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PRODUCTION OF HAPLOID PLANTS AND HOMOZYGOUS DIPLOID LINES
- 1. A PRESENTATION OF PLANT BIOTECHNOLOGY ON PRODUCTION OF HAPLOID PLANTS AND HOMOZYGOUS DIPLOID LINES SESSION – 2021 – 2022 SUBMITTED TO: SUBMTTED BY: Prof. S. S. Sandhu Ambika Prajapati Molecular Biology and Biotechnology of Fungi M.Sc. Biotechnology III Semester DEPARTMENT OF P. G. STUDIES AND RESEARCH IN BIOLOGICAL SCIENCES RANI DURGAVATI VISHWAVIDYALAYA, JABALPUR, MADHYA PRADESH.
- 2. PRODUCTION OF HAPLOID PLANTS Haploid plants are characterized by possessing only a single set of chromosomes (gametophytic number of chromosomes i.e. n) in the sporophyte. This is in contrast to diploids which contain two sets (2n) of chromosomes. Haploid plants are of great significance for the production of homozygous lines (homozygous plants) and for the improvement of plants in plant breeding programme. There are two approaches for the production of haploid plants. The two approaches are: (1) In vivo approach and (2) In vitro approach.
- 3. GROUPING OF HAPLOIDS Haploids may be divided into two broad categories: 1. Monoploids (mono-haploids): These are the haploids that possess half the number of chromosomes from a diploid species e.g. Maize, barley. 2. Poly-haploids: The haploids possessing half the number of chromosomes from a polyploid species are regarded as poly-haploids e.g. Wheat, potato. It may be noted that when the term haploid is generally used it applies to any plant originating from a sporophyte (2n) and containing half the number (n) of chromosomes.
- 4. IN VIVO TECHNIQUES FOR HAPLOID PRODUCTION There are several methods to induce haploid production in vivo. Some of them are listed below: 1. Androgenesis: Development of an egg cell containing male nucleus into a haploid is referred to as androgenesis. For a successful in vivo androgenesis, the egg nucleus has to be inactivated or eliminated before fertilization. 2. Gynogenesis: An unfertilized egg can be manipulated (by delayed pollination) to develop into a haploid plant.
- 5. 3. Distant hybridization: Hybrids can be produced by elimination of one of the parental genomes as a result of distant (interspecific or inter-generic crosses) hybridization. 4. Irradiation effects: Ultra violet rays or x-rays may be used to induce chromosomal breakage and their subsequent elimination to produce haploids. 5. Chemical treatment: Certain chemicals (e.g., Chloramphenicol, colchicine, nitrous oxide, maleic hydrazide) can induce chromosomal elimination in somatic cells which may result in haploids.
- 6. IN VITRO TECHNIQUES FOR HAPLOID PRODUCTION In the plant biotechnology programs, haploid production is achieved by two methods. 1. Androgenesis: Haploid production occurs through anther or pollen culture, and they are referred to as androgenic haploids. There are two approaches in androgenesis— anther culture and pollen (microspore) culture. 2. Gynogenesis: Ovary or ovule culture that results in the production of haploids, known as gynogenic haploids. Haploid plants can be developed from ovary or ovule cultures. It is possible to trigger female gametophytes (megaspores) of angiosperms to develop into a sporophyte. The plants so produced are referred to as gynogenic haploids.
- 7. IDENTIFICATION OF HAPLOIDS Two approaches based on morphology and genetics are commonly used to detect or identify haploids. Morphological Approach The vegetative and floral parts and the cell sizes of haploid plants are relatively reduced when compared to diploid plants. By this way haploids can be detected in a population of diploids. Morphological approach, however, is not as effective as genetic approach. Genetic Approach Genetic markers are widely used for the specific identification of haploids. Several markers are in use. i.) ‘a1‘ marker for brown colored aleurone. ii.) ‘A’ marker for purple color. iii.) ‘Lg’ marker for ligule less character.
- 8. DIPLOIDIZATION OF HAPLOID PLANTS (PRODUCTION OF HOMOZYGOUS PLANTS) Haploid plants are obtained either by androgenesis or gynogenesis. These plants may grow up to a flowering stage, but viable gametes cannot be formed due to lack of one set of homologous chromosomes. Consequently, there is no seed formation. Haploids can be diploidized (by duplication of chromosomes) to produce homozygous plants. There are mainly two approaches for diploidization— colchicine treatment and endomitosis.
- 9. 1. Colchicine treatment: It acts as an inhibitor of spindle formation during mitosis and induces chromosome duplication. There are many ways of colchicine treatment to achieve diploidization for production of homozygous plants. When the plants are mature, colchicine in the form of a paste is applied to the axils of leaves. Now, the main axis is decapitated. This stimulates the axillary buds to grow into diploid and fertile branches. 2. The young plantlets are directly treated with colchicine solution, washed thoroughly and replanted. This results in homozygous plants. 3. The axillary buds can be repeatedly treated with colchicine cotton wool for about 2-3 weeks.
- 10. 2. Endomitosis: Endomitosis is the phenomenon of doubling the number of chromosomes without division of the nucleus. The haploid cells, in general, are unstable in culture with a tendency to undergo endomitosis. This property of haploid cells is exploited for diploidization to produce homozygous plants. The procedure involves growing a small segment of haploid plant stem in a suitable medium supplemented with growth regulators (auxin and cytokinin). This induces callus formation followed by differentiation. During the growth of callus, chromosomal doubling occurs by endomitosis. This results in the production of diploid homozygous cells and ultimately plants. Doubled Haploids in Rice Improvement