The effect of colchicine on triticale anther-derived plants: Microspore pre-treatment and haploid-plant treatment using a hydroponic recovery system

In cereals, chromosome doubling of microspore-derived haploid plants is a critical step in producing doubled haploid plants. This investigation was undertaken to study the effect of incorporation of colchicine in the induction medium for anther culture, and the effect of colchicine on anther culture-derived plants of triticale grown under controlled greenhouse conditions. In the latter case, chromosome doubling of adult sterile plants derived from anther culture of fourteen triticale populations was attempted, where androgenetic plants with non-dehiscent anthers were cloned and subjected to the colchicine treatment, and then grown with the aid of hydroponics. The hydroponic system provided optimal conditions for recovery of the affected haploids from the toxic effects of colchicine treatment and all colchicine-treated plants survived. A topcross-F₁ (TC₁F₁) population with timopheevii cytoplasm produced the highest percentage of plants with seed-set either due to chromosome doubling by colchicine (98%) or spontaneous doubling of chromosome number (15%). Colchicine-treated anthers performed inferior than control in both induction and regeneration phases. One of the key observation of this study was the reversal from reproductive stage back to the vegetative stage which in turn enabled further cloning of haploid plants under hydroponic conditions once they were identified as sterile. The one hundred percent survival rate of in vitro-derived plants, 100% survival rate of colchicine treated haploid plants and the high chromosome doubling success rate (X = 82.3) observed in this study imply that a temperature-controlled greenhouse with an hydroponic system provides an efficient environment for inducing chromosome doubling of haploid plants in cereals. This revised version was published online in August 2006 with corrections to the Cover Date.     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. I. Ability of 1B—-1R wheat-rye translocation forms for androgenesis 45 winter wheat varieties or F₁ hybrids, F₂ populations and lines with 1B—1R wheat-rye translocation were tested for their anther culture ability. A total of 48058 anthers was cultured on Potato-2 medium. When averaged over all genotypes and the two experimental years frequencies of embryoid formation of 5.4—6.8 per 100 anthers were observed. Plant regeneration efficiency from embryoids ranged from 5.3—9.1 % or a mean of 4—5 green plants per 1000 anthers plated. The results confirmed the preferential regeneration frequency of gametes with the 1BL—1RS chromosome compared to the gametes with the 1BL—IBS chromosome. Multiple peroxidase were used as marker. The effect of cold pretreatment or of media on the androgenetic response and productivity was not important. On the contrary the variability between the anther response from single ears of the same genotype was noticeable. Examples are presented for the transferability of the androgenetic ability to breeding material. Most green plants obtained were haploid or spontaneous doubled haploid. By cloning it was guaranteed, that progenies were obtained from most of the haploids after colchicine treatment.     

Comparative analysis of in vitro anther- and isolated microspore culture in hexaploid Triticale (X Triticosecale Wittmack) for androgenic parameters

Two haploid induction media (190-0 and W14mi) were tested in isolated microspore culture of two triticale (X Triticosecale Wittmack) genotypes. The W14mi medium proved superior for the production of green plantlets in both genotypes. This basic medium (W14) was used to compare two doubled haploid production methods (isolated microspore culture and anther culture) with the same genotypes. The induction of androgenesis was more effective in isolated microspore culture than in anther culture. The number of embryo-like structures was 9.2 times higher in microspore culture (511.0/100 anthers) compared to anther culture (55.5/100 anthers) and the number of regenerant plantlets was also 3.4 times higher (anther culture—20.15/100 anthers; isolated microspore culture—67.6/100 anthers). However, the regenerant plantlets from isolated microspore culture were mainly albinos while predominantly green plantlets were regenerated from anther culture. The production of green plantlets from anther culture (16.8/100 anthers) was 2.9 times higher than from isolated microspore culture (5.8/100 anthers). The efficiency of anther culture was tested with eight winter triticale genotypes. The phenomenon of albinism did not hinder the green plant production in anther culture. Mean green plantlet production was 10.87/100 anthers. This value was two times higher than the number of albinos (5.01/100 anthers) and higher than previously published reports. The anther culture protocol described in this study is an efficient tool for the production of microspore-derived green plantlets in triticale.     

Spike pretreatments, anther culture conditions, and anther culture response of 17 German varieties of spring wheat (Triticum aestivum L.)

The aim of this work was to establish an in vitro regeneration system from anther cultures of different German varieties of spring wheat (Triticum aestivum L.). Using ‘Nandu’ the most widely grown spring wheat cultivar in Germany, different culture conditions were investigated with regard to their influence on anther culture response. The best results were obtained when applying a cold pretreatment to the donor spikes and using the synthetic L3 induction medium, liquid or solidified with gelrite. The highest rates obtained in these experiments with ‘Nandu’ were 8.6% responding anthers, 22.3% embryoid induction, 15.3% albino regeneration and 5.5% green plant regeneration (all rates related to the number of cultured anthers). Of the ‘Nandu’ plants analysed, 51.1% were haploid and 44.3% were diploid, probably as a consequence of spontaneous chromosome doubling. When screening a further 16 commercial German varieties of spring wheat, 10 exhibited good anther culture response and four of these (‘Eta’‘Jondolar’, ‘Mieka’, and ‘Star’) proved to be highly responsive, reaching embryoid induction rates between 4.3 and 10.3% and rates of green plant regeneration between 5.4 and 10.7%.     

Plant regeneration from anther culture in Canadian cultivars of flax (Linum usitatissimum L.)

The effect of culture of anthers at 35 °C for one to four days prior to culture at 25 °C in darkness, genotype, anther orientation on callus induction and shoot regeneration in anther culture of flax was investigated. The influence of type and concentrations of cytokinins in the regeneration medium on shoot regeneration was also investigated. The results suggested that culture of anthers at 35 °C prior to continuous culture at 25 °C in darkness did not significantly improve the percentage of anthers producing calli. However, culture of anthers at 35 °C for one day significantly increased the overall efficiency of regeneration compared to no culture temperature treatment. Genotypic effects were significant for the percentage of anthers producing calli and the overall efficiency of regeneration. Anther orientation showed no significant differences. The regeneration medium containing 4.5 μM zeatin had significantly higher percentage of calli forming shoots than the same basal medium containing 0.01 μM TDZ. The importance of these findings for flax breeding was discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Production of Doubled Haploids by Anther Culture and Wheat X Maize Method in a Wheat Breeding Programme

Utilization of the doubled haploid method of breeding usually shortens the time to cultivar release, and methods of haploid production need evaluation in a breeding programme. Thirty-eight different three-way crosses were tested for anther culture response. On average 5.8 percent of the anthers cultured produced calli. Three crosses were found recalcitrant for callus induction. Overall, the anther culture method produced 0.6 plantlet per 100 anthers cultured. Five crosses with an average of 5.8 and 2.8 percent of anthers producing calli and plantlets, respectively, were compared using anther culture and wheat × maize crosses. Non-responsive genotypes for callus induction and plantlet formation in the anther culture method proved to be good parental material in wheat × maize crosses. The average percentages of embryo formation and plantlet production in wheat × maize crosses were 10.3 and 4.7, respectively. Anther-derived plants were cytologically unstable, whereas all the plants regenerated from wheat × maize crosses were haploids (n = 21 chromosomes). The chromosome numbers of the polyhaploids were doubled with a colchicine treatment. Improvement of the two haploid production methods to facilitate their efficient use in a breeding programme is discussed.     

An improved protocol for androgenesis in cauliflowers (Brassica oleracea var. botrytis)

The genotypic responsiveness to androgenesis and the effect of two exposure times of cultured anthers to auxins (12 days and the entire period of culture) were studied in three lines of cauliflower (one spring - and two winter-types). The anthers of all genotypes responded to the protocol by producing embryos (0.2 -25.3%), 44.2% of which regenerated plantlets through organogenesis on a regulator-free B5 medium containing 40 g/l of sucrose and 800mg/l of L-glutamine. Embryo yield improvement, recently achieved on solid medium with the addition of 125mg/l silver nitrate, was not observed in liquid cultures. Mean frequencies of androgenic embryos, as affected by the duration of auxin supply to the culture, were always significantly higher (6.6- and 8.1-fold.) in the‘12 days of exposure’treatment than in the‘entire period of culture’treatment. The resolution of the parameter‘embryo percentage’into its two components (% of responsive anthers and number of embryos per responsive anther) gave evidence of a highly significant increase in the percentage of responsive anthers, whereas the number of embryos per responsive anther did not change statistically. The percentage of embryos regenerating plants was not influenced by the duration of auxin supply during anther culture. Chromosome count analysis revealed a haploid/diploid/polyploid plant ratio of 6:79:15. Of the 78 androgenic plants (RO generation) grown in the field, only 14 were fertile and produced seeds. RAPD analysis showed that none of the seven polymorphic loci out of 23 analysed expressed polymorphism within the RI progenies of 11 anther culture-derived lines.     

Androgenic capability among genotypes of winter and spring barley

Variable efficiency of androgenesis remains a serious problem in many species of cereals. It is still unclear what makes certain genotypes more amenable to androgenesis than others. This study was undertaken to quantify the previously suspected advantage of winter barley genotypes over spring ones with regard to regeneration efficiency in anther culture. The material consisted of 40 barley hybrids originating from Polish breeding companies. The number of androgenic structures per 100 anthers did not differ significantly between analysed groups (119 vs. 152 non‐significant), but the average regeneration of green plants per 100 anthers was five times higher in winter genotypes (6.4 vs. 1.3). The incidence of albinism was lower for the winter than for the spring materials (70% vs. 90%), while the rate of spontaneous chromosome doubling was similar in both groups (58% vs. 56%). The results strongly support the notion that winter genotypes are more amenable to androgenesis and this may be a consequence of their better adaptation to stress conditions.     

开发实用的染色体加倍体系构建成烟草DH群体

通过对4种染色体加倍方法(烟草浸花药法、浸花培苗法,叶片再生法及浸腋芽法等)的比较研究表明：以4g／L浓度的秋水仙碱浸泡花药法加倍率最高达47．5％～75％,其次为叶片再生法(30％～36．67％)和浸苗法(16．67％～32．35％),而浸腋芽法较低(17．78％)。前两种方法加倍率虽高,但有较高畸形苗比率,叶片再生法工序较繁琐。作者认为烟草加倍单倍体产生,应以采用浸苗法为主。在使用秋水仙碱浸苗加倍时,添加DMSO可明显促进秋水仙碱的加倍效率,且促进作用随时间延长而提高：从高效、快捷和节约等原则考虑,我们开发了烟草有效的染色体加倍体系,以4g／L的秋水仙碱 20g／LDMSO溶液浸苗48h的效果最好。对两个组合烟草单倍体苗浸12h以上,其染色体加倍效率达到对照的2．30-2．93倍。本试验用较低浓度秋水仙碱添加DMSO有利于节约实验费用。通过完善染色体加倍技术程序已构建成2个DH群体,供基因定位研究。     

葫芦科植物单倍体离体诱导研究进展

重点对葫芦科植物离体诱导单倍体的研究进行综述,包括花药培养、离体雌核培养、辐射花粉诱导单倍体培养等在育种中的应用,以及供体基因型、培养基、预处理方式、胚囊发育时期等因素对离体诱导再生频率的影响。关于离体培养发育机制的生理生化研究主要集中在离体雌核发育的形态及细胞学过程以及培养早期生理、生化的变化研究。同时总结了葫芦科植物DH育种研究进展,指出葫芦科植物离体诱导单倍体研究中存在的问题并提出思路和建议     

Die Nutzung der Antherenkulturmethode im Zuchtprozeß von Winterweizen

Anther culture in the breeding process of winter wheat. III. Ability of winter wheat F₁ populations with the two heterozygous 1AL–IAS/1AL–IRS and 1BL–1BS/1BL–IRS chromosome pairs Application of anther culture to four F₁ hybrids between the IBL–IRS (‘Amigo’) and several 1BL–IRS wheat-rye translocation forms yielded 129 green pollen plants in an average embryo induction frequency of 17.6 %. A total of 2632 anthers was inoculated. 25 % and 42 % of the regenerated plants were haploid and spontaneously doubled haploid, and 33 % had abnormal chromosomal structure. After chromosome doubling treatment 87% of all pollen plants set seeds. By means of multiple peroxidases and Giemsa C-banding patterns, the anther culture progeny could be further classified into 16 plants without the short arm of IR-chromosome of rye, 21 IAL–IRS and 50 1BL–IRS translocation lines and into 16 IAL–IRS, IBL–IRS double translocation lines according to the four possible characteristic types of F₂ gametes of the tested F₁ hybrids. Advantages of the haploid technique for the selection of desirable traits and the meaning of the IRS genes in wheat are discussed.     

Relative Efficiency of Anther Culture and Chromosome Elimination Techniques for Haploid Induction in Triticale × Wheat and Triticale × Triticale Hybrids

Summary The study was undertaken to evaluate the relative efficiency of anther culture and chromosome elimination (by crosses with maize) techniques of haploid induction in intergenotypic triticale and triticale × wheat hybrids. For this, 15 triticale × wheat and 8 triticale × triticale F₁ hybrids were subjected to anther culture and were also simultaneously crossed with the `Madgran Local' genotype of maize (Zea mays L.) to induce haploids through the chromosome elimination technique. The haploid embryo formation frequency through the chromosome elimination technique was significantly higher in both, triticale × wheat (20.4%) and triticale × triticale (17.0%) F₁ genotypes, as compared to the calli induction frequencies through anther culture (1.6 and 1.4%, respectively). Further, four triticale × wheat and three triticale × triticale F₁ genotypes failed to respond to anther culture, whereas, all the F₁ genotypes formed sufficient number of haploid embryos through the chromosome elimination technique with no recovery of albino plantlets. The haploid plantlet regeneration frequencies were also significantly higher through the latter technique in both triticale × wheat (42.7%) and triticale × triticale (49.4%) F₁s as compared to anther culture (8.2 and 4.0%, respectively), where the efficiency was drastically reduced by several constraints like, high genotypic specificity, low regeneration frequency and albinism. The overall success rates of obtaining doubled haploids per 100 pollinated florets/anthers cultured were also significantly higher through the chromosome elimination technique (1.1% in triticale × wheat and 1.5% in triticale × triticale hybrids), proving it to be a highly efficient and economically more viable technique of haploid induction as compared to anther culture, where the success rates were only 0.2% and 0.1%, respectively.     

Individual reaction of <Emphasis Type="Italic">Capsicum</Emphasis> F<Subscript>2</Subscript> hybrid genotypes in anther cultures

The present study evaluated the individual plants reaction of F₂ hybrid generation of C. annuum: ATZ1 × PO and ATZ1 × CDT as well as two interspecific hybrids: C. frutescens × C. annuum ATM1 and C. frutescens × C. chinense on androgenesis conditions in in vitro anther cultures. The experiment was carried out following a modified method of Dumas de Vaulx et al. (Agronomie 1:859–864, 1981). There were demonstrated clear differences in the effectiveness of androgenesis both between the pepper hybrid forms as well as among individual plants of all the genotypes tested. The highest effectiveness of androgenic embryos development was observed for the cultivated form of C. annuum: (ATZ1 × PO)F₂. Anthers of most of the plants of this hybrid produced embryos at the level higher than 5%, while in anther cultures of the second C. annuum hybrid (ATZ1 × CDT)F₂ almost 3-fold fewer embryos and plants were produced. Anthers isolated from flower buds of interspecific hybrids formed much lower number of embryos. A positive reaction was recorded for five hybrid plants of (C. frutescens × C. annuum ATM1)F₂, while in case of (C. frutescens × C. chinense)F₂ androgenic embryos were obtained from anthers of two plants. Only in the case of a one of these plants did the effectiveness of androgenesis exceed 5%. The ploidy level of the regenerants was determined by flow cytometry. Among the regenerants there were observed both haploid forms and the plants with the diploid number of chromosomes.     

基因型和环境条件对小麦花药培养效果的影响

为进一步提高小麦花培育种效率,明确花药培养力的遗传控制基础,以11个小麦品种及其组配的20个F₁杂种为材料,探讨了基因型、培养基和环境条件对愈伤组织诱导率的影响。在W14D、W14gD、W14GD培养基上,Alondra、Verry、石4185、新春9号和百农3217的花药易被诱导产生愈伤组织,诱导率为25.3%~51.9%,其中石4185是目前公认的花培育种优良亲本,新春9号为新发现的优良花培基因型。以宁春4号配制的部分F₁杂种的愈伤组织诱导率较高,大多数组合高于10.0%,表明宁春4号与供试品种间具有较高的花药培养配合力。小麦花培育种技术要求亲本之一具有较高的花药愈伤组织诱导率或较高的花药培养配合力。小麦花药培养力的遗传控制复杂,表现为数量性状遗传,亲本花药培养力很高,其F₁组合花药培养力不一定很高,这与双亲配合力有关。小麦花药培养中,供体植株生长和愈伤组织诱导的适宜条件为较长的营养生长期、适宜的前期(分蘖期)温度和较高的中期(拔节后期)温度。在添加低浓度生长素和葡萄糖的液体培养基中发现小麦花药直接成苗现象,2,4-D诱导花药直接成苗效果优于Dicamba。随着年度间气候升高的影响,相同基因型花药愈伤组织诱导率呈现增加趋势。     

Chromosome doubling of androgenic haploid plantlets of rice (Oryza sativa) using antimitotic compounds

The regeneration of haploid plantlets is considered as a bottleneck in rice anther culture. In this study, an antimitotic chromosome doubling method, simple and efficient, of androgenic haploid plantlets resulted in an efficient doubled haploid obtainment. Through chromosome doubling capacity comparison of the three antimitotic compounds (colchicine, trifluralin and oryzalin), colchicine at 500 and 625 mg/L without supplementing with DMSO was found to be the best antimitotic treatment, with a chromosome doubling capacity of 40%. Furthermore, the in vitro growth of plantlets was followed to analyse the effects of antimitotic compounds. Colchicine treatments were more toxic than dinitroanilines, and colchicine DMSO-supplemented treatments had significant lower values on shoot growth. On the other hand, dinitroaniline compounds impeded root growth, provoked helical growth of shoot and caused the apparition of white nodules in the base of the plantlet due to sprouting abortion. In this study, a protocol for doubled haploid plant recovery was established taking advantage from androgenic haploid plantlets in order to increase the number of doubled haploid plantlets produced after an anther culture protocol.     

Identification of proteins related to microspore embryogenesis responsiveness in anther cultures of winter triticale (×<Emphasis Type="Italic">Triticosecale</Emphasis> Wittm.)

For a better understanding of the physiological background of microspore embryogenesis (ME), the protein profile was analyzed in four winter triticale DH lines, which show extremely different embryogenic potential. The analysis were conducted with anthers at the phase of development optimal for ME induction and then after low temperature (LT, 3 weeks at 4 °C) ME-inducing tillers treatment. The sub-proteome of anthers was mapped by two-dimensional gel electrophoresis (2-DE). The protein species significantly more abundant (at least 2-fold) in responsive DH lines after LT treatment were chosen for identification by MALDI-TOF/TOF analysis. In total, 31 protein species were successfully identified as involved in the determination of microspore competence, stress response and in the regulation of ME induction. Microspore competence required sufficient energy supply and efficient system of cell protection that determine survival under prolonged LT stress treatment. LT stress was associated with increased accumulation of proteins typical for cell defence against oxidative stress (e.g., l-ascorbate peroxidase), chaperons (e.g., HSP70) and other enzymes/factors ensuring protein biosynthesis, stability and active cell divisions. Also here, effective cell defence required undisturbed energy supply. Among proteins that accumulated differentially in accordance with microspore embryogenic potential again the most important role seems to be played by the enzymes ensuring energy production and determining ability of plant stress adaptation. Two protein species (enolase, 12S storage protein), proposed earlier as candidates for markers of embryogenesis in other in vitro plant culture systems confirmed their utility for triticale anther cultures.     

Genetic,quantitative and microscopic evidence for fusion of haploid nuclei and growth of somatic calli in cultured <Emphasis Type="Italic">ms10</Emphasis><Superscript><Emphasis Type="Italic">35</Emphasis></Superscript> tomato anthers

In plant breeding, androgenic doubled haploids represent powerful tools to save time and resources for pure line generation. While in many species efficient protocols are known, in tomato (Solanum lycopersicum), the knowledge on the induction of androgenesis is still very scarce, and little is known about the particularities of this highly recalcitrant species. The only known method capable of yielding haploid/doubled haploid tomato plants is anther culture. However, this method has important limitations, including low efficiency of haploid induction and a low proportion of spontaneously doubled haploids. To understand these limitations better, we have analyzed the process of callus formation in anthers of tomato lines carrying the ms10 ³⁵ gene for male-sterility, using light and electron microscopy, flow cytometry and genetic analysis with morphological and molecular markers. Our results demonstrate that haploid, doubled haploid and diploid calli occur in tomato anthers, although at different frequencies. Diploid calli derived either from somatic cells or from the fusion of two genetically different haploid nuclei account for more than 90% of the total of calli produced. Somatic calli are derived from the stubs of connective tissue present in the interlocular septa of anthers. This growth is markedly increased in the ms10 ³⁵ mutants, which explains their higher callogenic rates than standard tomato lines. Together, our results reveal serious drawbacks that explain the low efficiency of anther-derived, doubled haploid production in tomato, and stress the need for alternatives towards doubled haploidy.     

Influence of genotype and culture conditions on the production of embryos from anthers of tetraploid wheat (Triticum turgidum)

Summary Anther culture of 10 tetraploid wheat (Triticum turgidum) genotypes and two backcross lines representing a wide range of genetic variation was studied in a randomized block design with three replications. Each replication consisted of 2 pots with 3 plants. The day length was 16h and temperature 25° C/15°C for day/night in a controlled greenhouse where the anther donor plants were grown. Two different treatments were used for anther culture. The first one was potato 2 medium (Chuang et al., 1978) modified by adding 0.5 mg/l glutamine and solidified by gelrite (4g/l) (Henry & De Buyser, 1981). Cultures were incubated in light (15 E m^–2 S^–1) at 26°C at 16h day length. The second medium was described by Fadel & Wenzel (1990), differing from the first by the nature of the sugar (maltose) and consistency of the medium (semiliquid by ficoll). Anther cultures were incubated in the dark at 28°C. The study of about 1300 anthers per genotype and treatment showed that both genotype and treatment affected embryo formation of tetraploid wheat. The backcross lines exhibited significant differences for androgenic abilities when compared to their common parent. Most of the genotypes were medium dependent for androgenesis and revealed significant interactions with the two treatments. Five green plantlets were regenerated and fertile doubled haploid plants were obtained from three out of the 12 studied genotypes.     

High frequency of plant regeneration from anther culture in flax, Linum usitatissimum L.

The effects of induction medium compositions on flax anther culture were investigated in order to improve the efficiency of callus induction and plant regeneration. Anthers were inoculated onto the modified MS medium supplemented with five different combinations of plant growth regulators. The medium containing the combination of 2mg/l 2,4- dichlorophenoxy-acetic acid (2,4-D) and 1 mg/1 6-benzylaminopurine (BAP) produced a significantly higher number of calli forming shoots/100 responded anthers and a significant increase in overall efficiency of regeneration than the same basal medium containing 1 mg/1 a-naphthalene-acetic acid (NAA) and 2 mg/1 BAP (CK). Among the five levels of thiamin hydrochloride tested, the modified MS medium containing 10 mg/1 thiamin hydrochloride significantly increased the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration compared with the same basal medium containing 0.1 mg/1 thiamin hydrochloride. Maltose concentration had a significant effect on the percentage of anthers producing call, the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration. The medium containing 6% or 9% maltose produced the highest overall efficiency of regeneration among the five levels of maltose evaluated. Sucrose concentration significantly affected the number of calli forming shoots/100 responded anthers and the overall efficiency of regeneration, and dramatically affected the frequency of microspore-derived plants and the frequency of spontaneous chromosome doubling in microspore-derived plants. The efficiency of doubled haploid line production obtained in this study appears adequate for applied breeding programmes.     

Improving androgenesis‐mediated doubled haploid production efficiency of FCV tobacco (Nicotiana tabacum L.) through in vitro colchicine application

Production of doubled haploid plants through androgenesis in flue‐cured Virginia (FCV) tobacco is a promising and convenient alternative to conventional selfing techniques for the generation of absolute homozygous lines. Here, we show a robust in vitro haploid and doubled haploid development protocol in FCV tobacco with major emphasis on improving the efficiency of chromosome doubling using in vitro colchicine treatment. We used five FCV tobacco hybrids for comparison of colchicine treatments. The anther culture response varied with developmental stages of the buds, and the highest response was observed in stage 2 buds. The effect of cold pretreatment was significant, and 4 days of pretreatment was optimum for gametic embryogenesis. Among the methods used for determining the ploidy status of plants, flow cytometry was found to be easy, fast and reliable for high‐throughput screening of haploids. Doubled haploids regeneration percentage varied from 6.77 to 11.95 in in vivo treatment, while the range of variation was 22.11% to 28.40% in in vitro colchicine treatment. We observed a pronounced increase in plant survival and the proportion of doubled haploid plants in in vitro treatment compared with the standard in vivo approach.