent and seven hybrids had plain green pads like the pod parent.

One best plant from each of the three flower coloring types from the pink-flowered group and the other one from the blue-flowered group were subjected for hybridity testing. The selected samples were constant pink coloring, un-named hybrid H11; lighter pink in inner petals with dark pink at tip, N. ‘Siam Pink Tips’ H8; darker pink in outer petals with lighter pink at lower part of inner petals, N. ‘Siam Pink’ H9 and blue-purple coloring, N. ‘Siam Blue Hardy’ H3. The methods used were morphological comparison (Fig. 3) and molecular marker analysis (Fig. 4). 3.6. Morphological comparison The results from the comparison of morphological characteristics in the four hybrids with those of the pod and pollen parent indicated that all four hybrids were true intersubgeneric hybrids (Fig. 3). The hybrids inherited genetic traits from both the pod (Nymphaea) and pollen (Brachyceras) parent. The petal colors of the hybrids were either pink like pod parent or blue-purple like pollen parent. The height of flowers at blooming stage of the pod parent was at the surface to above water level (0–7 cm), whereas that of the pollen parent was above water. All four hybrids flowered above water level and three of the four hybrids selected (except H11) had flecked pad like the pollen parent. The hybrids did possess many hardy waterlily (Nymphaea) characteristics. They had horizontally

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Fig. 4. DNA fingerprints of the parent and their hybrids based on PCR-RFLP technique of ITS sequence. P1 = Subgenus Nymphaea (N. ‘Supranee Pink’). P2 = Subgenus Brachyceras (O.P. hybrid #11). H3 = N. ‘Siam Blue Hardy’. H8 = N. ‘Siam Pink Tips’. H9 = N. ‘Siam Pink’. H11 = Un-named hybrid. M = DNA ladder.

grown rhizome rootstock, entire pad margins and syncarpous carpels, which were characteristics of the pod parent (Nymphaea). 3.7. Verification using DNA markers

the cross-pollination was performed between Nymphaea (Syncarpiae) and Brachyceras (Apocarpiae). The results from both DNA fingerprint and morphological comparison of the four hybrids and the parent confirmed that they were true intersubgeneric hybrids.

DNA fingerprints were generated based on a co-dominant PCRRFLP marker specific to ITS of nrRNA gene after the amplified products of about 870 bp were cut with three restriction enzymes (AluI, RsaI, and MseI), then electrophoresed in 1% agarose gel, followed by ethidium bromide staining and visualization under UV light (Fig. 4). Each parent did possess distinct DNA patterns or alleles that were found to be common in all the hybrids tested. 4. Discussion Our results represent the only successful cross-pollination between the subgenus Nymphaea: N. ‘Supranee Pink’ with the subgenus Brachyceras: O.P. hybrid #11 from a total of 1230 pollinations between our selected cultivars. It proved difficult to get the right combinations that had genetic compatibility to produce well-formed seeds following the pollination. The parents of the successful hybridization are our former hybrids. N. ‘Supranee Pink’ is the hybrid of N. ‘Mayla’  N. ‘Perry’s Fire Opal’. It should be N. odorata with chromosome numbers 2n = 6x = 84 (Pagels, 2000). O.P. hybrid #11 is our open pollinated hybrid, which might be N. capensis (2n = 2x = 28) or N. nouchali/N. stellata (2n = 28 (2x), 56 (4x), 84 (6x); Pagels, 2000; Hossain et al., 2007), according to its phenotypic characters. We had performed a preliminary study on chromosome numbers of the parents by comparing chromosomes at metaphase. Chromosome numbers were significantly less in pollen parent (ca. 2n = 28–56) than in the pod parent (ca. 2n = 84) (unpublished data). The hybrids from this parent might be sterile. We had carried out about 50 pollinations using the hybrid N. ‘Siam Blue Hardy’ as either pod or pollen parent, but no successful hybridization was obtained. Fertility and cytogenetic studies of the hybrids derived from intersubgeneric hybridization should be conducted. Doran et al. (2004) and Les et al. (2004) reported the successful cross-pollination between different subgenera. However, their success was on crossing between subgenera Anecphya and Brachyceras which were in the Apocarpiae group. In our study,

Fig. 5. Flowers of Nymphaea ‘Siam Blue Hardy’.

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Since the blue-flowered hybrid resulted from the crosspollination between subgenera Nymphaea and Brachyceras, the name should indicate the combination of both hardy and tropical waterlily. However, the specific characteristics, for example the syncarpous ovary carpel, entire pad margin, rhizome rootstock and horizontal growth, of the hybrids are mostly found in the hardy waterlily (Nymphaea), especially the syncarpous which is the important characteristic used to distinguish hardy waterlily from Brachyceras. The only pollen parent characteristics of the blueflowered hybrid are flecked pad and blue petals (Fig. 3). So this waterlily hybrid is considered to be a hardy waterlily for its important characteristics and phenotype. One of our hybrids, N. ‘Siam Pink’, had been sent to test for the ability to sustain winter outdoors in non-tropical climates. It has been successfully grown and survived through winter. Three hybrids from our cross had blue-purple flower color, the ‘‘new color’’ that has never been seen in hardy waterlily. So, it could be claimed that these hybrids are the first group of blue hardy waterlily in the world, especially the blue-purple flower hybrid number 3 that produces numerous and completely formed flowers (Fig. 5). This unique one is selected to be the representative of ‘‘the first blue-flowered hardy waterlily of the world’’ which has been developed in Thailand in the year 2007. For honoring the birth place of the blue hardy waterlily, it is named Nymphaea ‘Siam Blue Hardy’. Our future study is to create more waterlily cultivars from the successful hybridization and to try reciprocal cross as well as backcross to the blue-flowered parent and select the ones with the dark blue color with numerous numbers of petals. The multiplication of the hybrids through tissue culture should also be investigated. Acknowledgements The authors would like to thank Dr. Slearmlarp Wasuwat for knowledge and support in the study, Dr. Jinda Jan-orn for manuscript preparation, Khun Wirot Hiranyuprakhon for

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providing the testing areas, Khun Primlarp (Wasuwat) Chukiatman for materials and place for hybridization, Mrs. Kit Knotts (the manager of www.victoria-adventure.org) for information and advices, Dr. N. Nopchai Chansilpa for consultant on hybridization and seedling establishment, Dr. Yongyut Jiamchaisri for advice and solving the problem of algae control, Khun Phishet Prommoon and Associate Professor Dr. Amara Thongpan for encouragement and advising in writing the manuscript. At last, thanks to Khun Gantapon Prommoon for help in article translation and others (names have not been mentioned) who have given assistance. References Anon, 2008. Genera of Nymphaeaceae, Germplasm Resources Information Network, GRIN Taxonomy for Plants. , 17/02/2008www.ars-grin.gov. Conard, H.S., 1905. The Waterlilies, A Monograph of the Genus Nymphaea. The Carnegie Institute of Washington, Washington, DC, pp. 86–87, 126. Doran, A.S., Les, D.H., Moody, M.L., 2004. Nymphaea ‘William Phillips’, a new intersubgeneric hybrid. HortScience 39 (2), 446–447. Doyle, J.J., Doyle, J.L., 1987. A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem. Bul. 19, 11–15. Gupta, P.P., 1980. Cytogenetics of aquatic ornaments. VI. Evolutionary trends and relationship in the genus Nymphaea. Cytologia 45, 307–314. Hossain, A., Kabir, G., Ud-deen, M.M., Alam, A.M.S., 2007. Cytological studies of Nymphaea species available in Bangladesh. J. Bio-sci. 15, 7–13. Jacobs, S.W.L., Porter, C.I., 2007. Nymphaeaceae. Flora of Australia 2, 259–275. Latour-Marliac, J.B., 1893. The new hardy water lilies. The Garden 12 (23), 582–584. Latour-Marliac, J.B., 1899. Hardy Hybrid Water Lilies. The Garden 3 (18) 20/04/ 2003www.victoria-adventure.org. Les, D.H., Moody, M.L., Doran, A.S., 2004. A genetically confirmed intersubgeneric hybrid in Nymphaea L. (Nymphaeaceae Salisb.). HortScience 39 (2), 219–222. Nash, H., 1996. Perry D. Slocum: An American Legacy. Water Gardening 20/04/ 2003www.victoria-adventure.org. Pagels, W., 2000. Chromosome counts of waterlilies and other Nymphaeaceae. , 25/ 05/2003www.victoria-adventure.org. Reditt, J., 1989. The Quest for the Hardy Blue. Cyprus Weekly. Christmas Edition 1989. www.victoria-adventure.org, 20/04/2003. Slocum, P.D., 2005. Waterlilies and Lotuses: Species Cultivars and New Hybrids. Timber Press, Inc, Portland, OR, USA, 260 p. Speichert, G., Speichert, S., 2004. Encyclopedia of Water Garden Plants. Timber Press, Inc, Portland, OR, USA, 388 p. Vandaveer, C., 2003. How does this waterlily protect its seeds? , 15/09/2003In: www.killerplants.com.