SIGNIFICANCE OF HAPLOIDS,DIPLOIDIZATION AND BULBOSUM TECHNIQUE.pptx

Significance of Haploids. Diploidization, and bulbosome technique- introduction, methods, significance, limitations,

1. SIGNIFICANCE OF HAPLOIDS,DIPLOIDIZATION AND BULBOSUM TECHNIQUE

2. CONTENT Introduction Methods of haploid production Diploidization Bulbosum technique Significance Limitations Conclusion Reference

3. INTRODUCTION Haploids are sporophytic plants that are autonomous and possess only a single set of chromosomes. These are in contrast of diploids containing two sets of chromosomes. Haploids may be originated from a gametic cell in the embryo sac or in the pollen grain. Haploids may be divided into two categories :

4. 1. Monoploids ( monohapioids ): These are the haploids that possess half the number of chromosomes from a diploid species e.g. Maize, Barley. 2. Polyhaploids : The haploids possessing half the number of chromosomes from a polyploid species are regarded as polyhaploids 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.

5. The existence of haploids was discovered by Bergner in Datura stramonium . Plant breeders have been conducting extensive research to develop haploids. The Indian scientists Guha and Maheshwari (1964) reported the direct development of haploid embryos and plantlets from microspores of Datura innoxia by the cultures of excised anthers. Subsequently, Bourgin and Hitsch (1967) obtained the first fully-pledged haploid plants from Nicotiana tabacum . Thereafter, much progress has been made in the anther cultures of Wheat, Rice, Maize, Pepper and a wide range of economically important species.

6. METHODS OF HAPLOID PRODUCTION There are two way for the production of haploid plants. They are: (1)  In Vivo  and (2)  In Vitro . The process of apomixis or parthenogenesis is  responsible for producing spontaneous natural haploids. Many techniques are followed both by in vivo and in vitro methods for haploid production. In vitro techniques are more applicable for much haploid production.

7. IN VIVO APPROACH Androgenesis :     In the process of androgenesis , haploid plants are mainly derived from male gamete. Development of a haploid plant from an egg cell containing male nucleus is referred to as androgenesis .  Before fertilization this egg nucleus has to be inactivated or eliminated for successful haploid production in vivo techniques. 2. Gynogenesis :   A haploid plant can be produced from an unfertilized egg cell as a result of delayed pollination. e.g. cross between  Solanum tuberosum  and  Solanum phureja  produced diploid potatoes.

8. 3. Distant hybridization:   Haploid hybrids can be produced by elimination. In this process, one of the parental genomes have to be eliminated to produce haploid hybrids.  Distant hybridization may be continued by inter-specific or inter-generic crosses. Gersenson (1928) obtained 7 haploids out of 35 offspring by the crossing between  Solanum nigrum  and  Solanum luteum .  4. Parthenogenesis :   In plants, parthenogenesis is a component process of apomixis . Haploids may be developed from an unfertilized egg cell. In this case, no fertilization is needed. 5. Apogamy :   In apogamy , haploids may be developed from other cells of the mega-gametophyte.

9. 6. Shocks with high or low temperatures :   Haploid plants may also be produced by applying high or low temperature. Blakesle et al (1927) got haploid plant by the application of high and low temperature. 7. Irradiation with X-rays or UV light:   For producing haploids,  x-rays or ultra violate rays may be used to induce chromosomal breakage and their subsequent elimination. Arenberger (1948) got haploid  Antirrhinum majus  .   8. Chromosome elimination :   Chromosome elimination occurs in some intergeneric and interspecific crosses to produce haploids. e.g. Cross between  Hordeum vulgare  and  H. bulbosum .

10. IN VITRO APPROACH Anther culture : Most research has been carried out on isolated anthers which has been isolated on to solid or on liquid nutrient media 2. Pollen grain culture:   This is less used technique due to technical problems 3. Inflorescences:   Useful with grasses and other plant species which have small flowers, e.g. Hordeum

11. 4. Embryo culture:   Hordeum vulgare is crossed with H. bulbosum . Soon after fertilization, the chromosome of H. bulbosum are eliminated. Haploid embryo of H. vulgare is produced with no endosperm and without embryo culture this embryo would abort. Another example is crossing between Triticum aestivum and H. bulbosum which resulted in the production of a haploid Triticum aestivum embryo. 5. Pseudo-fertilization:   Hess and Wagner (1974) pollinated Mimulus luteus in vitro with alien pollen of Torenia fournieri by prickle pollination a haploid cell of Mimulus developed into a haploid plant.

12. 6. Development of unfertilized ovules without pseudo-pollination: The main advantage of ovule culture appears to be that the majority of regenerants are green and are more genetically stable, e.g. rice, wheat, maize, Hordeum vulgare , etc. 7. Gametophytic irradiation and ovary culture: In Petunia, the in vitro ovary culture of plants pollinated with irradiated pollen is so far the most efficient techniques for haploid production. The irradiated pollen acts not only in stimulating gynogenesis but also takes part in fertilization.

13. DIPLOIDIZATION Haploids may grow normally up to flowering stage but in the absence of homologous chromosomes meiosis is abnormal and, consequently viable gametes are not formed. To obtain fertile, homozygous diploids for analyzing the progenies and the breeding behavior of the pollen plants the chromosome complement of the diploids must be duplicated. Spontaneous duplication of the chromosomes in pollen derived plants has been observed but its frequency fairly very low.

14. In wheat and barley 20-50% and 20- 70% of the pollen plants respectively, exhibited spontaneous diploidization . However, generally, the rate of spontaneous duplication of chromosomes may be very low. This can be significantly enhanced by using artificial means. For Nicotiana tabacum only 0.4% solution of colchicine is recommended to diploidize the pollen plants. In practice, the young pollen-derived plants are immersed in a filter- sterlized solution of colchicine for about 96 hours and then transfer to a culture medium to allow their further growth.

15. Alternatively the treatment is given in form of a lanolin paste. It is applied to the axils of the upper leaves and the main axis is duplicated to stimulate the axillary buds to grow into diploid and fertile branches. Lichter et al. (1989) induce chromosome doubling in B. napus by injecting 2% solution of colchicine into secondary buds. Besides bringing about chromosome duplication, colchicine treatment may also result in chromosome and gene instabilities.

16. Endomitosis : Haploids cells are unstable in culture and have tendency to undergo Endomitosis . i.e chromosome duplication without nuclear division. This property can be used for obtaining homozygous diploid plants. In this process, a small explant of stem from a haploid plant is cultured on auxin-cytokinin added medium where the segment forms the callus tissue. During callus growth, diploid homozygous cells are produced by endomitosis . Now large number of isogenic diploid plants can be obtained by organogenesis..

17. iii) Fusion of Pollen Nuclei: Homozygous diploid callus or embryos may form by the spontaneous fusion of two similar nuclei of the cultured pollen after first division. In Brassica , the frequency of spontaneous nuclear fusion in microspore is high in culture

18. BULBOSUM TECHNIQUE The production of haploid plants was not the only mechanism obtained by pollen or anther culture. There are other alternatives to haploid production. Selective elimination of chromosome of one species during interspecific crosses of two different species of the same plant and retaining one chromosome of one species is being utilised to obtain haploids.

19. The tetraploids of Hordeum vulgare and Hordeum bulbosum were crossed. The products of nearly all the plants were dihaploids . These dihaploids were again crossed, the progeny comprised of haploids and morphologically it is like hordeum vulgare . kasha et.al.(1978) treated fertilised florets with low concentration of GA3 for a couple of days considerably enhanced the frequency of seed set and the recovery of haploid plants.

20. Detailed cytological studies have revealed that in these crosses double fertilization occurs normally but elimination of bulbosum chromosomes during the early stages of development of the hybrid embryo leads to the formation of embryos with only vulgare chromosomes. The chromosome elimination is controlled by genes present on chromosome 2 and 3 of H.vulgare . In these crosses the embryo normally aborts 10 days after pollination. For raising full plants it is essential to excise the immature embroys and rear them on an artificial nutrient medium.

21. SIGNIFICANCE In haploid plants, as a chromosome possesses dominant and recessive genes, only one set of homologous chromosomes present derived from pollen grains. All the receissive genes will be expressed phenotypically in the progeny as there is no masking effect of recessive gene by dominanat genes. So in the haploidS , it is easy to identify recessive characters. Haploid plants are sterile as there is no zygotene pairing in the first meiotic division and all the chromosome appear as univalent.

22. Significance in Early Release of Varieties: As time taken for getting the homozygous line is less so ultimately many new hybrid varieties can be obtained in less time applying this technique. Hybrid Sorting in Haploid Breeding: Haploid breeding involves the hybrid sorting and it is considered superior to pedigree and bulk method as the superior haploid plants are selected and hybridized.

23.   Induction of Mutagenesis: Haploid cell cultures are useful material for induction of mutations and to study the effect of mutation. This method can overcome the masking effect of presence of dominant gene. The screening method for detection of muta­tional effect is also easier in this technique. Ex: the anther derived haploids of Hyoscymus niger when treated with x ray achieved mutant plants which show higher alkaloid contents.

24.   Induction of Genetic Variability: The pollen/microspore are easy explant for pro­duction of genetically variable types by introducing the different foreign genes through different transformation procedure. These transformed or transgenic hap­loids can be used further in breeding programme.

25. Generation of Exclusively Male Plants: By haploid production of some dioecious male plant, the dihaploid plant containing both Y chromosomes can produce supermale . In case of Asparagus officinalis , male plants give more yield than female plants, so if haploids are produced from anthers of male (XY) plants and by chromosome doubling the Y chromosome containing plant will produce super male plant (YY) which can be subsequently vegetatively propagated.

26. Development of Disease Resistant/Insect Resistant Varieties: Haploid production can be used for the introduction of disease resistance genes into cultivars. An established cultivar is crossed with a donor disease resistant species and either F 1  or F 2  anthers are cultured. Haploids are developed and screened for resistance and then diploidized . New hybrids are produced in tobacco which exhibited resistance to bacterial wilt.

27. Development of Salt Tolerant Varieties: Salt tolerant breeding lines have been developed in different crop species which have been further integrated in convention­al breeding procedure. Ex: Datura innoxia from haploid culture derived from anther culture In raising isogenic pure lines which takes 7to8 years by conventional methods in cross pollinated crops, haploid culture of f1 plants, this may be achieved in single generation.

28. Development of Aneuploids : Haploids have been used in the production of aneuploids like monosomies in wheat, trisomies in potato. In tobacco nullisomics were derived from haploids obtained from monosomies which could not produce nullisomics on selfing . Pollen culture allows a greater survival rate of embryos. This technique is effective in induction frequency of the economically important plants.

29. LIMITATIONS The practical applications of androgenic haploids are still limited as the desired success with anther and pollen culture has been largely restricted to only a few families, such as Brassicaceae , Poaceae and Solananceae . Low yields Generally only 5 to 8 % of the total pollen grains in a responding anther under go androgenic development, and a very small proportion of these androgenic grains develop into full sporophytes . This turns out to be a serious limitation in obtaining full range of gametic segregants of interest of plant breeders.

30. Conversion of pollen embryos into plants One of the major problems in anther/pollen culture of brassica species is the structural, physical and biochemical abnormalities of pollen embryos so that 70 to 80% of the embryos are incapable of normal germination. Albinism in cereals The degree of albino formation may vary with the variety. The different variety of rice shows different rate of formation albino. The albino plants are morphologically normal except for the absence of chlorophyll. Since the albino plants cannot survive in nature there is no nagronomic value.

31. Genetic changes Instabilioty of genetic material during androgenesis could be an hindrance in the use of haploids. In tobacco the androgenic plants exhibited growth depression, reduced agronomic performanced and abnormalities such as reduced leaf and flower size and tumerization of leaf cells, which could be correlated to amplification of DNA.

32. CONCLUSION Haploids have been produced for a very large number of angiosperms, and the technique of anther/pollen culture for haploid production has become an integral part of plant breeding programs. The main factors that hinder the application of anther and pollen culture to cereals are low rates of androgenesis and high frequency of albinism( upto to 80%).

33. REFERENCE Bhojwani,S.S .,& Razdan,M.K .,(2013). Plant tissue culture: theory and practice. Elsevier pvt.ltd. Netherland pp-167-214 Singh,s.k ., & Srivastava,S .,(2012). Plant tissue culture. Campus Books International.New delhi . pp-46-56 Narayaswamy,S ,.(2017). Plant cell and tissue culture. Tata McGraw Hill Education Private Limited.New delhi pp- 340-347 https://www.biologydiscussion.com/plants/haploid-plants/top-10-applications-of-haploid-plants-biotechnology/61373 https://www.slideshare.net/jannatiftikhar/haploid-culture-final https://plantlet.org/haploid-breeding-development-of-pure-homozygous-line/

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