Available Online at http://www.recentscientific.com International Journal of CODEN: IJRSFP (USA) Recent Scientific

International Journal of Recent Scientific Research Research Vol. 11, Issue, 01(D), pp. 36941-36949, January, 2020 ISSN: 0976-3031 DOI: 10.24327/IJRSR RESEARCH ARTICLE

DOUBLED HAPLOIDS IN GENETIC IMPROVEMENT: A REVIEW

1Seeja, G and 2*Sreekumar, S

1Department of and Genetics, College of Agriculture, Kerala Agricultural University, Ambalavayal, Wayanad Pin 673593 2Saraswathy Thangavelu Extension Centre, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Puthenthope, Thiruvananthapuram, Pin 695586

DOI: http://dx.doi.org/10.24327/ijrsr.2020.1101.5029

ARTICLE INFO ABSTRACT

Article History: Doubled haploidy is a powerful tool to induce genetic improvement leading to economic

Received 6th October, 2019 benefits especially among perennial crops with critical breeding duration. It represents Received in revised form 15th recombinant products of parental genomes in a completely homozygous state which have November, 2019 more relevance in horticultural crops that are either perennial in nature or out crossing Accepted 12th December, 2019 plants with severe inbreeding depression. The efficiency of doubled haploid recovery in Published online 28th January, 2020 various horticultural crops is one of the best breeding techniques which has to be explored extensively. Now-a-days, it can be efficiently combined with other plant biotechnological Key Words: techniques, enabling several novel breeding achievements, such as improved mutation Double haploidy, Polyploid, breeding, backcrossing, hybrid breeding and genetic transformation. The present reviews focussed on the genesis and application of doubled haploids in plant breeding. Population, Genetics, Crop, Breeding

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INTRODUCTION depression. Selection can be expected for traits caused by recessive deleterious genes that are associated with vegetative Genetic improvement is the basis of plant breeding, which growth. Induction of doubled haploidy and its relevance in actually expect some desirable variability in plant traits either plant breeding are briefly reviewed here. by utilization of existing natural variability or by creating artificial variability via conventional methods such as heterosis Origin of Doubled Haploids breeding, recombination breeding, mutation breeding, Haploids are plants with a gametic chromosome number (n) breeding and non-conventional methods like and produced from diploid species (2n=2x), known as somatic hybridization, genetic engineering etc. Polyploidy is monoploids, contain only one set of chromosome in the one of the best options to induce genetic variability with sporophytic phase (2n=x). They are smaller, exhibit lesser plant desirable character especially among perennial crops. Based on vigour and are sterile due to the inability of their chromosomes the variation in chromosome number polyploidy can be to pair during meiosis. Therefore, to propagate through seed classified into different types such as haploids, diploids, and to include in breeding programs, their fertility has to be triplods etc,. Haploids can be modified into doubled haploids restored. A plant or line obtained by doubling the chromosome (DHs ) in which have homozygous alleles at all loci. Hence, it number of a haploid plant or individual is doubled haploid. can be represented as new varieties (self-pollinated crops) or as Doubled haploid technique is a valuable method for genetic parental inbred line for the production of hybrid varieties cartography of complex traits. (cross-pollinated crops). In fact, cross pollinated species often express a high degree of inbreeding depression due to The best method to shortcut breeding technology is induction heterozygosity and hence the induction of doubled haploids of haploid lines. Haploid plants are intensively utilized for process per se can serve not only as a fast method for the investigation and improvement of many agricultural crops. production of homozygous lines but also as a selection tool for Haploids are unique plants and can provide researchers with the elimination of genotypes expressing strong inbreeding

*Corresponding author: Sreekumar, S Saraswathy Thangavelu Extension Centre, Jawaharlal Nehru Tropical Botanic Garden and Research Institute, Puthenthope, Thiruvananthapuram, Pin 695586 Seeja, G and Sreekumar, S., Doubled Haploids in Genetic Improvement: A Review genetic information not possible with normal diploid is needed, since kernels containing haploid embryos exhibit a individuals. normal germination rate and lead to viable haploid seedlings.

In general, haploids are classified into three types. Maternal Haploid embryos can be selected early in the breeding process, based on morphological and physiological markers. haploids contain only nuclear material and cytoplasm from the maternal parent. In vitro androgenic haploid obtained through In vitro induction of maternal haploids is gynogenesis.i.e., the anther or microspore culture and contain both the through in vitro culture of un-pollinated flower parts, such as cytoplasm and nucleus of the developing microsporophyte. The ovules, placenta attached to ovules, ovaries or whole flower microspore developed haploids are better and accurate. In vivo buds. Regenerants show higher genetic stability and lower rate androgenic haploid develop from an egg cell or any other cell of albino plants compared to androgenetic ones. It is used in of the embryo sac and later chromosomes of the maternal plants in which other induction techniques, such as androgensis parent being lost during embryogenesis. It contains the and the pollination methods are unsuccessful. Gynogenesis is cytoplasm of maternal plant and only the chromosomes of the successful in several species10, such as onion, sugar beet, paternal parent. cucumber, squash, gerbera, sunflower, wheat, etc.,

History however, the success is greatly influenced by several biotic and abiotic factors. The genotype of donor plants, combined with The first report of the haploid plant was published in 1992 by growth conditions, is the crucial factor. In onion, marked 1 Blakeslee et al. in Datura stramonium. Subsequently, haploids differences in embryo yields have been recorded among 2 were reported in many other species . In 1964 Guha and accessions and among plants within accessions. The average 3 Maheshwari developed an anther culture technique for the frequencies of induced embryos (calculated from ovaries production of haploids in the laboratory. Haploid production by possessing 6 ovules) varied between 0% in non-responding 4 5 wide crossing was reported in barley and tobacco . Tobacco, accessions to 18.6-22.6% in extremely responsive accessions, rapeseed and barley are the most responsive species for with individual donor plants producing up to 51.7% embryos. doubled haploid production. Doubled haploid methodologies The high haploid production frequency was tested in two have now been applied to over 250 species. Haploids can consecutive years and showed to be stable over years11. originate spontaneously in nature or as a result of various Induction rates were even higher in preselected onion induction techniques like chromosome doubling. Spontaneous genotypes, achieving frequencies of 196.5% embryos from a haploid plants are reported in several crop species, like tomato, doubled haploid line12 or 82.2% for an inbred line13. potato, soybean, maize, barley, wheat, rice, rye etc. Androgenic haploid embryos are produced by or Chromosome elimination after wide hybridization is an pseudogamy. In 1969 Kermicle6reported the possibility of alternative method for producing haploids which is commonly obtaining androgenic haploids in maize. Pollination of plants utilized in wheat. In this approach, pollen from either Hordeum containing the homozygous gene ig1 (indeterminate bulbosum or maize is applied to the silks of an emasculated gametophyte 1) produced 1-3% of seeds with an androgenic wheat spike. The application of maize pollen has proven to be haploid embryo. In vitro androgenesis is a process of induction the most successful approach by providing the highest and regeneration of haploids and doubled haploids originating frequency of haploids. It was discovered that haploid plants of from male gametic cells. Due to its high effectiveness and Hordeum vulgare could be obtained on a large scale following the hybridization of Hordeum vulgare with Hordeum applicability in numerous plant species, it has outstanding 14 potential for plant breeding and commercial exploitation. It is bulbosum . When H. vulgare and H. bulbosum are crossed, a well established for plant breeding, genetic studies and/or normal double fertilization event occurs. However, during seed induced mutations of many plant species, including barley, development, chromosomes of H. bulbosum are eliminated in wheat, maize, rice, , rye, tobacco, rapeseed and both the embryo and endosperm. At approximately 10 days cabbage7.Gynogenesis is production of haploid embryos post-fertilization, most dividing cells in the embryo are haploid. exclusively from a female gametophyte. In situ induction of Colchicine, a mitotic inhibitor is applied to the haploid maternal haploids includes, pollination with pollen of the same seedlings generating fertile spikelet/seed sectors with double species (e.g., maize), pollination with irradiated pollen, the chromosome number. Haploids with these fertile sectors pollination with pollen of a wild relative (e.g., barley, potato) generate seed that have a normal diploid chromosome number. or unrelated species (e.g., wheat). Pollination can be followed The ‘bulbosum’ method was the first haploid induction method by fertilization of the egg cell and development of a hybrid to produce large numbers of haploids across most genotypes embryo, in which paternal chromosome elimination occurs in and quickly entered into breeding programs. Pollination with early embryogenesis or fertilization of the egg cell does not maize pollen could also be used for the production of haploid occur, and the development of the haploid embryo is triggered barley plants, but at lower frequencies. In Nicotiana tabacum by pollination of polar nuclei and the development of wide crossing with Nicotiana africana,0.25 -1.42% of the endosperm. In maize, maternal haploids can occur progeny survive and can readily be identified as either F1 spontaneously. Their rate is usually about one haploid maize hybrids or maternal haploids. This method of interspecific per one or two thousands of normal diploid plants. However, pollination serves as a practical way of producing seed-derived the low frequency of natural haploid generation prevents an haploids of N. tabacum, either as an alternative method or efficient use of this approach in breeding programs. An complementary method to anther culture. Paternal chromosome alternative approach to obtain and investigate maternal elimination has also been observed after interspecific crosses haploids in maize was reported8 following the discovery of a between wheat (Triticum aestivum) and maize. After line called "Stock 6". Today, modern haploid inducing lines pollination, a hybrid embryo between wheat and maize showed high induction rates9 of 8 to 10%. No in vitro culture develops but, in the further process, the maize chromosomes are eliminated so that haploid wheat plantlets can be obtained. 36942 | P a g e International Journal of Recent Scientific Research Vol. 11, Issue, 01(D), pp. 36941-36949, January, 2020

Such haploid wheat embryos usually cannot develop further medium, showed a significant increase in embryo formation when left on the plant, because the endosperm fails to develop and green plant regeneration in wheat16. Duplication treatments in such seeds. By applying growth regulator 2,4- are applied after regeneration at either embryo, shoot or plantlet dichlorophenoxyacetic acid embryo growth is maintained to the level. Since chromosome segregation is driven by stage suitable for embryo isolation and further in vitro culture. microtubules, colchicine is also used for inducing polyploidy in The maize chromosome elimination system in wheat enables plant cells during cellular division by inhibiting chromosome the production of large numbers of haploids from any segregation during meiosis; which results half of the gametes genotype. Pollination with maize is also effective for inducing without chromosomes and other half contains double the haploid embryos in several other cereals, such as barley, number of chromosomes. It leads to embryos with double the triticale (Tritico secale), rye (Secale cereale) and oats (Avena usual number of chromosomes (i.e., tetraploid instead of sativa)15. diploid). In plant cells it is not only usually well tolerated, but also frequently results in larger, hardier, faster-growing, and in Generating doubled haploids where the haploid embryo is general more desirable plants than the normally diploid parents; rescued cultured and chromosome is doubled through the use for this reason, this type of genetic manipulation is frequently of colchicine or other mitotic inhibitors, such as nitrous oxide used in breeding plants commercially. The concentration and gas and some herbicides. Following treatment of the apical duration of treatments must be always determined in relation to meristem by a mitotic inhibitor, chromosome numbers are two effects: the percentage of doubled plants and the doubled in small sectors of the haploid plant. Normally the percentage of survival. Optimization treatments often require doubled sectors produce seeds. These seeds are doubled large experimental units (such as 300 explants per treatment) haploids, pure line . Of the foregoing different due to the substantial variation of response. High methods for doubled haploid production androgenesis is the doses/durations can lead to tetraploidization. Treatment with most preferred one. Different haploid production methods are nitrogen oxide (N2O), which was developed for maize summarized in figure 1. 17 seedlings is a special case. Chemical treatment might be avoided by using in vitro adventitious somatic regeneration, which itself frequently leads to increased ploidy. Such an approach was efficient in onion18. No potentially damaging chemicals are used in the process and the regenerants do not for the most part show a mixoploid character. Up to 100% doubling efficiency in relation to individual line treatment can be achieved using this method19. After a successful haploid induction and the regeneration procedure, evaluation of regenerants is needed to distinguish between desired haploids (or spontaneously doubled haploids) and redundant

heterozygous diploids. Several direct and indirect approaches Figure 1 Different haploid production methods for chromosome doubling are available for determining the ploidy level of regenerated plants. Chromosome Doubling Indirect Approaches Haploid plants obtained from either anther or ovule culture may grow normally under in vitro conditions or can even be Based on comparisons between regenerated and donor plants in acclimatized to form vital mature plants. Such plants often terms of plant morphology (plant height, leaf dimensions and express reduced vigor but in some crops such as onion, even flower morphology), plant vigor and fertility, number of haploid plants might grow vigorously, however at the chloroplasts and their size in stomatal guard cells. They are flowering stage, haploid plants form inflorescences with fairly unreliable and subject to environmental effects but do not evident malformations. Due to the absence of one set of require costly equipment. homologous chromosomes, meiosis cannot occur, so there is no seed set. Therefore, duplication of the chromosome Direct methods for ploidy determination are more robust and reliable and include conventional cytological techniques, such complement is inevitable in these plants. Generally, during the 20 synchronous division of two or more nuclei in early stages of as counting the chromosome number in root tip cells and measurement of DNA content using flow cytometry13. embryo development might leads to develop common spindle. The nuclear fusions might be associated with delayed cell wall The latter provides a rapid and simple option for large-scale formation which resulted chromosome doubling. ploidy determination as early as in the in vitro culturing phase.

Colchicine (originally extracted from autumn crocus It also enables detection of mixoploid regenerants (having cells Colchicum autumnale) is the most widely used chromosome with different ploidy) and the determination of their proportion. doubling agent, which inhibits microtubule polymerization by Several markers can be used for assessing the origin of binding to tubulin and ultimately inhibiting chromosome diploids, depending on their availability for a particular plant segregation. Although colchicine is highly toxic, used at a species. In the past, evaluation of regenerants mainly relied on millimolar concentration and known to be more efficient in phenotypic markers, progeny testing after self-pollination and isozyme analysis. plant tissues. Other options are treatment with oryzalin, amiprophosmethyl (APM), trifluralin and pronamide, all of Now-a-days, DNA molecular markers, such as AFLP which are used as herbicides and are effective in micromolar (Amplified Fragment Length Polymorphism), RAPD (Random concentrations. Colchicine application on anther culture Amplified Polymorphic DNA), SCAR (Sequence 36943 | P a g e Seeja, G and Sreekumar, S., Doubled Haploids in Genetic Improvement: A Review

Characterized Amplified Regions) or SST (Simple Sequence development of molecular markers provides an easier method Repeat), are commonly used for homozygosity testing and of selection based on the genotype (marker) rather than the assessment of plant origin. phenotype. Conventional methods combined with doubled haploidy selection becomes more effective. There is a considerable difference in interpretation between dominant or co-dominant electrophoretic profiles. Co-dominant In marker assisted backcross conversion, a recipient parent is molecular markers, as well as I sozyme markers, have the crossed with a donor line and the hybrid (F1) backcrossed to advantage that a single locus, when heterozygous in donor the recipient. The resulting generation (BC1) is backcrossed plants, might be used for homozygosity determination. In and the process is repeated until the desired genotypes are contrast, a more complex profile is analyzed with dominant produced. The combination of doubled haploidy and molecular markers. In such a case, bands missing from the donor profile marker provides less time for the development of homozygous indirectly indicate homozygosity. A fast and reliable haploid lines23. In barley, marker assisted backcross conversion with identification method is needed for large scale production of doubled haploidy of BC1 individuals was applied to select DHs. Morphological markers expressed at the embryo, seed or stripe rust resistant lines24. early seedling stages are preferentially used. In maize, the most Bulked Segregant Analysis (BSA) efficient haploid identification marker is the ‘red crown’ kernel trait, which causes deep pigmentation of the aleurone layer in BSA is a popular method in marker assisted breeding. the crown region (endosperm) and scutellum (embryo tissue)[9]. Population is screened for a trait of interest and the genotypes at the two extreme ends form two bulks. These are tested for DH plants are normally selfed for maintenance and for further the presence or absence of molecular markers. DH population, multiplication. In cross-pollinated species with strongly the genotypes at the two extreme ends form two bulks and expressed self-incompatibility, various techniques are used to hence can be tested accurately and repeatedly. So DH overcome the incompatibility reaction. In Brassicas, bud populations are commonly used in bulked segregant analysis. pollination is enhanced by treatment in a CO2 enriched 21 This method has been applied in rapeseed and barley. atmosphere or by application of gibberellic acid, sodium chloride, urea or ammonium sulphate on stigmas22. Genetic Maps Alternatively, DH lines might be clonally propagated, in which Genetic maps used to understand the structure and organization case micropropagation is often the best choice. of genomes from which evolution patterns and syntenic Factors Affecting Haploid Induction and Subsequent relationships between species can be deduced. It provides a Regeneration of Embryos framework for the mapping of genes of interest and estimating the magnitude of their effects and used in understanding of Genotype of the donor plants, physiological condition of donor genotype/phenotype associations. In DH populations it is plants (i.e., growth at lower temperature and high illumination), possible to produce a genetic map within two years of the developmental stage of gametes, microspores and ovules, pre- initial cross in any type of species. DH populations have now treatment (i.e., cold treatment of inflorescences prior to culture, been used to produce genetic maps of barley, rapeseed, rice, hot treatment of cultured microspores), composition of the wheat, and pepper. culture medium (including culture on “starvation” medium low with carbohydrates and/or macro elements followed by transfer Genetic Studies to normal regeneration medium specific to the species), Genetic ratios and mutation rates can be read directly from physical factors during tissue culture (light, temperature, etc). haploid populations. A small doubled haploid (DH) population Genetics of Doubled Haploid Population was used to demonstrate that, a dwarfing gene in barley is located chromosome 5H. In another study, the segregation of a In DH method only two types of genotypes occur for a pair of range of markers has been analyzed in barley. alleles, A and a, with the frequency of ½ AA and ½ aa, while in diploid method three genotypes occur with the frequency of ¼ Genomics AA, ½ Aa, ¼ aa. If n loci are segregating, the probability of QTL analysis generate a vast amount of information on gene getting the desirable genotype is (½)n by the haploid method locations and the magnitude of effects on many traits, the and (¼)n by the diploid method. Hence the efficiency of the identification of the genes involved has remained elusive. This haploid method is high when the number of genes concerned is is due to poor resolution of QTL analysis. Here, backcrossing is large. Adoption of doubled haploidy does not lead to any bias carried out until a desired level of recombination has occurred of genotypes in populations23. and genetic markers are used to detect desired recombinant Application of Doubled Haploids in Plant Breeding chromosome substitution lines in the target region, which can be fixed by doubled haploidy. In rice, molecular markers have Mapping of Quantitative Trait Loci been found to be linked with major genes and QTLs for Quantitative traits are controlled by genes with small but resistance to rice blast, bacterial blight, and sheath blight in a cumulative effects. DH use in identifying loci controlling map produced from DH population. quantitative traits. It is because of their true breeding nature 23 Elite Crossing and it can conveniently be produced in large numbers . Traditional breeding methods are slow and take 10–15 years Backcross Breeding for development. It has inefficiency of selection in In backcross conversion, genes are introgressed from a donor early generations because of heterozygosity. These two cultivar or related species into a recipient elite line. The 36944 | P a g e International Journal of Recent Scientific Research Vol. 11, Issue, 01(D), pp. 36941-36949, January, 2020 disadvantages can be overcome by DHs, and more elite crosses base material for Targeting Induced Local Lesions in can be evaluated and selected within short time. Genomes (TILLING) populations. Cultivar Development  Production of stable transgenic plants and reverse breeding. Uniformity is a general requirement of cultivated line in most Disadvantages of DHs species, which can be easily obtained through DH production. There are various ways in which DHs can be used in cultivar  Selection cannot be imposed on the population. But in production. The DH lines themselves can be released as conventional breeding selection can be practised for 25 cultivars. More than 200 varieties have been developed . It can several generations; thereby desirable characters can be be used as parents in hybrid cultivar production, creation of improved in the population. breeder lines and for germplasm conservation. Doubled  In haploids produced from anther culture, it is observed haploidy already plays an important role in hybrid cultivar that some plants are aneuploids and some are mixed production of vegetables, and the potential for ornamental haploid-diploid types. So identification of the ploidy production is being vigorously examined. level is important.

According to published information there are currently around  Induction of DH through non-conventional method 300 DH derived cultivars in 12 species worldwide. Barley has (tissue culture) involve high cost for facility over 100 direct DH cultivars26. The relevance of DHs to plant establishment. breeding has increased markedly in recent years owing to the  The over-usage of doubled haploidy may reduce genetic development of protocols for 25 species. DHs are also being variation in breeding germplasm. Hence one has to take developed in the medicinal herb Valeriana officinalis to select several factors into consideration before deploying lines with high pharmacological activity. Another development doubled haploidy in breeding programmes. In fact, under is that fertile homozygous DH lines can be produced in species optimal conditions, doubled haploids (DH) have been that have self-incompatibility systems. routinely used in breeding for several decades, although their common use is still limited to selected species. Advantages of DHs Reason is biological, based on plant status (annual,  The ability to produce homozygous lines after a single biannual, perennial, autogamous, allogamous, round recombination saves a lot of time for the plant vegetatively propagated) and flower morphology or breeders. technical, which are the result of the feasibility and  Efficient in genetic analysis and development of markers efficiency of DH induction protocol. for useful traits in much less time.  DH induction protocols substantially vary not only  The possibility of seed propagation as an alternative to among species but also among genotypes of the same species. vegetative multiplication in ornamentals.  In tree species with long life cycles and inbreeding Achievements depression preclude traditional breeding methods, doubled haploidy provides new alternatives. DH varieties have been developed via various methods in all over the world and severaln species such as asparagus, barley,  Possibility of screening breeding material for the mustard, eggplant, melon, pepper, rapeseed, rice, tobacco, presence of advantageous genes. triticale, wheat etc. commercial cultivar production was  Giving an immediate product of stable recombinants achieved. Perusal of the literature indicated that more than 290 from species crosses or fixation of heterotic DH varieties have already been released combination. (http://www.scri.ac.uk/assoc/). The list comprises some of the  No masking effects because of high homogeneity. High commercially exploited DH varieties from various countries efficiency in stacking specific targeted genes in and the DH inductions successfully achieved through various homozygous line. methods are given in table 1 and 2 respectively.  Increased performance per se due to selection pressure in the haploid phase or during first generation of DHs. Table 1 List of doubled haploid varieties developed in various  For self-incompatible species, dioecious species and crops

species that suffer from inbreeding depression due to Haploid production Crop Varieties Country self-pollination, haploidy may be the only way to method develop inbred lines. Tanfeng 1, Tan Fong 1, Hua Yu 1, Hua 03, Anther culture China  Simplified logistics for seed exchange between main and Xin Xiu, Xhongua 8, off season programmes since each line is fixed and can Ta Be 78, Guan 18 be represented by a single plant. Anther culture Dama Hungry Rice  Natural selection on haploids can be utilized as a genetic Anther culture Parag 401 (ACR 401) India CR Dhan 801 (CRAC alter to identify or remove harmful mutant genes. Anther culture 2224-1041; IET CRRI,India  Used to transfer sterile cytoplasm of CMS lines into a 18720) desirable background in a single step avoiding many Anther culture Patei and Moccoi Argentina generations of backcrossing. Hua Pei 1, Lung Hua Anther culture 1, Jinghua 1, Yunhua China  To study inheritance of quantitative traits, QTL 1, Yunhua 2 Wheat mapping, functional genomics, gene identification, as Anther culture Kharoba Morocco Anther culture Florin France Wheat x Maize Glosa, Faur F, Liter, Romani 36945 | P a g e Seeja, G and Sreekumar, S., Doubled Haploids in Genetic Improvement: A Review

Miranda raised in vitro for breeding multiple virus resistance and Tan Yu 1, Tan Yu 2, Anther culture China treatment of colchicine among 167 micropropagated haploid Tobacco Tan Yu 3, Anther culture F211 Japan shoots resulted10 diploid and 100 mixoploid plants. High Barley H. bulbosum Mingo, Gwylan Canada percentages of viable pollen when stained with acetocarmine Table 2 Induction of double haploids through various was found not only in the diploids but also in >60% of the 39 methods in different crops plants scored as mixoploid or haploid by flow cytometry .

Sl. No. DH produced crops SI.No DH produced crops Fruit crops -Musa balbisiana (BB) DH via callus DH via androgenesis 1 Cynara scolymus27 34 Hordeum vulgare 68 The production of 41 haploid (n=x=11) plants from anther 2 Ephedra foliata27 35 Solanum tuberosum69 43 27 70,71 culture of banana [Musa balbisiana (BB)], was reported , of 3 Solidago virgaurea 36 Triticum aestivum L. DH via callus and plants 37 Secale cereale72 these, 18 from the genotype Pisangklutuk, 12 from Pisangbatu, 4 Psoralea corylifolia27 38 Brassica napus73 7 from Pisangklutukwulung and 4 from Tani. The frequency of 5 Valeriana officinalis27 39 Chrysanthemum74 75 callus induction was 77% and about 8% of anthers developed DH via anther culture 40 Petunia 6 Prunus persica (L.) Batsch28,29,30 41 Camalia76 embryos after 6 months of culture. The frequency of embryo 7 Citrus31 42 Dianthus caryophyllus L.77 formation was genotype-dependent. 8 Prunus armeniaca L.32 43 Zantedeschia sp.78 33,34 79 9 Carica papaya 44 Lupinus Carica papaya L 35 80 10 Vitis 45 Primula (Baby primrose) 11 Pyrus36,37 DH via gynogenesis The main breeding systems of papaya, a polygamous species, 12 Cucumis38,39 46 Actinidia deliciosa81 40 82 using true-bred lines, benefit greatly from haploid induction 13 Allium cepa L. 47 Morus alba Citrullus lanatus (Thunb.) Matsum. through anther culture. Haploid plantlets and pollen-derived 14 DH via microspore &Nakai41 embryos were obtained from papaya anthers cultured at the 15 Musa42,43 48 Helianthus annuus L.83 105 44 84 uninucleate stage . 16 Rubus 49 Helianthus annuus L. 17 Cucumber45,46 50 Rosa85 47 86 Medicinal plants - Chlorophytum 18 Prunus 51 Vaccaria 19 Cucurbita48 52. Diplacus aurantiacus87 49 88 Haploids and DH in Chlorophytum borivilianum Santa Pau 20 Prunus avium (L.) L. 53. Zantedeschia 21 Olea europaea L.50 54. Ornamental Brassica89 &Fernandes, an endangered medicinal crop, was achieved 51 through in vitro anther culture and out of 9% rooted plantlets 22 Hepatica 55. Hibiscus sabdariffa L.90 isolated 5% of them were haploid and 4% DH106. (Prabha 23 Citrus reticulata Blanco52 56. Anemone 91 24 Anthurium53,54 57. Iris92 et.al., 2012). DH via ovule culture DH via irradiated pollens 25 Gerbera 55,56 58. Pyruscommunis36,93 Ornamental plants – Orchids 26 Spathiphyllum57 59. Citrus 94,95,96 27 Chrysanthemum58 60. Actinidi adeliciosa97,98 An US patent(US 2005/0289663 A1) was filed to induce 28 Tagetes erecta L.59 61. Malus domestica99,100 haploids in orchids (Cattleya) by dropping any one of the 101 DH via androgenesis 62. Theobroma cacao auxins such as Indoleacetic acid (IAA), 4-chloro-indoleacetic 29 Lily 60 DH via embryo/plant 30 Oryza sativa61 63. Gentian triflora27 acid, phenylacetic acid, 2,4-dichloro-phenoxyacetic acid (2,4- 31 Nicotianatabacum62,63,64 64. Gentian102 D), C.-naphthaleneacetic acid (NAA), 2,6-dichloro-benzoic 65 27 32 Brassica oleracea 65. Hyoscyamussp acid, indolebutyric acid (IBA), 4-chloro-phenoxyacetic acid, 5- 33 Asparagus officinalis66 66. Fagopyrum esculentum27 chloro-indazole ethyl acetate and 2,4,5-trichloro-phenylacetic acid between concentration 0.1 to 5.0% on unfertilized flowers Case studies which induced parthanogenesis and production of haploid Rice plants.

In vitro production of DH plants through anther culture in two CONCLUSION AND FUTURE PERSPECTIVES rice species (Oryzasativa L. and Oryzaglaberrima Steudt.) was achieved for the rapid development of new breeding material. Doubled haploids is emerging as a powerful tool to enhance All O. glaberrimag enotypes areregenerated to plants whileO. genetic gain per cycle, which is of more relevance in Sativag enotypes, only japonica variety IKP produced plants. horticultural crops that are either perennial in nature or Many albino plants were obtained from the culture. Only one outcrossing with inbreeding depression or with great O. glaberrimag enotype (6202 Tog) produced green plants. commercial value where breeding duration is critical. Overall, Total of 93 plants were regenerated with 14 green plants and 79 there is a growing interest in haploidy for horticultural crop albino plants. Anther culture response is largely species and improvement in India and the prospects for utilization of this 103 technology appear to be bright. genotype dependent .

Vegetables - Onion and Melon References

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Seeja, G and Sreekumar, S.2020, Doubled Haploids in Genetic Improvement: A Review. Int J Recent Sci Res. 11(01), pp. 36941-36949. DOI: http://dx.doi.org/10.24327/ijrsr.2020.1101.5029

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