Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029

International Journal of Current Microbiology and Applied Sciences ISSN: 2319-7706 Volume 9 Number 1 (2020) Journal homepage: http://www.ijcmas.com

Review Article https://doi.org/10.20546/ijcmas.2020.901.114

Double Haploid: An Overview

Omesh Kumar* and Madhu Choudhary

Department of Genetics and , Institute of Agricultural Sciences, Banaras Hindu University, Varanasi, U.P., 221005

*Corresponding author

ABSTRACT

A doubled haploid (DH) is a genotype developed when haploid cells undergo chromosome doubling. Artificial production of doubled haploids is important in plant breeding. Double haploid (DH) breeding can helps in

speedup conventional plant breeding programmes and make early release of K e yw or ds with superior and desirable traits along with greater utility in other research aspects of plant breeding, genetics and genetic engineering. DH’s Doubled haploid (DH) Genomics, helps in complementing back cross breeding by transferring genes of Quantitative Trait interest from wild relatives thus breaking genetic barriers. The unique Loci (QTL) complete homozygous nature of DH’s, less time requirement to produce a

large number of DH’s, absence of heterozygosity, efficiency over Article Info

conventional systems and absence of gametoclonal variation in DH’s make

Accepted: them very valuable material for very important genetic and molecular 15 December 2019 studies. So, DH’s are extensively used for genetic studies like studying Available Online: 20 January 2020 inheritance of quantitative traits, Quantitative Trait Loci (QTL) mapping, Genomics, gene identification, whole genome mapping and production of

stable transgenic plants.

Introduction haploids. Haploid production by wide crossing was reported in (Kasha and Kao, 1970) A plant or line obtained by doubling the and tobacco (Burk et al., 1979) chromosome number of a haploid plant is called double haploid. The first report of the Doubled haploid methodologies have now haploid plant was published by Blakeslee et been applied to over 250 species. DHs are al. (1922) in Datura stramonium and Guha very valuable material for very important and Maheshwari (1964) developed anther genetic and molecular studies (Hussain et al., culture technique for the production of 2012). Haploid line can be developed by the different methods such as distant 1012

Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029 hybridization, , anther culture, Approaches for double haploid production ovary culture etc. In wheat, distant hybridization methods like Bulbosum The importance of haploids in the field of technique, wheat x maize, and wheat x plant breeding and genetics was realised long Imperta cylidrica were used. Out of which ago. Their practical application, however, has wheat x Imperta cylidrica system is more been restricted due to very a low frequency (< efficient as compared to other methods. 0.001%) of their formation in nature. The Bulbosum technique in wheat was no longer process of or parthenogenesis effective due to the presence of Kr1 and Kr2 (development of embryo from an unfertilized genes situated on the 5A and 5B wheat egg) is responsible for the spontaneous natural chromosomes which markedly reduce production of haploids. Many attempts were crossability between wheat and bulbosum made, both by in vivo and in vitro methods to (Niroula et al., 2009). There are three develop haploids but the success was much outstanding double haploids lines of rice were higher by in vitro techniques. selected from ten doubled haploid lines (DHL’s) generated through anther culture In vivo techniques for haploid production from F1506, a cross between C40442B22 and IR1354056321 were had high yield, and There are several methods to induce haploid uniformity (Lapitan et al., 2004). production through in vivo techniques.

Three outstanding DHL’s of rice generated Some of them are listed below: from the popular IR64 variety utilizing the o Distant hybridization crosses followed anther culture technique had resistance to by chromosome elimination different soil related stresses such as P, K, Zn, o Bulbosum Technique S, and Cu deficiencies. Evaluation of these o Parthenogenesis variants showed improvements on their root o Inducer based approach system in terms of root length and weight as compared to the seed of IR64 variety (Lapitan Distant hybridization methods et al., 2004). Hybrids can be produced by elimination of Using Triticum x Zea crosses for producing one of the parental genomes as a result of DH lines proved to be efficient in accelerating distant (inter-specific or inter-generic crosses) the release of new cultivars, in the wheat hybridization. There are some methods for breeding program. producing double haploid in wheat. Cytological analyses performed to reveal the The advantages of the DH system, particularly mechanism of chromosome elimination the advancement of newer, superior crosses, enunciated similar mitotic abnormalities in could counterbalance the limitations in the many hybrids (Laurie and Bennett, 1988, number of crosses and progenies. The utility 1989; Mochida et al., 2004). Chromosomes of DHs in development, back being eliminated tend to be apart from the crossing, genome mapping, hybrid sorting, equatorial plate at metaphase. Sister mutation research, evolutionary studies, gene chromosomes fail to move toward the poles at identification, cytoplasmic research and anaphase and are retained in the cytoplasm to transgenic plant development make them the be eliminated from the nuclei. These best material for genetic improvement and chromosomes form extra-micronuclei and are genetic studies. finally degraded. In addition to mitosis

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Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029 dependent elimination, another pathway of demonstrate that the maize pollen normally chromosome elimination was proposed in germinates and grows into the wheat embryo wheat × pearl millet crosses (Gernand et al., sac where the wheat egg is fertilized by the 2005). Micronuclei containing pearl millet maize sperm nuclei. A hybrid zygote with 21 chromatin were directly budded and wheat chromosomes and 10 maize eliminated from interphase nuclei. There are chromosomes is produced. The hybrid zygotes the some distant hybridization methods which are karotypically unstable; therefore, maize are used for the production of haploids in chromosomes fail to move to the spindle poles wheat viz., Wheat X Maize Method (Maize during cell divisions. Possibly, their Pollen Method), Wheat X Imperta cyllindrica centromeres fail to attach to the spindle and Wheat X Pearl millet Method. These microtubules due to progressive loss of Inter-generic crosses have been found to be centromere activity, which is seen as reduction effective for the production of dihaploid plants in size and finally the loss of, the primary in wheat. Polyhaploids induction from such constriction as reported for H. vulgare x H. crosses is possible because of the preferential bulbosum hybrid. The maize chromosomes are chromosomes elimination of the pollen parent rapidly eliminated after a few cell divisions during embryo development and haploid and thus forming a haploid embryo with 21 plants can be recovered following embryo wheat chromosomes. Wheat x maize system rescue. of crosses following chromosome elimination is an effective and handy tool among available Kasha and Kao for the first time reported methods for haploid induction in wheat. Its haploid barley through barley x Hordeum superiority over other techniques includes bulbosum crosses (H. bulbosum system). Later higher efficacy (2-3 times more efficient for this system was extended to wheat. This green plant production than the anther system was also no longer effective due to the culture), simple, less genotype dependent presence of Kr1 and Kr2 genes situated on the response, less gametoclonal variation and less 5A and 5B wheat chromosomes which time consuming markedly reduce the crossability between wheat and bulbosum. Despite the limited Wheat X Imperta cyllindrica success, the efficiency of haploid production in wheat through anther/microspores and This novel haploid induction method has two wheat x bulbosum techniques are highly great advantages in practical application, (1) genotype-dependent which limits the use of high frequency of haploid induction these techniques in practical wheat breeding. irrespective of wheat genotypes, and (2) natural coincidence of flowering period of Wheat X Maize Method (Maize Pollen wheat and I. cylindrica under subtemperate Method) condition. Wheat genotypes affect the crossability with alien species (Inagaki and It was 1984 for the first time Zenkteler and Mujeeb-Kazi, 1995; Pratap et al., 2005). For Nitzsche reported that embryos are frequently example, wheat varieties with dominant Kr formed when hexaploid wheat pollinated with genes, which inhibit alien pollen tube growth, maize. This raised the considerable interest show greatly reduced crossability with many among wheat breeders and Laurie and Bennett alien species (Riley and Chapman, 1967; at Plant Breeding Institute, Cambridge started Snape et al., 1979; Falk and Kasha, 1981, systematic study to confirm the previous 1983; Sitch et al., 1985). This restricts the reports. They were cytologically able to application of haploid induction method by

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Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029 crossing with H. bulbosum or rye to only some myo-inositol, 1 mg/l thiamine HCl, 0.5 mg/l wheat varieties. Although maize pollen is pyridoxine HCl, 0.5 mg/l nicotinic acid, 2 relatively insensitive to wheat genotypes, and mg/l glycine and 8 g/l agar, and adjusted to pH therefore maize system has been a practical 5.8 with NaOH. When the regenerated plants haploid induction method by chromosome grew enough to transplant, they were elimination, it is necessary to adjust the transferred to soil in a field. flowering period using greenhouse to obtain fresh maize pollen for crossing in Bulbosum method of double haploid subtemperate condition, which increases the production costs of this method. I. cylindrica also shows genotype nonspecific crossability with wheat, The first to be reported in the early 1970’s was and the efficiency of haploid induction is the interspecific hybridization of cultivated comparable to that in using maize. In addition, barley (Hordeum vulgare L.) with H. the coincidence of flowering period between bulbosum L. (Kasha and Kao, 1970). Haploid wheat and I. cylindrica enables us to obtain plantlets of H. vulgare are generated as a fresh pollen of I. Cylindrical freely. From result of gradual elimination of H. bulbosum these points, I. cylindrica is expected to be chromosomes from hybrid embryos between employed in wheat haploid production the two species. H. bulbosum is a perennial alternative to maize (Chaudhary et al., 2005; outcrossing species found in the Pratap et al., 2005). Mediterranean region. Although there are two cytotypes, diploid (2n = 2x =14) and tetraploid Procedure (2n = 4x = 28), only the diploid form has been used to generate haploid plants of barley. In Hexaploid wheat, Triticum aestivum L. cv. the procedure the donor plant should be grown ‘Chinese Spring’ (2n = 6 x = 42) and Imperata in favoulable condition. Emasculation of cylindrica (2n = 2 x = 20) were used as female barley florets is carried out by forming a slit and pollen parents, respectively. The wheat with a forceps in the lemma, through which spikes were emasculated before anthesis. The the three anthers are removed. A freshly upper small spikelets of the wheat spikes and collected H. bulbosum pollen mixture from the central florets of each spikelet were four genotypes is applied to receptive stigmas removed, and then anthers were detached from with a paint brush 2-3 days later. Longevity of remaining florets. A few days after developing seeds in intergeneric and emasculation, the top of the florets was cut off interspecific hybrids in the Poaceae has been to expose stigmas. Fresh pollen of I. improved by the application of gibberellic acid cylindrica was collected from wildly growing e.g. GA3 and other plant growth regulators to plants around the field and applied on the florets after fertilization. Without GA3 the stigmas with a soft brush. The uppermost developing seed degenerate before embryo internodes of wheat stems were injected with rescue. GA3 (Sigma G-7645) at 75 mg/L + 100 mg/l 2,4-D solution, and 20 mg/l 2,4-D 0.05% Tween 20 applied as a spray to florets solution was dropped onto the ovaries 24 h 1-2 days after pollination to enhance seed after pollination. The 2,4-D treatments were development and embryo size. Early in spring repeated for two more consecutive days. and in the fall, the same treatment is usually Embryos were excised from immature seeds repeated one day after the first one. GA3 + 13 to 16 days after pollination and grown at 2,4-D + Dicamba at 75 mg/L; 2 mg/L and 1 20°C for several weeks on the MS medium mg/L, respectively, has been used on the day supplemented with 30 g/l sucrose, 100 mg/l after pollination to promote effectively seed

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Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029 longevity. Eleven (summer) to 20 (early and the other half the hap gene. Only the latter spring and late autumn) days after pollination, will be useful since they will not produce spikes covered by their labelled brown paper haploids (sterile) again in their progeny. bags are cut off from the plants, immersed to a depth of 5-6 cm in a beaker containing tap In vitro techniques for haploid production water. If seeds are not dissected on the same day, the spikes can be stored up to 15 days in a In the plant biotechnology programmes, fridge at 4°C in the dark. Seeds are removed haploid production is achieved by two from spikes and by the tissue culture methods. technique haploid plant can be develop. The haploid plant can be convert into double Androgenesis haploid by colchicines treatment. Haploid production occurs through anther or Inducer based approach pollen culture, and they are referred to as androgenic haploids. There are at least two important examples of specific genes that can induce haploidy in Anther culture plants : (i) the indeterminate gametophyte (ig) gene in Zea mays, which induces haploids of The selected flower buds of young plants are both male (androgenetic) and female surface-sterilized and anthers removed along (gynogenetic) origin; and (ii) the haploid with their filaments. The anthers are excised initiator {hap) gene in barley (Hordeum under aseptic conditions, and crushed in 1% vulgare), which induces haploids of female acetocarmine to test the stage of pollen origin. The plants homozygous for ig when development. If they are at the correct stage, used as female parent, produced 3% haploids each anther is gently separated (from the in the progeny and these haploids are found in filament) and the intact anthers are inoculated 2 : 1 ratio with respect to maternal and on a nutrient medium. Injured anthers should paternal origin. It has been shown that not be used in cultures as they result in homozygosity for ig gene induces multiple callusing of anther wall tissue.The anther egg cells (five egg cells) and polar nuclei cultures are maintained in alternating periods (seven). Marker genes, r* (colourless seed, of light (12-18 hr.) and darkness (6-12 hrs.) at green plants) and RnJ (purple pigmented kernel 28°C. As the anthers proliferate, they produce and seedlings) have been combined with ig to callus which later forms an embryo and then a allow identification of maternal or paternal haploid plant. After this haploid plant are haploids in different crosses. In paternal subjected to the colchicine treatment for the haploids, the genome of male parent is production of double haploid plants. transferred to the cytoplasm of the female parent so that cytoplasmic male steriles with Pollen (microspore) culture desired nucleus can be easily obtained.In barley, plants homozygous for hap gene Haploid plants can be produced from produce l%-40% haploid progeny obtained immature pollen or microspores (male either on selfing or in crosses as female gametophytic cells). The pollen can be parent. The plants heterozygous for hap gene extracted by pressing and squeezing the (obtained from a cross using hap homozygous anthers with a glass rod against the sides of a plant as female) produce 0.1% to 10% beaker. The pollen suspension is filtered to haploids, of which half will carry hap gene remove anther tissue debris. Viable and large

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Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029 pollen (smaller pollen do not regenerate) are pedicel, is inserted in the solid culture concentrated by filtration, washed and medium. Whenever a liquid medium is used, collected. These pollens are cultured on a solid the ovaries are placed on a filter paper or or liquid medium. The callus/embryo formed allowed to float over the medium with pedicel is transferred to a suitable medium to finally inserted through filter paper. The commonly produce a haploid plant, and then a diploid used media are MS, White’s, N6 and Nitsch, plant (on colchicine treatment). supplemented growth factors. Production of gynogenic haploids is particularly useful in Comparison between anther and pollen plants with male sterile genotype. For such cultures plant species, this technique is superior to another culture technique. Anther culture is easy, quick and practicable. Anther walls act as conditioning factors and Limitations of Gynogenesis promote culture growth. Thus, anther cultures are reasonably efficient for haploid In practice, production of haploid plants by production. The major limitation is that the ovary/ ovule cultures is not used as frequently plants not only originate from pollen but also as anther/ pollen cultures in crop improvement from other parts of anther. This results in the programmes. population of plants at different ploidy levels (diploids, aneuploids). The disadvantages Delayed pollination associated with anther culture can be overcome by pollen culture. This has been observed to enhance the frequency of occurrence of spontaneous Gynogenesis haploids in a number of crops. Kihara (1940) while working on T. monococcum raised the Haploid plants can be developed from ovary frequency of haploids by delaying the time of or ovule cultures. It is possible to trigger pollination and showed that the two female gametophytes (megaspores) of phenomena have a definite correlation. No angiosperms to develop into a sporophyte. The haploids were produced in 41 individuals plants so produced are referred to as pollinated 3-5 days after emasculation, gynogenic haploids. Gynogenic haploids were whereas there were three haploids amongst 8 first developed by San Noem (1976) from the individuals pollinated 9 days after ovary cultures of Hordeum vulgare. This emasculation. technique was later applied for raising haploid plants of rice, wheat, maize, sunflower, sugar Use of abortive pollen beet and tobacco. In vitro culture of un- pollinated ovaries (or ovules) is usually As would be expected, the abortive pollen of employed when the anther cultures give the same species would be capable of unsatisfactory results for the production of providing the hormonal stimulus for the haploid plants. The procedure for gynogenic development of the egg and the endosperm haploid production is briefly described. The though it may not be able to bring about flower buds are excised 24-48 hr. prior to fertilization. anthesis from un-pollinated ovaries. After removal of calyx, corolla and stamens, the When this results in the successful stimulation ovaries are subjected to surface sterilization. of the egg, a haploid would be the natural The ovary, with a cut end at the distal part of result. In a number of plants aberrant pollen

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Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029 has been successfully used for this purpose as Detection of haploids per example in Rice (Nakamura 1933); Nicotiana glutinosa (Webber 1933). The haploids can be detected by the using of following techniques:- High and low temperatures Morphological techniques By subjecting plants to higher as well as lower temperatures than the plant usually We have learnt that the vegetative and floral experiences, the egg may be stimulated to parts and the cell size in haploids are reduced develop parthenogenetically. By higher relative to diploids in maize. Greenblatt and temperature treatment i.e. 41 o C for 45 Bock (1967) selected 45 seedlings out of minutes for 21 hours after pollination 10,000 seedlings, on the basis of size (i.e. thin Nordenskiold (1939) obtained a haploid plant coleoptiles, etc.) and five of them were found of rye. to be monoploids showing some success in screening for haploids. Stomata size, pollen Povolochko (1937) obtained haploids of size and pollen abortion can be other criteria Nicotiana tabacum by subjecting the female to help initial screening, although in each case parent to high as well as low temperatures and cytological confirmation will' be essential. pollinating it with N. alata. by low temperature nearly at the time of fertilization Genetically techniques Blakeslee et al. (1922) obtained haploids in Datura stramonium. Many genetic marker systems are used for recognizing the haploids. The basic principle Radiation treatment involved is that the hybrids and non-hybrids should differ, so that any failure of The first known haploid to be recognized as fertilization and parthenogenetic development such in angiosperms i.e. Datura stramoniwm of embryo can be recognized in the progeny at was obtained by Blakeslee et al. (1922) by the seed stage, seedling stage or adult plant applying X-rayed pollen to emasculated stage. For instance, if a female parent in a flowers. Later, Stadler (1931) obtained cross carries recessive markers and is ha69ploids in Zea mays by a similar method. pollinated by dominant stock, then any Randolph (cfr. Smith 1946), working on the recessive progeny would be generally a same crop, found that the use of X-ray maternal haploid, although possibility also irradiated pollen raised the frequency of exists of its being a diploid of one of the haploids from 1:1550 (normal untreated following types- pollen) to 1:1040. In wheat, treatment with X- rays has been tried quite extensively and with (i) diploid hybrids, mutant or deficient for fair degree of success. the marker gene in parent (ii) diploid hybrids with suppression of Chemical treatment dominant marker, due to suppressor gene or a disease Deanon (1957) tried various concentrations of (iii) diploids which are maternal due to aqueous maleic hydrazide, a concentration of apomictic development. If the recognition 50 ppm., being mostly effective when silks of needs to be done at the seed stage, then Golden Cross Bantom Sweet Corn were the marker should be such that it treated, 24 hours before pollination. expresses at the late stage of embryo

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development, such as aleurone colour from microspore culture. Hence it provides a marker. If screening is to be done at rapid and simple option for large-scale ploidy seedling stage, then the marker should determination in early phases. clearly express in the seedling (e.g. the purple seedling colour). Flow cytometry also allows the detection of mixoploid (plants with unequal number of This technique of genetic markers has been chromosome sets in adjacent cells or tissues) extensively used in developing maize regenerants. Although flow cytometry is an monoploids. Most extensively used markers efficient technique with high degree of include 'at' for brown colour in aleurone, plant accuracy, the preparation of high quality plant and root and ‘lg’ for liguleless, their samples for ploidy analysis is the hurdle that corresponding dominant markers being ‘A’ for accounts for its low utilization in plant purple colour and ‘Lg’ for liguled characters. breeding. If the female parent carries a and/or lg, then the monoploids will be recognized by lack of Mechanism of chromosome doubling purple colour and/or ligule, in crosses with (diploidization) male parent carrying A and/or Lg. Another critical marker gene is R allele (Rni-cudu) which Endomitosis produces deep pigmentation of aleurone, and thus allows screening at the dormant seed It is described as chromosome multiplication stage. and separation but failure of spindle leads to one restitution nucleus with chromosome Chromosome counting number doubled It has also been called ‘Nuclear Restitution’. This is one of the direct and precise methods for ploidy level determination. Haploids and Endoreduplication DH plants can be identified by chromosome counting during mitotic or meiotic cell It is a phenomenon of DNA or Chromosome division. Chromosome counting during doubling without Cytokinesis. mitotic division is easy and fast, and can be done by using root tips or other meristamatic C-mitosis tissues. Determination of ploidy by counting mitotic chromosomes is, however, time An artificially induced abortive nuclear consuming and difficult especially in crops division in which the chromosome number is with small chromosomes such as Brassica doubled (as that caused by exposure of cells to napus. colchicine)

Flow cytometry Nuclear fusion

Flowcytometry using DNA selective It occurs when two or more nuclei divide flourochromes has been considered to be the synchronously and develop a common spindle. fast and reliable method for the measurement Thus, two or more nuclei could result with of nuclear DNA content (Dolezel et al., 2007; doubled, polyploid or aneuploid chromosome Ochatt, 2008). Flow cytometry enables the number. measurement of ploidy level in an early developmental stage of plantlets emerging

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Other chromosome doubling agents is desirable genotype, the probability of obtaining this genotype is higher in haploid 1. Acenanaphthene method than in diploid method. If n loci are 2. Chloramphenicol segregating, the probability of getting the 3. Nitrous oxide desirable genotype is (1/2)n by the haploid 4. Parafluorophenyl alanine method and (1/4)n by the diploid method. 5. Hydroxyquinone Hence the efficiency of the haploid method is high when the number of genes concerned is Colchicine large.

It is a alkaloid isolated by French chemists Studies were conducted comparing DH P.S. Pelletier and J. Caventon in 1820. It is a method and other conventional breeding toxic natural alkaloid and secondary methods and it was concluded that adoption of metabolite, extracted from seed and corn of doubled haploidy does not lead to any bias of plants of the genus Colchicum (autumn genotypes in populations, and random DH’s crocus, Colchicum autumnale, also known as were even found to be compatible to selected "meadow saffron"). line produced by conventional pedigree method. How colchicine work Application of double haploids When plant parts were treated with appropriate media, the chromosome of treated In plant breeding double haploid can be used cells replicated properly, but spindle formation in many ways as describe below. was inhibited and cytolasmic phase of cell division did not take occurs. Cultivar development

Methods of colchicine application Uniformity is a general requirement of cultivated line in most species, which can be 1. Seed and seedling treatment- 0.1 to 0.4 easily obtained through DH production. There % colchicine for 2 to 10 hrs. to 24 hrs. are various ways in which DHs can be used in in vegetables cultivar production. The DH lines themselves 2. Meristematic cell- 0.05 to 0.1 % for 2 can be released as cultivars, they may be used to 10 hrs. as parents in hybrid cultivar production or 3. Presoaked seed with – 0.2% colchicine more indirectly in the creation of breeders solution for 2 to 8 hrs. lines and in germplasm conservation. Barley 4. Among all methods of colchicine has over 100 direct DH cultivars. According to application, shoot apex treatment at the published information there are currently seedling stage is most effective. around 300 DH derived cultivars in 12 species worldwide. Genetics of double haploid population The relevance of DH’s to plant breeding has In DH method only two types of genotypes increased markedly in recent years owing to occur for a pair of alleles, A and a, with the the development of protocols for 25 species. frequency of ½ AA and ½ aa, while in diploid Doubled haploidy already plays an important method three genotypes occur with the role in hybrid cultivar production of frequency of ¼ AA, ½ Aa, ¼ aa. Thus, if AA vegetables, and the potential for ornamental

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Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029 production is being vigorously examined. DHs phenotyping with replicated trials is needed. are also being developed in the medicinal herb This is possible with doubled haploidy Valeriana officinalis to select lines with high organisms because of their true breeding pharmacological activity. Another interesting nature and because they can conveniently be development is that fertile homozygous DH produced in large numbers. Using DH lines can be produced in species that have self- populations, 130 quantitative traits have been incompatibility systems. mapped in nine crop species. In total, 56 DH populations were used for QTL detection. Wheat (Triticum aestivum L.). Several cultivars of wheat have been released in China Backcross breeding as a direct result of anther culture (Han, 1986). The development of ‘Jinghua No. 1’ has been In backcross conversion, genes are most completely described (Daofen, 1986). introgressed from a donor cultivar or related From the F1 of a cross between F1 (Lovrin 18 species into a recipient elite line through x 5238-036) x Hongliang No. 4, 400 anthers repeated backcrossing. A problem in this were cultured. Fortysix anther-derived green procedure is being able to identify the lines plants were obtained, from which 28 selffertile carrying the trait of interest at each generation. plants were produced after chromosome The problem is particularly acute if the trait of doubling by colchicine. Seed was increased interest is recessive, as it will be present only for these 28 lines, and one line was selected in a heterozygous condition after each for good performance in field evaluations. backcross. The development of molecular markers provides an easier method of After 3 years of regional testing, just 6 years selection based on the genotype (marker) after the initial cross had been made to rather than the phenotype. generate the anther donor, the selected line was released as ‘Jinghua No. 1’. More Combined with doubled haploidy it becomes recently, ‘Anther Culture 28’ has been more effective. In marker assisted backcross released in China following a similar protocol, conversion, a recipient parent is crossed with a except by selection among the anther-derived donor line and the hybrid (F1) backcrossed to plants in the greenhouse for resistance to the recipient. The resulting generation (BC1) Erysiphe graminis DC. ex Mérat ; ‘Anther is backcrossed and the process repeated until Culture 28’ demonstrated immunity in the the desired genotypes are produced. The field to the same pathogen (Zhao et al., 1990) combination of doubled haploidy and molecular marker provides the short cut. In the Mapping quantitative trait loci back cross generation one itself a genotype with the character of interest can be selected Most of the economic traits are controlled by and converted into homozygous doubled genes with small but cumulative effects. haploid genotype. Chen et al. (1994) used Although the potential of DH populations in marker assisted backcross conversion with quantitative genetics has been understood for doubled haploidy of BC1 individuals to select some time, it was the advent of molecular stripe rust resistant lines in barley. marker maps that provided the impetus for their use in identifying loci controlling Hybrid sorting quantitative traits. As the quantitative trait loci (QTL) effects are small and highly influenced One of the essential steps in haploid breeding by environmental factors, accurate involves selection of superior plants among

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Int.J.Curr.Microbiol.App.Sci (2020) 9(1): 1012-1029 haploids derived from F1 hybrids through resistance to various drugs, amino acid anther culture. It is popularly described as analogues, pathotoxins, salts, herbicides, hybrid sorting and virtually means election of chilling, viruses and nematodes. In tobacco a recombinant superior gametes. The haploid cell line resistant to methionine-sulphoximine method of breeding involving hybrid sorting is and wild fire disease (caused by Pseudomonas considered superior over pedigree and bulk tabaci) has been developed. After methods, firstly because the frequency of differentiation and diploidization, such useful superior gametes is higher than the frequency mutations can be incorporated in the of corresponding superior plants in F2 regenerated plants in homozygous condition. generation, and secondly because haploid Varieties may thus be produced whik show breeding reduces significantly the time resistance to diseases, salinity and required for development of a new variety. For environmental stresses along with better instance, if we assume that the frequency of nutritional qualities. superior F, gametes is one in one hundred, then the frequency of homozygous F2 plants Haploids in evolutionary studies derived from the fusion of two such superior similar gametes would be one in ten thousand. A comparison of dihaploids in potato with Therefore, smaller populations of doubled diploid wild species proved useful in deriving haploids derived from haploids will need to be conclusions about the origin of cultivated handled. potato. Since, production of 2n gametes is common both in dihaploids as well as in The above technique of hybrid sorting has diploid species, it is suggested that Andigena, been successfully utilized, particularly in the progenitor of Tuberosum must have China and Japan, for development of new originated many times from different but varieties in several crops. In Japan, an related 2x populations in different locations excellent tobacco cultivar, ‘F-21V bred through sexual polyploidization. Haploids in through anther Culture, is resistant to bacterial potato have also been used for crosses with wilt and has a mild smoking quality. In China, diploid species to conduct genome analysis. more than 100 rice varieties developed using The meiosis in hybrids and further generations the same technique, gave an increased yield suggested that wild species differ from and, therefore, occupy significant areas of rice cultivated potato in a small number of genes. cultivation A study of pachytene chromosomes in potato dihaploids and tomato (diploids, 2n = 24) Haploids in other fields suggested closer evolutionary relationship between potato and tomato. Haploids in mutation research Haploids in genetics Haploids are also of great interest in basic and applied research on induced mutations, Disomic inheritance and reduced chromosome because due to the presence of only one set of number in polyhaploids (e.g. dihaploids in chromosomes, even a recessive mutation is potato) offer special advantage for their use in immediately expressed. Further, the isolated a variety of genetic studies. Using potato as an pollen and single cells can be plated in small example, following uses of haploids in genetic Petri plates and screened in large numbers. studies in higher plant illustrated: (i) Using Mutant cell lines in some crops using haploids electrophoresis, number of genes for several have actually been isolated, which showed isozymes determined for potato (Simon and

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Peloquin, 1980; Quiros and McHale, 1985). of trisomics using one of the following two progress has also been made in determining procedures : (i) isolation of parthenogenetic the number of genes for other traits like aneuhaploid offspring following Ax x 2x disease resistance and other agronomic traits, crosses; (ii) isolation of aneuploids from 3x x (ii) Using haploids in potato, heritability was 2x crosses, where 2x is a haploid. In both the also determined for several traits like specific schemes, a numbeti of trisomics with 2n = 25 gravity, vine maturity, glucose concentration, were obtained. In tobacco several nullisomics tuber dormancy, total yield and tuber weight, were derived from haploids obtained from (iii) The genotype of a tetraploid can also be sterile monosomies. Haploids have also been' determined by extracting and analysing the used for the production of alien addition and possible haploids, as has been done for acid alien substitution lines in a crop like wheat. gel proteins, several isozymes and self incompatibility in potato, A study of meiosis in haploids also gives evidence for the original base number ; in a The recessive alleles are also un-masked in species or a genus. For instance in bajra these haploids. Intralocus interactions could be (Pennisetum typhoides), occurrence oil some studied using haploids in potato, because at a pairing (2 bivalents in some cells) suggested tetraallelic locus (A1A2A3A4) in potato, one that the original base number may be x = 5. expects six first order interactions (A1A2, Similar evidence is also available in maize, A1A3,A1A4,A2A3,A2A4,and A3A4), three which is sometimes considered to be an second order intractions (A1A2A3, A1A3A4, archaic tetraploid. In potato also, chromosome A2A3A4) and one third order intraction behaviour of dihaploids (2n = 2x = 24) (A1A2A3A4). As opposed to this, dihaploid suggested that the base number is x = 12 and will have only one intraction, which partly that potato is an autotetraploid. Chromosome explains loss of vigour. And recently behaviour of monoploids in potato (2n = x = dihaploid X wild species crosses were also 12) formed only univalents confirming this used for the prepration of RFLP Linkage conclusion. maps. Doubled haploids derived from F1 hybrids are also being out to increasing use for Advantages of DH’s prepration RFLP maps and are considered to be superior over F2 progenies. The ability to produce homozygous lines after a single round recombination saves a lot of Haploids in cytogenetic research time for the plant breeders. Studies conclude that random DH’s are comparable to the Haploids have also been found to be useful in selected lines in pedigree inbreeding. The various areas of cytogenetic research, other advantages include development of large including their use in production of number of homozygous lines, efficient genetic aneuploidsand determining the basic analysis and development of markers for chromosome number or the nature of useful traits in much less time. More specific . In wheat, initially monosomies benefits include the possibility of seed were produced by E.R. Sears, when haploids propagation as an alternative to vegetative were pollinated by normal wheat pollen. The multiplication in ornamentals, and in species monosomies, presumably resulted due to such as trees in which long life cycles and functioning of egg with restitution nuclei inbreeding depression preclude traditional deficient for any individual chromosome. In breeding methods, doubled haploidy provides potato haploids were used for the' production new alternatives.

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Disadvantages of DHs mixed haploid-diploid types. Another disadvantage associated with the double The main disadvantage with the DH haploidy is the cost involved in establishing population is that selection cannot be imposed tissue culture and growth facilities. The over- on the population. But in conventional usage of doubled haploidy may reduce breeding selection can be practised for several genetic variation in breeding germplasm. generations: thereby desirable characters can Hence one has to take several factors into be improved in the population. In haploids consideration before deploying doubled produced from anther culture, it is observed haploidy in breeding programmes that some plants are aneuploids and some are

Table.1 Varieties developed through double haploid technology

Crop Method followed Varieties Country

Rice Anther culture Tanfeng 1, Tan Fong 1, Hua Yu 1, Hua China 03, Xin Xiu, Xhongua 8, Ta Be 78, Guan 18 Anther culture Dama Hungry Anther culture Parag 401(ACR 401) MH, India Anther culture CR Dhan 801 CRRI, India Anther culture Patei and Moccoi Argentina Wheat Anther culture Hua Pei 1, Lung Hua 1, Jinghua 1, China Yunhua 1, Yunhua 2 Anther culture Kharoba Morocco Anther culture Florin France

Wheat x Maize Glosa, Faur F, Liter, Miranda Romania

Tobacco Anther culture Tan Yu 1, Tan Yu 2, Tan Yu 3, China

Anther culture F211(wilt resistant & mild smoking) Japan

Barley H. bulbosum Mingo, Gwylan Canada

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Table.2 Pest and disease resistance lines developed using double haploid technique

Crop & line Resistence to Reference

Barley- Mingo Barley yellow mosaic virus Foroughiwer & Friedt,1984 Q-21681 Stem rust, leaf rust and PM Stoffenson et al.,1995

Rice- Zhonghua no-8 & 9 Blast , high yielding and good quality DDBeeong-geun et al., 1997 Hwasambye Leaf blast, BLB, & rice stripe tenui virus. Hwachengbyeo BPH & cold tolerance Lee et al., 1989 Bacterial blight, rice tenui virus. Shirayukhine High yielder, tolerant to major pest and Disease Kazahiro &Terahiro., 2002 (Japan ) Tobacco- Tan yu 3 Necrotic strain of potato virus Witherspoon et al., 1991

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How to cite this article:

Omesh Kumar and Madhu Choudhary. 2020. Double Haploid: An Overview. Int.J.Curr.Microbiol.App.Sci. 9(01): 1012-1029. doi: https://doi.org/10.20546/ijcmas.2020.901.114

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