Development of doubled haploids in wheat
Authors: Vikas Gupta, Satish Kumar, CN Mishra and Raj Pal Meena
Wheat (Triticum aestivum, 2n=6x=42), is the second most important staple food for about two third of world's population. Varietal development in wheat is very time-taking as it takes around 10-12 years for releasing a variety. Conventional plant breeding involves crossing followed by repeated cycles of selfing to generate homozygous progenies. Doubled haploid (DH) production technique offers great opportunity in producing completely homozygous lines in one generation thereby reducing breeding cycle. The selection efficiency for both qualitative and quantitative traits can be precisely performed as all alleles are fixed in DH lines. DH can be induced by anther culture as well as through wide hybridization involving wheat x maize (WxM) crossing. The WxM system is more efficient as compared to anther culture as it is free from any genotypic differences and less number of albinos formed. The WxM system is based on chromosome elimination technique. This method involves crossing wheat with maize pollen and in the first few mitotic divisions maize chromosomes got eliminated as they fail to attach spindle apparatus forming haploid embryo. These haploid embryos are rescued, regenerated and colchicine treated to produce doubled haploid plants.
Advent of wheat x maize system was manifested by the observation of microscopic, early stage embryos in crosses between hexaploid wheat and maize (Zenkteler and Nitzsche 1984). Later, Laurie and Bennett (1986) at Plant Breeding Institute (PBI), Cambridge studied the early post-pollination events in wheat x maize crosses and demonstrated that both the wheat and maize chromosomes were present in the zygote, but the maize chromosomes were eliminated during the initial cell divisions. Another significant observation from their study was that in such crosses the endosperm was absent and resulted in embryo abortion before the embryo could develop to a rescueable size. After this discovery, refinements in DH technology utilizing WxM system were studied by various workers across the world. Different steps involved in WxM system of DH production are
1. Synchronising wheat and maize flowering: For conducting wheat x maize crosses, maize is planted in staggered sowing at 1-10 days interval, starting from second fortnight of August. Growing maize under a polyhouse is essential for proper growth and timely flowering. Raising of wheat for this special season requires that seed be sown in small cups in the beginning of September. The wheat plants are kept outdoors in sunlight or partial shade for about 8 hours and moved to air-conditioned growth chamber maintained at 20-22oC in dark for about 16 hours each day. These conditions are maintained for 20-25 days after which plants can be shifted to bigger pots maintained outdoors.
2. Emasculation technique: The spikes that are partially emerged from the boot are selected for emasculation. The central florets are removed so as to facilitate better development of embryos in the primary and secondary florets. Anthers are removed without cutting the lemma and palea. This is in contrast to the conventional method, which involves cutting the lemma and palea before removing anthers. Emasculated spikes are covered with glassine bags to avoid any cross-pollination. These bags are marked with date of emasculation, pollination, and cross. Tags are also attached to each crossed spike carrying the relevant information.
3. Method of pollen collection: Pollen is collected by tapping the tassels onto a large butter paper (to avoid pollen wastage). Pollen collected should be fresh which is indicated by its light yellow colour and fine powdery form and is dusted over the mature ovary by opening the floret with the help of forecep. It is desirable to discard collected pollen after half an hour's use and collect a fresh lot for continuing the pollinations.
4. Hormonal application: The pollinated tillers are given 200-ppm 2,4-D (2,4-dichloro phenoxy acetic acid) treatment as a spray on the pollinated spikelets. 2,4-D is applied for 3 consecutive days starting one day after pollination i.e. 24h, 48h and 72hrs after pollination.
5. Detached tiller culture: Twenty four hours after pollination the crossed tillers are detached below the second internode from top and cultured in media containing sucrose and 2,4-D (100ppm). Prior to culture, the culms are sterilized in 0.1% mercuric chloride and only the sterilized portion is inserted in the culture vessel. Before culturing, a second cut below the internode is given with a sharp blade under water by placing the tiller in a shallow tray containing autoclaved water.
6. Embryo rescue: Caryopses are harvested 15-18 days after pollination. The crossed caryopsis are retained and selfed ones discarded on basis of presence of endosperm. The embryo carrying caryopses are surface sterilized with 0.1% mercuric chloride for eight minutes. This is followed by treatment with absolute ethanol for 2 minutes and subsequent 3-4 washings with sterile distilled water to remove any toxic effect of these treatments before dissecting out the embryos. The embryos are dissected from each caryopsis and transferred to test tubes containing Murashiege and Skoog medium .The medium is maintained at pH 5.8. The embryos are given cold treatment for 24h and incubated in dark till germination. The regenerated plants are maintained at 250C and photoperiod of 8-10h.
7. Colchicine treatment: After washing away the media sticking to the roots, plants are transferred to small cups containing vermiculite. For about one week plants are covered with small transparent polythene bags to prevent dessication shock. If the conditions are conducive outside, plants can be shifted to soil, after a total stay of about 15 days in vermiculite cups. At 2-3 tiller stage the haploid seedlings are given colchicine treatment (0.1% colchicine + 2% DMSO (Dimethylsulphoxide) + 0.05% Tween 20) by cutting the roots (3/4 th) for 6hrs. The treated haploid seedlings are then shifted to pots containing vermiculite. Treated tillers generally die and new tillers come up with doubled sectors.
Application of double haploids in wheat breeding:
Doubled haploid production can play an important role in wheat breeding programmes by reducing the breeding cycle. It also increases the selection efficiency by easier identification of the superior lines. In addition doubled haploid breeding is used in molecular mapping. Doubled haploid also provides a way of combining and fixing the desirable features of diverse wheat genotype into common genetic background. Double haploids play an important role in mutation studies as it allows expression and fixation of recessive alleles in one generation. The WxM approach of doubled haploid production is finding increasing use in wheat breeding programmes world wide. In Canada, Superb, Snowbird and Alvena bread wheat varieties have been released by Agriculture and Agri Food Canada. Colorado State University released wheat variety Bond CL in 2004 which was developed through using WxM system. In Japan, wheat cultivar Sanukinoyume 2000 is one of the cultivars developed by using the wheat x maize system. DT801 (2010) first DH of durum wheat was released (De Pauw et al 2010).
1. Laurie D A and Bennett M D (1986) Wheat x maize hybridization. Can J Genet Cytol 28:313-16.
2. DePauw R M, Knox R E, Thomas J B, Humphreys D G, Fox S L, Brown P D, Singh A K, Randhawa H S, Hucl P, Pozniak C, Fowler D B, Graf R J and Braule-Babel A (2010) New breeding tools impact Canadian commercial farmers fields. Proc of the 8th Internal Wheat Conference. St. Petersburg, Russia
3. Zenkteler M and Nitzsche W (1984) Wide hybridization experiments in cereals. Theor Appl Genet 68:311-15.
About Author / Additional Info:
I am working as a Scientist in Indian Institute of Wheat and Barley Karnal, Haryana under ICAR, New Delhi.