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Hardy Waterlily Hybridization
An Illustrated Tutorial

Article and photos by Pairat Songpanich, Thailand 
Click images to enlarge
     
Hybridization, Imagination and Objectives


Hardy waterlily flower and pod with seeds
Natural pollination to produce seed in sexual reproduction of the waterlily occurs via wind and insect. Natural cross-pollination or hybridization between cultivars has yielded many new interesting colorful waterlily identities/cultivars. However, for developing a cultivar with desired characteristics, for example a new flower shape and color, controlled pollination (especially cross-pollination for fertilization made by hand) is necessary before making a selection that involves both science and art.
Hardy waterlilies (Nymphaea sp. subgenus Nymphaea) with blue flowers are rarely seen; therefore, many crosses (involving cross-pollination among many cultivars, including between species) of hardy waterlilies and others were made before the selection for interesting beautiful waterlilies, including blue ones.  

Nature of the hardy waterlily
Hardy waterlilies have ovate and entire leaves, with flowers that float on the surface of the water. The majority of rootstocks are rhizomes that run horizontally under the soil surface. Flower colors are white, pink, red, yellow, and orange. Each flower blooms approximately three days. Flowers bloom from about 6:00 am to 2:00 pm in the Thai climate. Nymphaea mexicana displays only two day’s bloom, open from 11:00 am to late afternoon.

The stigma, located at the center of flower, contains stigmatic fluid or nectar. Nectar can be found only on the first day of the blooming. The nectar is surrounded by stamens. The outer surrounding organs are petals and sepals, respectively.  

Waterlilies naturally have a solitary perfect flower that can’t be self-pollinated within a single flower. However, the “protogynous” phenomenon in the Nymphaea genus 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 shed or pollinate. This results in the stigma of the Nymphaea genus becoming ready to be cross-pollinated during the first bloom day with the pollen shed from another flower/plant on the second day and third day of blooming. However, exceptions exist for some species in which the pollen is ready to shed for fertilization in the first bloom day according to the stigma receptivity, enabling pollination or fertilization to occur within the same single flower. This exception is not found in hardy waterlilies.

 
The Protogyny
of the Waterlily
Readiness of stigma and stamens
(stigma becomes receptive on first
day while stamens are ready to
shed pollen on second day
and third day)

Flower parts

Prior to performing hand cross-pollination, it is important to distinguish the first day flower, second day flower, and third day flower. Characteristics of stigma and stamen should be considered for the flower as follows:

The first day flower
- stamens are straight up
- easy to see the stigmatic disc
- nectar is present
The second or third day flower
- stamens curve to the center of the stigma
- unable to see the stigma


In the photo to the right, the flower on the left is in the first day bloom and the flower on right is in the second day or the third day bloom. The inner rings of anthers are open in the second day bloom in which pollen can be seen easily, compared to the third day bloom. Pollen of second and third day flowers can be used for hand cross-pollination. 

First day bloom and third day bloom >


Emasculation

Emasculation is the process for eliminating the anthers prior to performing hand cross-pollination. This process leads to the prevention of self-pollination. However, most waterlilies have the system of cross-pollination prevention called protogyny. To prove that hardy waterlilies are protogynous, DNA testing of cross-pollinated offspring (obtained from pods or fruits derived from flowers without emasculation) was employed in 2007. The results showed that all of offspring displayed male parent and female parent DNA bands.

In early 2009, one hundred samples of the possible self-pollination of N. ‘Supranee Pink’ (a cultivar with prolific pod development) were done by bagging the flowers before blooming. The results showed that none of the bagged flowers had pod development. These two studied cases indicate that emasculation is not necessary for controlled cross-pollination by hand in hardy waterlilies.

< Self-pollination of the hardy waterlily by bagging the flower
to prevent cross-pollination


Suitable time for controlled cross-pollination by hand

The time for controlled hand cross-pollination depends on both male and female parents. The female parent flower should be used for cross-pollination on the first day of blooming when its stigma is receptive to pollination and fertilization. Nectar is necessary for pollen germination.

Pollen and/or anthers for controlled cross-pollination should be obtained from the second or third day male parent, the flower previously bagged before blooming to eliminate possible contamination. At this stage, the anthers are open and release plenty of pollen. The pollen is yellow in color and is on top of the anthers.

Success of controlled hand pollination

There is a wide range of pollination success. The degree of success depends on parental cultivars. The information of parents can be seen at http://www.victoria-adventure.org/waterlilies/parents_for_hybrids.html.


Hand pollination equipment

1) scissors
2) spoon
3) tag
4) pencil
5) paintbrush
6) thin cloth bag
7) string
8) recording book

     

Steps of hand cross-pollination

To hand cross-pollinate, bring pollen from male parent flower into the nectar of female parent flower. There are many methods for pollen collecting, for example, using scissors or forceps cutting whole stamens, or attaching pollen grains to a paintbrush.

Steps of hand cross-pollination are as follows:


1) Choose a good flower of the pod or female parent plant, to be used in cross-pollination on the first day of blooming.

 
First day bloom
of the pod parent


Cover the flower
 
2) Cover the flower of the pod parent plant with a thin cloth bag before the cross-pollination. The stamens do not need to be removed because it is not ready to shed pollen on the same day as pistil receptivity, according to the protogynous behavior of the plant. Nectar can be observed in the stigmatic disc.
3) Choose a desired male or pollen parent plant that has been blooming for two or three days, as the anthers should be open to release the pollen. The appropriate time would be around 8:00-10:00 am. To prevent contamination from other flowers, the flower should be covered with a thin cloth bag before it blooms.

 
Second day bloom
of the pollen parent


Cut the anthers into a spoon
 
4) Cut the anthers into a spoon, observing the yellow pollen on the anthers.
5) Pour or shake the pollen into the stigmatic disc of the female or pod parent flower and lightly stir with a brush to spread the pollen evenly.


Pour anthers onto
the stigmatic disc

 
 

6) Cover the cross-pollinated flower with a thin cloth bag, close and tie up the end of the thin cloth bag with string.

< Cover the cross-pollinated flower

7) Label the details of pollination (date, time and pod parent cross pollen parent) on a tag attached to the pollinated flower peduncle.


Label the details on a tag

 
 

8) Five days after blooming, the cross-pollinated flower will begin to sink beneath the water surface. Leave the cross-pollinated flower for two weeks and observe any change. If the flower rots, there is no seed set or cross-fertilization, but if the sepals and petals remain, the ovaries will enlarge to be a pod.

< The pollinated flower will sink beneath the
water surface and peduncle will twist.

The ovaries will enlarge to make a pod. >
9) About 3 to 4 weeks (in the Thai climate) after the pollination, the pod will contain seeds covered with gelatinous arils (the arils help the seeds travel along the water and spread out). Put the seeds in the thin cloth bag into a water-filled container for two days. Wash the arils off the seeds as they begin to dissolve; separate the healthy seeds from the abnormal ones and debris.

 
The outer wall of the mature
pod will come off

 
< Seeds inside the arils 
 

 Select only the healthy seeds >

 
Recording table
 
10) Record pollination date and time, pod and pollen parents details, pod setting result, pod maturity date, number of seeds, date that the seeds are first kept in the fridge, for future reference.
11) Keep the healthy seeds in water inside small sealed containers and stick on the pod number label.

 
Seeds in the sealed small
bottle containing water


Seeds germinate after
cold storage
 
12) Bring the seeds into the stratification process by keeping them in a temperature controlled environment of 4-5°C (39-41°F) for at least two months. Then break the dormancy of the seeds, and plant them for germination.

Additional considerations and cautions for cross-pollination
1) Use genetically diverse pairs of parents to obtain diverse or broad characteristics in the offspring.
2) Besides using a thin cloth bag to cover flower to prevent contamination from undesirable pollen, clean or change to new equipment are the other techniques to prevent contamination. 
3) Two weeks after the controlled hand cross-pollination, check the success of pollination by observing the curvature of flower stalk or peduncle and enlargement of flower base; the increment of flower base size varies, depending on cultivars used as parents and the number of seeds.


Curved and twisted peduncles
indicate successful fertilization
 
4) If the amount of nectar in female parent flower is low, the addition of nectar from another female flower is necessary; however, contamination prevention from the other plant is necessary. 

Developed hardy waterlily hybrids

To date, I have selected 17 promising cultivars from my hybridization of waterlilies. Among these cultivars are crosses of hardy waterlily cultivar x hardy waterlily cultivar, intersubgeneric hybrids, and open-pollinated crosses of tropical night bloomers. The 17 cultivars are as follows:

     
1) N. ‘Mayla’ x N. ‘Perry’s Fire Opal’
- N. ‘Supranee Pink’
- N. ‘Pink Ribbon’
- N. ‘Miss Siam’
2) N. ‘Mayla’ x N. ‘Madame Wilfron Gonnère’
- N. ‘Tanpong’
- N. ‘Rattana Ubol’
- N. ‘Siam Jasmine’
3) Subgenus Nymphaea x Subgenus Brachyceras
- N. ‘Siam Blue Hardy’
- N. ‘Siam Pink’
- N. ‘Siam Pink Tips’
4) N. ‘Perry’s Fire Opal’ x N. ‘Sirius’
- N. ‘Tan-khwan’
5) N. ‘Odorata Luciana’ x N. ‘Madame Wilfron Gonnère’
- N. ‘Manee Red’
6) N. ‘Mayla’ x N. ‘Splendida’
- N. ‘Prapunt Red’
7) N. ‘Madame Wilfron Gonnère’ x N. ‘Perry’s Pink’
- N. ‘Manee Siam’
8) N. ‘Perry’s Pink’ x N. ‘Splendida’
- N. ‘Sri Siam’
9) N. ‘Madame Wilfron Gonnère’ x N. ‘Perry’s Fire Opal’
- N. ‘Prapunt White’
10) (N. ‘Mayla’ x N. ‘Madame Wilfron Gonnère’) x Unknown (Open pollination)
- N. ‘Siam Nymph’
11) N. ‘Maroon Beauty’ x unknown (open pollination)
- N. ‘Rojjana Ubol’ 


N. ‘Tanpong’


N. ‘Siam Pink’


N. ‘Siam Nymph’

Conclusion

Besides knowledge and skill in waterlily breeding, controlled hand cross-pollination to obtain as many as genetically diverse pairs of parents is a factor for success. Consequently, aiming to obtain new attractive characteristics is the major goal of waterlily breeders.

Acknowledgements

The author would like to thank Dr. Slearmlarp Wasuwat for knowledge, Dr. Jinda Jan-orn for manuscript preparation and Dr. Karsedis Distabanjong for help in article translation. 

Bibliography

Anon. 2008. General knowledge about plant hybridization, Available source: www.sc.chula.ac.th, 18/12/2008. (in Thai)
Chomchalow, N. and Chansilpa, N.N. 2009. The role of the ‘Suthasinobon’ complex in introgressive hybridization. Thai Journal of Genetics 2(1): 22-29.
Conard, H.S. 1905. The Waterlilies, a Monograph of the Genus Nymphaea. The Carnegie Institute of Washington, Washington, D.C. 279 p.
Slocum, P.D. 2005. Waterlilies and Lotuses: Species Cultivars and New Hybrids. Timber Press, Inc., Portland, Oregon, U.S.A. 260 p.
Samphantharak, K. 1985. Plant Breeding. Thai Watanapanich Co., Ltd., Bangkok, Thailand. 168 p. (in Thai)
Vandaveer, C. 2003. How does this waterlily protect its seeds? Available source: www.killerplants.com, 15/09/2003.
Slearmlarp Wasuwat, 1994. Waterlilies: Ornamental Plants. Amarin Printing & Publishing Public Company, Limited, Bangkok, Thailand. 297 p. (in Thai)
 


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