DH Technology in Maize Research and Breeding
Homozygous inbred lines are the basis of hybrid maize breeding. Two inbred lines are crossed to produce high yielding hybrids. Hybrid varieties exploit heterosis and is highly successful in increasing the agriculture production worldwide. Inbred lines are formed by continuous selfing for 6 to 8 generations. Each generation the line vigour reduce due to inbreeding depression. Inbreeding depression is the reduction or loss in vigour as a result of continuous inbreeding.
A method speeds up the inbred line development and is currently developing maize breeding and research “the double haploid technique”. In this technique an inducer genotype is used as a pollinator which activates the female flower to produce kernel with haploid embryo(n). These haploid inducers were discovered in 1950’s. Now-a-days haploid induction technique is used is maize for large scale DH line development.
In-vivo haploid induction method:
It requires 4 steps:
- Inducing haploids 2. Identifying kernels with haploid embryos 3. Doubling haploid chromosomes 4. Selfing of doubled plants.
The first step is the induction cross, which induces specific lines that can induce haploildy. To identify the haploids dominantly inherited purple coloured marker is used (endosperm and embryo markers are used to differentiate haploid or diploid embryo). Pollens from haploid inducers are collected, these pollens are used to pollinate the female flowers of the donor plants. Donor ears are harvested, some kernel contain the haploid embryo with only donor parent chromosomes, majority of the kernel will be normal crossing kernel with diploid embryos. These diploid embryos contain chromosomes from both the donor and inducer parent. Normal crossing kernels are of no use for DH line production.
The second step is kernel identification, phenotypic identification is used this is based on dominantly inherited purple colouration of the kernel scutellum and aleurone which act as embryo and endosperm marker.
Basically three groups are formed.
Group 1: contains non-coloured embryo, non-coloured endosperm these may resulted from pollen contamination. Group 2: contains all normal crossing kernel having purple coloured embryo and purple coloured endosperm this results from the dominantly inherited coloration present in the inducer. Group 3: represents the kernel with haploid embryo, coloured endosperm indicates that they are the result of induction cross but non-coloured embryo indicates that embryo contains only donor gene. Small quantity of group 1 is obtained (contaminated kernels). Large amount of F1 crossing kernels are obtained 90-95% kernels belong to this group. Group 3 is the haploid only this group is used in the double haploid line development.
The third step is artificial chromosome doubling of haploids so one copy of each chromosome is made so that the subsequent plants are diploid and homozygous. For doubling of chromosomes colchicine is used it works as a mitotic inhibitor. Colchicine disrupts mitosis by binding to tubulin, in this way formation of microtubules and polar migration of chromosomes is inhibited as a result single cell with doubled chromosomes number is obtained.
A fungicide treatment is applied to the kernels and they are placed in trays for germination. Each tray is clearly labelled with entry number, trays are covered with slightly perforated aluminium foil. Kernels are allowed to germinate for 2 to 3 days. Germination room is kept at a temperature of 260C and moderate humidity. Trays are checked on daily basis and moisture is applied if necessary. When the coleoptile length is 2cm then seedlings are ready for colchicine treatment. Mesh bags and plastic bags are prepared, seedlings with correct coleoptile length are taken from the trays and the tips of the seedling are cut to ensure greater penetration of colchicine. Mesh bags containing the prepared seedling are placed in the steel tank. Tap water is then added to the tank until all the seedlings are well submerged. Water from the tank is then transferred to a measuring tank to estimate the amount of colchicine needed for the seedlings. An electric pump automatically transports the colchicine to the steel tank. After the treatment seedlings are rinsed with tap water.
The treated seedlings are transplanted into the pots filled with soil, special care must be taken to avoid damage during this process. These pots are kept in green house for several days. 2 weeks after treatment seedlings are ready for the transplantation in the field. Transplanting can be done manually or through machine.
The fourth step is selfing of doubled haploid. Selfing of colchicine treated plants produces newly developed completely homozygous doubled haploid inbred line. Colchicine treated plants produces low number of seeds, because of this low seed rate ears are harvested with husk and kept inside pollination bags to minimize loss of seed during handling. In the warehouse ears are carefully dehusked.
Colchicine treated plants can be differentiated from the F1 plants because colchicine treated plants are short weak and produce small amount of pollens while the F1 plants are usually vigorous, have high amount of pollens, many tillers, highly branched tassel and exhibit purple stalk.
In-vivo method of haploid induction is currently favoured in maize breeding and research. And the current success rate at CIMMTY is about 3-5%.
Major: Plant breeding and genetics