Haploid Plants Research Articles

Use of nurse endosperm for the culture of haploid embryos produced by durum wheat x maize crosses

The use of doubled-haploids in plant breeding programs enables accelerating the release of new varieties adapted to climate change. The durum wheat x maize crosses technique is a method of choice for producing durum wheat haploid plants. The haploid embryos produced by this method develop without albumen and their survival is ensured by post-pollination hormonal treatments. In this study, nine post-pollination treatments with 2,4-Dichlorophenoxyacetic acid (2,4-D), Picloram and Dicamba at the concentrations of 10, 50 and 100 mg.L-1 were applied to 7 durum wheat genotypes. The effects of genotype and post-pollination treatment on durum wheat haploid embryos produced by durum wheat x maize crosses and the use of the endosperm nursing technique for haploid plantlets regeneration were investigated. The haploid induction parameters varied with the durum wheat genotypes as well as the post-pollination treatments. The phenomenon of polyembryony resulting from durum wheat x maize crosses is reported for the first time in this article. The durum wheat genotypes showed different abilities to produce monoembryo and polyembryos. The post-pollination treatments with 2,4-D (10 mg.L-1) and Picloram (10 and 100 mg.L-1) gave a higher embryo formation frequency than the treatments with Dicamba. The embryo conversion to plantlet was greatly improved, especially in recalcitrant genotypes using the durum wheat endosperm as supplemental nourishment in combination with the Gamborg B5 regeneration medium.
 Keywords: Durum wheat; Haploid embryo; Maize; Nurse Endosperm Technique; Polyembryony

From Petri Dish to Field: Plant Tissue Culture and Genetic Engineering of Oats for Improved Agricultural Outcomes

Oats (Avena sativa) hold immense economic and nutritional value as a versatile crop. They have long been recognized as an exceptional choice for human consumption and animal feed. Oats’ unique components, including proteins, starches, and β-glucans, have led to its widespread use in various food products such as bread, noodles, flakes, and milk. The popularity of oat milk as a vegan alternative to dairy milk has soared due to the increasing number of vegetarians/vegans and growing environmental awareness. Oat milk offers a sustainable option with reduced greenhouse gas emissions during its production, rendering it an appropriate choice for individuals who are lactose-intolerant or have dairy allergies. To ensure improved adaptability and enhanced nutrition, the development of new oat varieties is crucial, considering factors like cultivation, climate, and growing conditions. Plant cell culture plays a crucial role in both traditional and contemporary breeding methods. In classical breeding, plant cell culture facilitates the rapid production of double haploid plants, which can be employed to accelerate the breeding process. In modern breeding methods, it enables genetic manipulation and precise genome editing at the cellular level. This review delves into the importance of oats and their diverse applications, highlighting the advantages of plant cell culture in both classical and modern breeding methods. Specifically, it provides an overview of plant tissue culture, encompassing genetic transformation, haploid technology, protoplast technology, and genome editing.

Microsatellite-based identification of doubled haploid plants by androgenesis in Anemone coronaria L.

Microsatellite-based identification of doubled haploid plants by androgenesis in <i>Anemone coronaria</i> L.

Paclitaxel and Caffeine-Taurine, New Colchicine Alternatives for Chromosomes Doubling in Maize Haploid Breeding.

The doubled haploid (DH) technology is employed worldwide in various crop-breeding programs, especially maize. Still, restoring tassel fertility is measured as one of the major restrictive factors in producing DH lines. Colchicine, nitrous oxide, oryzalin, and amiprophosmethyl are common chromosome-doubling agents that aid in developing viable diploids (2n) from sterile haploids (n). Although colchicine is the most widely used polyploidy-inducing agent, it is highly toxic to mammals and plants. Therefore, there is a dire need to explore natural, non-toxic, or low-toxic cheaper and accessible substitutes with a higher survival and fertility rate. To the best of our knowledge, the advanced usage of human anticancer drugs "Paclitaxel (PTX)" and "Caffeine-Taurine (CAF-T)" for in vivo maize haploids doubling is being disclosed for the first time. These two antimitotic and antimicrotubular agents (PTX and CAF-T) were assessed under various treatment conditions compared to colchicine. As a result, the maximum actual doubling rates (ADR) for PTX versus colchicine in maize haploid seedlings were 42.1% (400 M, 16 h treatment) versus 31.9% (0.5 mM, 24 h treatment), respectively. In addition, the ADR in maize haploid seeds were CAF-T 20.0% (caffeine 2 g/L + taurine 12 g/L, 16 h), PTX 19.9% (100 μM, 24 h treatment), and colchicine 26.0% (2.0 mM, 8 h treatment). Moreover, the morphological and physiological by-effects in haploid plants by PTX were significantly lower than colchicine. Hence, PTX and CAF-T are better alternatives than the widely used traditional colchicine to improve chromosome-doubling in maize crop.

Induction of Triticale (×Triticosecale Wittmack) In Vitro Androgenesis in Anther Cultures of F1 Hybrid Combinations, Varieties and Homogeneity Testing of Offspring Generation.

In cereal breeding, in vitro androgenesis methods are frequently applied to achieve doubled haploid (DH) plants. The aim of this study was to determine the effects of genotype (three registered varieties and eight F1 crossing combinations) and induction medium (W14mf and P4mf) on anther cultures (ACs) of triticale (×Triticosecale Wittmack). Androgenesis was induced in the treatment of each tested genotype, and the genotype significantly influenced the efficiency of AC, including in embryo-like structures (ELSs), albinos, green plantlets, and transplanted plantlets. The utilized medium also had a significant effect on the number of ELSs, albinos, and transplanted plantlets. Both media were suitable for AC in triticale DH plant production. The efficiency of AC was higher when using the P4mf medium (103.7 ELS/100 anthers, 19.7 green plantlets/100 anthers) than when using the W14mf medium (90.0 ELS/100 anthers, 17.0 green plantlets/100 anthers). However, the green plantlet regeneration efficiency of microspore-derived structures was 18.0% when using the W14mf medium, while this value was 15.9% in the case of ELSs induced with the P4mf medium. After nursery seed evaluation and propagation (DH1), the genetic homogeneity of the offspring generation (DH2) was tested using a molecular genetic method. Most of the tested DH lines showed homogeneity and were progressed into a breeding program after agronomic selection. Some DH lines showed inhomogeneity, which could be explained by the outcross inclination of triticale. We would like to call breeders' attention to the outcross character of triticale and emphasize the vigilant propagation and maintenance of the triticale DH lines in breeding programs. Due to the outcross nature of triticale, even in self-pollinated genotypes, breeders should focus on careful maintenance, along with isolation in the case of line propagations, in triticale breeding programs.

Haploid Induction in Indica Rice: Exploring New Opportunities.

Haploid plants are of significant interest to crop breeders due to their ability to expedite the development of inbred lines. Chromosome-doubling of haploids, produced by either in vitro or in vivo methods, results in fully homozygous doubled haploids. For nearly five decades, in vitro methods of anther and microspore culture have been attempted in many crops. In rice, in vitro methods are used with some success in japonica cultivars, although indica types have remained recalcitrant to a large extent. This review aims to explore the reasons for the lack of success of in vitro methods in indica rice and discuss new advancements in in vivo haploid induction protocols in other cereals and their relevance to rice. In particular, the current level of understanding of in vivo haploid inducer systems that utilize MTL and CENH3 mutants is analyzed in detail. One notable advantage of in vivo haploid induction systems is that they do not require tissue culture competence. This makes these methods more accessible and potentially transformative for research, offering a pragmatic approach to improving indica rice cultivars. By embracing these in vivo methods and harnessing the power of gene editing technologies like CRISPR/Cas9 systems, breeders can reshape their approach to indica rice improvement.

Production of haploid and doubled haploid plants of Allium tuncelianum (Kollman) Özhatay, Matthew and Şiraneci via in vitro gynogenesis

Production of haploid and doubled haploid plants of Allium tuncelianum (Kollman) Özhatay, Matthew and Şiraneci via in vitro gynogenesis

Morphological, Histological, and Transcriptome Analysis of Doubled Haploid Plants in Poplars (Populus simonii × Populus nigra)

In this study, the poplar doubled haploid (DH) plants were used as the experimental material to explore the huge phenotypic differences between homozygous DH plants and the paternal plants, and the molecular regulation mechanism of the differential phenotypes. In this experiment, through morphological and histological observation and statistics, we found that the double haploid plants had significantly reduced plant height and ground diameter, increased leaf aspect ratio, premature senescence phenotype of top bud, and significant changes in the shape and cell area of the shoot apical meristem. Significantly differentially expressed genes were obtained using RNA-seq transcriptome sequencing. They were subjected to GO enrichment and KEGG analysis. Transcription factors with key functions were screened out for qRT-PCR to verify gene expression changes to predict gene function. The results showed that after the IAA and ABA treatment, the expression levels of some hormone-responsive genes in wild type plants were significantly changed with different treatment time. In the dihaploid plants, the corresponding genes also changed to different degrees, which reflected the changes in the response of the dihaploid plants to hormones. Compared to in WT, the differential expressed genes in the double haploids were involved in multiple physiological process such as response to oxidative stress, response to salicylic acid, plant pathogen interaction, and plant hormone signal transduction. A TF–centered gene regulatory network for phytohormone synthesis and plant senescence was constructed with the expression patterns of differentially expressed transcription factors (TFs). This study increases researchers’ understanding of the regulation of poplar growth and development and provides new research ideas for the creation of new species of poplar.

Induced chromosome doubling in microspores and regenerated haploid plants of Brassica napus

Induced chromosome doubling in microspores and regenerated haploid plants of Brassica napus

ECS1 and ECS2 suppress polyspermy and the formation of haploid plants by promoting double fertilization.

The current pace of crop plant optimization is insufficient to meet future demands and there is an urgent need for novel breeding strategies. It was previously shown that plants tolerate the generation of triparental polyspermy-derived plants and that polyspermy can bypass hybridization barriers. Polyspermy thus has the potential to harness previously incompatible climate-adapted wild varieties for plant breeding. However, factors that influence polyspermy frequencies were not previously known. The endopeptidases ECS1 and ECS2 have been reported to prevent the attraction of supernumerary pollen tubes by cleaving the pollen tube attractant LURE1. Here, we show that these genes have an earlier function that is manifested by incomplete double fertilization in plants defective for both genes. In addition to supernumerary pollen tube attraction, ecs1 ecs2 mutants exhibit a delay in synergid disintegration, are susceptible to heterofertilization, and segregate haploid plants that lack a paternal genome contribution. Our results thus uncover ECS1 and ECS2 as the first female factors triggering the induction of maternal haploids. Capitalizing on a high-throughput polyspermy assay, we in addition show that the double mutant exhibits an increase in polyspermy frequencies. As both haploid induction and polyspermy are valuable breeding aims, our results open new avenues for accelerated generation of climate-adapted cultivars.

Obtaining haploid plants by irradiated pollen culture in oil seed crops

Haploid plant production is of great importance to shorten the breeding period in plant breeding programs. Obtaining pure lines in plant growing programs require an intensive work with huge labor and time. Obtaining one hundred percent homozy-gous pure lines is a key point for the improvement and development of new cultivars. Haploid plants with a single set of homozygous chromosomes have become a valua-ble tool in plant breeding. Dihaploid plants that are homozygous at all loci with dou-bling of their chromosomes can be propagated by seed and reach full homozygosity in a single generation. Traditional methods take seven years to reach homozygosity. Dihaploidization methods provide significant advantages in terms of gaining homo-zygosity in a short period of one year and bringing pure lines into agriculture. Anther culture and irradiated pollen technique are among the most widely used techniques in this respect; where physical or chemical agents are used to induce mutated pollen grains and anthers that are subsequently employed to develop dihaploids through in vitro cultures. These techniques are a good source to facilitate gene mapping, cyto-genetic research, and evolutionary studies. Irradiated pollen culture techniques have been applied to many oilseed crops to obtain pure lines. This study highlights some salient features of producing dihaploids using irradiated pollen grains and their maintenance.

Application of Auxins in Haploid Embryo Induction in Hexaploidy Wheat

Doubled haploid (DH) plant production plays a crucial role in modern plant breeding programs, offering an efficient means to generate homozygous lines from heterozygous parents within a single generation. Different types of auxins have been utilized in wheat cross with maize DH production, with 2,4-D being the most widely used and effective hormone, followed by dicamba. Other auxins, including picloram, Indole-3-acetic acid (IAA), phenylacetic acid (PAA), silver nitrate, 1-naphthaleneacetic acid (NAA), kinetin, 6-benzyladenine (BA), and zearalenone, have also been tested for their potential role in haploid embryo induction. Various methods have been explored for the application of 2,4-D, such as spray, tiller injection, dipping, and spikelet culture methods. 2,4-D found to be most effective auxin treatment both alone and with combination with other phytohormones.

A NEW METHOD FOR OBTAINING HAPLOID PLANT SHED-MICROSPORTS CULTURE

In plant breeding studies, dihaploidization method is used to obtain homozygous pure lines used to develop varieties in a shorter time. Dihaploidization method is applied in most types of vegetables such as wheat and corn, as well as in vegetable species. Androgenesis methods (anther and microspore culture) and gynogenesis and parthenogenesis methods (ovula and ovary culture) are used to obtain haploid plants. One of the androgenetic methods, shed-microspore culture has been a new alternative technique to anther and microspore cultures, and successful results have been obtained. The most important feature of this technique is the presence of a double-layered nutrient medium in a single petri dish, and the culture of microspores in the anthers in solid and liquid nutrient medium. In this study, the definition and application of microspore culture and shed-microspore cultures, studies conducted in the world and in our country and their developments in recent years were examined.

Androgenesis in tomato (Solanum lycopersicum L.)-Effect of genotypes, microspore development stage, pre-treatments and media composition on induction of haploids

Doubled haploid (DH) technology remarkably accelerates the crop breeding by obtaining homozygous lines in a single generation. The present study was targeted in generating haploid plants through androgenesis. Anthers from immature flower buds of six tomato genotypes viz., LE-1230, LE-1236, LE-1256 TLCV 2, PKM 1 and TNAU tomato hybrid CO 3 were used for induction of haploids. A preliminary study based on callus induction frequency (CIF), more than 5% was helpful in short listing flower bud size, pre-treatments and growth regulator combinations. Subsequently, anthers from two different sized flower buds (4 and 6 mm length), dissected either from fresh or pre-treated flower buds (2 and 5 days in dark at 4 °C or gamma irradiated) were inoculated in MS medium fortified with different growth regulators for callus induction. Among the genotypes, TLCV 2 had recorded the maximum CIF (38.80%) from anthers of 4 mm long flower buds followed by TNAU tomato hybrid CO 3 (34.70%). Throughout the study, anthers from 4 mm long flower buds responded the best for callus induction. Among the pre-treatments, anthers from gamma irradiated flower buds recorded the highest CIF (31.90%) when compared to others. Cold shock (4 °C) in dark to flower buds for 2 days had improved the CIF of anthers when compared to fresh in LE 1230, LE 1238, TLCV2 and TNAU tomato hybrid CO 3, but when the cold shock was increased to 5 days, invariably there was a reduction in CIF in all the six genotypes. TA 8 (MS + 2iP (0.5 mg L-1) + NAA (0.5 mg L-1)) medium was found to be the best for maximum CIF in LE 1230 and PKM1, TA1 (MS + 2iP (1.0 mg L-1) + IAA (2.0 mg L-1)) in LE 1238, LE 1256 and TNAU tomato hybrid CO 3 and TA7 (MS + 2iP (0.5 mg L-1) + Kinetin (1.5 mg L-1) + NAA (1.0 mg L-1)) for TLCV 2 genotypes. The callus induced was sub cultured at monthly intervals in the same medium for proliferation and later transferred to regeneration medium. A good number of shoots got regenerated only from anther calli of TNAU hybrid CO 3 that was sub cultured in MS medium fortified with Zeatin (0.5 mg L-1). The clumps of shoots induced were separated and inoculated in MS medium supplemented with GA3 (0.5 mg L-1) for shoot elongation. After 4-6 weeks, the elongated shoots were transferred to half strength MS medium enhanced with IBA (1 mg L-1). Profuse rooting from the base of the shoot was noticed in 4-5 weeks. The stomatal count with leaves from the diploid plants and in vitro plants observed were 3-4 and 1 respectively indicating the haploidy nature of in vitro plants.

Induction of haploid plants for speed-up breedıng in sunflower (Helianthus annuus L.) by pollen irradiation

The creation of homozygous plants is essential for both genetic studies and the production of hybrid seeds, particularly in plants that were pollinated by an alien species. Dihaploid (DH) technology is an important application in obtaining homozygous lines used to plant speed breeding programs. The generation of haploid embryos by pollen irradiation and pollination of female flowers with irradiated pollen has been successfully applied. The influence of irradiated pollen on the induction of haploids in sunflower (Helianthus annuus L.) has been evaluated on 16 different sunflower lines from Turkey’s National Sunflower Breeding Program. In this study, pollen grains were irradiated with varying dosages of Gamma rays (500-1000 Gy) to generate parthenogenetic haploid embryos in oilseed sunflowers. At a period of 12–20 days following the process of pollination, embryos were plated into modified MS culture media under a 16/8 h photoperiod and a temperature of 25◦C. Parthenogenetic 18457 embryos at different developmental stages were rescued in vitro and 650 were germinated. The ploidy analysis of regenerants was made with chromosome count and flow cytometry. Except for 500 Gy, all dosages were effective for inducing embryos and haploid plants. Obtained results indicate that parthenogenetic haploid embryos can be produced in K3AD SN:8, IMI 069, and IMI 044 sunflower genotypes by selecting the appropriate Gamma ray dose and embryonic developmental stage. This study was focused to recover sunflower doubled haploids to accelerate the production of non-segregating lines for breeding.

Combining multiplex gene editing and doubled haploid technology in maize.

A major advantage of using CRISPR/Cas9 for gene editing is multiplexing, that is, the simultaneous targeting of many genes. However, primary transformants typically contain hetero-allelic mutations or are genetic mosaic, while genetically stable lines that are homozygous are desired for functional analysis. Currently, a dedicated and labor-intensive effort is required to obtain such higher-order mutants through several generations of genetic crosses and genotyping. We describe the design and validation of a rapid and efficient strategy to produce lines of genetically identical plants carrying various combinations of homozygous edits, suitable for replicated analysis of phenotypical differences. This approach was achieved by combining highly multiplex gene editing in Zea mays (maize) with in vivo haploid induction and efficient in vitro generation of doubled haploid plants using embryo rescue doubling. By combining three CRISPR/Cas9 constructs that target in total 36 genes potentially involved in leaf growth, we generated an array of homozygous lines with various combinations of edits within three generations. Several genotypes show a reproducible 10% increase in leaf size, including a septuple mutant combination. We anticipate that our strategy will facilitate the study of gene families via multiplex CRISPR mutagenesis and the identification of allele combinations to improve quantitative crop traits.

Estimation of the efficiency of &lt;i&gt;in vitro&lt;/i&gt; anther culture method for obtaining haploid triticale plants (×&lt;i&gt;Triticosecale&lt;/i&gt; wittmack)

Anther culture is one of the most popular methods for obtaining haploid triticale plants. The current paper has presented the study results of the efficiency of the method for obtaining haploid plants in promising winter triticale forms bred by the Federal Agricultural Research Center of South-East. There has been estimated haploproduction parameters for the studied genotypes on the nutrient media C-17 and P-2. There were no genotypes unresponsive to androgenesis. There has been found that the medium Potato-2 had a significant positive effect on the total number of embryogenic structures per 100 cultivated anthers in four of the six studied genotypes when the index increased in 1–3 times. The mean value of the EC/100KP indicator was 23.3 with a variation of 6.4-75.1. The largest number of ES (45.8 and 75.1) was found in genotype No. 96 (F5 Zimogor/win.br.wheat L.39) on induction media C-17 and P-2. The genotypes No. 95 and No. 97 were characterized by the lowest indicators F4 DH13/ win.br.wheat ‘Atkara’//Vodoley/ADP-2///Colina and F5 MAG/Vodoley//TI-17. Plant regeneration varied from 3.4 to 22.1 with an average value of 12.6 per 100 EC. The mean number of green plants per 100 EC was 3.9, varying from 0.4 to 12.1. The proportion of green plants, depending on the genotype, varied from 5.0 to 76.9 %. Two-way analysis of variance has identified the dominant role of the genotype at all stages of haploproduction. The proportion of effect of the nutrient medium was statistically significant, but insignificant in comparison with the genotype. There has been established that, along with genotypic dependence and albinism, the limiting factor of the method is a low frequency of regeneration of embryo-like structures. In total, there were obtained 949 regenerated plants out of 7538 embryogenic structures, of which 326 were green. The ratio of green plants to albinos was 1:2.

Molecular profiling of some blast resistance genes and analysis of physiological variability in rice plants produced by anther culture and hybridization

For genetic improvement of rice plants against blast fungus disease caused by Magnaporthe oryzae, eight rice genotypes including four Egyptian and four foreign genotypes which represented a broad range of variation for blast resistance and other several traits were used in this study through line X tester mating design to produce F1 hybrids used as source of anther culture. Results of analysis of variance showed highly significant differences for all studied traits indicating overall genetic variations among parents and hybrids suggested that specific combining ability referred heavily to the expression of studied traits. Data of anther culture experiment for parents (8 parents) and hybrids (15 crosses) showed highly significant differences for all studied traits which illustrate genetic variations through the used materials. The highest callus induction percentage was found for CR7 (18.3%) and P6 (14.0%) while, the lowest callus induction percentage was found for CR5 (2.0%) and CR1 (4.0%). On the other hand, nine out of 15 hybrids recorded regeneration percentage more than the highest parental genotypes. Anther culture-derived plants and their parental genotypes were evaluated for blast disease resistance according to artificial infection with 10 blast races. The results showed that resistance percentage varied between 10% and 100%. While all anther culture-derived plants showed high level of resistance (100%) against all tested blast races. For enzyme activities for catalase, peroxidase and polyphenol oxidase in anther culture-drived plants and their parent, results showed that line TC9 derived from CR9 gave the highest value for catalase activity (301). Also, CR11 and P4 recorded the high level of peroxidase and polyphenol oxidase activities, respectively. Moreover, genetic diversity among anther culture-derived plants and their parental genotypes using five SSR primers related to Pi5(t), Pikh, Pi-b, Pi-1 and Pi-ta blast resistance genes showed heterozygous bands in all tested genotypes. The highest PIC value recorded for RM 247 and RM 206 markers which had PIC values of 0.778 and 0.761, respectively. Out of the used five SSR markers, RM 247 was the most informative marker with amplicon size ranged from 134 to 242 bp. These markers are therefore useful markers for studying polymorphism concerning blast resistance in rice and hence can be used in the marker-assisted selection for blast resistance. While development of resistant genotypes is the most effective way to control rice blast disease, anther culture technique used in this study helpful in production of resistant double haploid plants used in rice preeding programe in Egypt.

Exceptionally high genetic variance of the doubled haploid (DH) population of poplar

Doubled haploid (DH) plants have been widely used for breeding and biological research in crops. Populus spp. have been used as model woody plant species for biological research. However, the induction of DH poplar plants is onerous, and limited biological or breeding work has been carried out on DH individuals or populations. In this study, we provide an effective protocol for poplar haploid induction based on an anther culture method. A total of 96 whole DH plant lines were obtained using an F1 hybrid of Populus simonii × P. nigra as a donor tree. The phenotypes of the DH population showed exceptionally high variance when compared to those of half-sib progeny of the donor tree. Each DH line displayed distinct features compared to those of the other DH lines or the donor tree. Additionally, some excellent homozygous lines have the potential to be model plants in genetic and breeding studies.

Protocol optimization and assessment of genotypic response for inbred line development through doubled haploid production in maize

BackgroundDoubled haploid technology offers the fastest route of inbred line development by rapidly fixing the desirable combinations in a single year. However, the differential response of haploid induction to genetic background of maternal lines accompanied with low induction rate and high mortality rate due to artificial chromosomal doubling of haploid seedlings creates hindrance in doubled haploid production on a commercial scale under tropical conditions. To speed up the hybrid breeding programme in sub-tropical maize, efforts are reported here to optimize the protocol for efficient production of fixed lines using haploid inducers. The second-generation haploid inducers i.e. CIM2GTAILs obtained from CIMMYT, Mexico were used for haploid induction in 13 F1s of diverse backgrounds. For standardization of chromosomal doubling protocol, various concentrations of colchicine and two seedling growth stages were used to determine the extent of chromosomal doubling and survival rate of doubled haploid plants.ResultsA high mean haploid induction rate is obtained from CIM2GTAIL P2 (10%) as compared to CIM2GTAIL P1 (7.46%). Out of four treatments, CIMMYT reported protocol of chromosome doubling in tropical maize comprising combination of 0.07% colchicine and 0.1% DMSO at V2 stage is highly effective for acquiring doubled haploid plants in sub-tropical adapted maize with high survival rate of 52.7%. However, increasing the colchicine concentration from 0.07 to 0.1% led to high mortality rate.ConclusionAccording to the findings, the haploid induction rate, survival rate and overall success rate varied depending upon the genotype of the inducer and the source population along with the concentrations of chemical used. The optimized protocol developed using CIMMYT haploid inducer CIM2GTAIL P2 for efficient doubled haploid production will not only fasten the breeding programme but will also reduce the production cost of doubled haploid with great efficiency in sub-tropical maize.