Haploid Production Research Articles

The effect of genotype, media composition, pH and sugar concentrations on oat (Avena sativa L.) doubled haploid production through oat \xd7 maize crosses

Doubled haploid (DH) technology in oat has not reached the same stage as in other cereals leading to its application in plant breeding. The objective of this investigation was to increase the effectiveness of Avena sativa L. haploid embryo germination obtained by the distant crosses with maize. Developed embryos (obtained from 22 genotypes) were transferred on five germination media: MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) with 3% sucrose, pH 5.8 (control medium), and 190-2 supplemented with 6 and 9% maltose. The pH of 190-2 was adjusted to 5.5 and 6.0. Of all tested genotypes, 591 haploid embryos were obtained, almost half of them (279) germinated. The rate of haploid embryo germination induced on 190-2 was 6.92%, while in MS it was 3.25%. The sugar and its concentration significantly affected the germination of haploid embryos. The highest percentage of haploid embryo germination (9.11%) and DH lines production (1.64%) was achieved on 190-2 with 9% maltose and pH 6.0. All DH lines are incorporated to breeding programs for the development of new cultivars.

Nitrous Oxide‐Induced Chromosome Doubling of Maize Haploids

Doubled haploid (DH) production is an important step in varietal development in maize (Zea mays L.), and the success rate of chromosome doubling determines a major portion of the production costs per DH line. Colchicine, the standard chemical used for chromosome doubling, is highly toxic and requires special disposal. In comparison, nitrous oxide (N2O) is a relatively safe gas with simple disposal requirements. Therefore, the objective of this study was to compare chromosome doubling treatment of maize seedlings with N2O with the standard colchicine treatment and a recently proposed alternative herbicide treatment containing amiprophos‐methyl and pronamide in two environments suitable for DH production (DH nursery and greenhouse). Various treatment factors, including different pressures and concentrations of N2O, O2, and air in the treatment chamber, imbibition duration, and treatment duration were analyzed for their effect on survival rate of germinated seedlings, reproduction rate measured as the proportion of haploid (D0) plants with seed set, and overall success rate (OSR) measured as the proportion of D0 plants with seed set obtained from the germinated seedlings. The OSR of the best N2O treatment ranged from 6.4 to 33.3%, whereas the OSR of colchicine ranged from 5.0 to 28.1%, depending on the year and environment. Nitrous oxide treatment did not have a significantly different OSR than herbicide treatment. In conclusion, N2O treatment is a competitive alternative to colchicine and herbicide treatments for chromosome doubling and offers safety and disposal‐related benefits.

Modification of important factors for efficient microspore embryogenesis and doubled haploid production in field grown white cabbage (Brassica oleracea var. capitata L.) genotypes in India

Abstract Cabbage is the most important member of brassica vegetables grown worldwide. Development of homozygous inbreds is pre-requisite in breeding programme and precision genetic studies in this highly heterozygous crop. Doubled haploids (DHs) developed through isolated microspore culture is one of the most effective methods for rapid development of completely homozygous lines in shortest time period. We report for the first time the role of various factors like bud developmental stages, culture density, heat shock treatments and flow cytometry on efficient microspore embryogenesis and development of DH in field grown plants of white cabbage. Among the 4 genotypes, the highest viability of microspores was recorded in bud size of 4.0–4.5 mm in genotypes, Golden Acre and KTCBH-2085 and 4.5–5.0 mm in KTCBH-6621 and KTCBH-6045. In most of the genotypes, buds with highest microspore viability also contained the maximum proportion of microspores at late uni-nucleate to early bi-nucleate stage. For effective conversion of gametophytic pathway to sporophytic pathway, a temperature shock treatment of 30 °C for 48 h + 25.0 °C (Golden Acre and KTCBH-2085) and 32.5 °C for 24 h + 25.0 °C (KTCBH-6621 and KTCBH-6045) were most suitable. The microspore density of 4 × 104/ ml induced the maximum number of embryos per plate in genotypes, KTCBH-2085, KTCBH-6621 and KTCBH-6045, whereas, the microspore density of 6 × 104/ ml was found optimum in the genotype, Golden Acre. Among the different factors, bud size and microspore developmental stage were found to be the most critical in successful microspore embryogenesis in cabbage. However, it was found that genotypes, bud size, culture density and heat treatments had significant effects on microspore embryogenesis. The interaction among these factors also played significant role in microspore embryogenesis of white cabbage. Flow cytometry analysis of microspore derived plants revealed that 41.9% of the plants were spontaneously diplodized in the process of in vitro culture. Among the 221 plants analyzed, 29.0% were haploids, 10.9% were tetraploids and 16.3% were mixoploids and aneuploids. This study will be instrumental in large scale application of DH based cabbage breeding programmes and genetic studies.

Effects of Temperature on the Meiotic Recombination Landscape of the Yeast Saccharomyces cerevisiae.

ABSTRACTAlthough meiosis in warm-blooded organisms takes place in a narrow temperature range, meiosis in many organisms occurs over a wide variety of temperatures. We analyzed the properties of meiosis in the yeast Saccharomyces cerevisiae in cells sporulated at 14°C, 30°C, or 37°C. Using comparative-genomic-hybridization microarrays, we examined the distribution of Spo11-generated meiosis-specific double-stranded DNA breaks throughout the genome. Although there were between 300 and 400 regions of the genome with high levels of recombination (hot spots) observed at each temperature, only about 20% of these hot spots were found to have occurred independently of the temperature. In S. cerevisiae, regions near the telomeres and centromeres tend to have low levels of meiotic recombination. This tendency was observed in cells sporulated at 14°C and 30°C, but not at 37°C. Thus, the temperature of sporulation in yeast affects some global property of chromosome structure relevant to meiotic recombination. Using single-nucleotide polymorphism (SNP)-specific whole-genome microarrays, we also examined crossovers and their associated gene conversion events as well as gene conversion events that were unassociated with crossovers in all four spores of tetrads obtained by sporulation of diploids at 14°C, 30°C, or 37°C. Although tetrads from cells sporulated at 30°C had slightly (20%) more crossovers than those derived from cells sporulated at the other two temperatures, spore viability was good at all three temperatures. Thus, despite temperature-induced variation in the genetic maps, yeast cells produce viable haploid products at a wide variety of sporulation temperatures.

Identification of BABY BOOM homolog in bread wheat

Modern breeding practice of small grain cereals necessitates the development of an efficient system for the large scale and reproducible production of the doubled haploid (DH) lines. It is believed that among the available DH generation techniques, only isolated microspore culture (IMC) can satisfy the demand of public and private breeding programs. Unfortunately, the IMC method is prone to several challenges that jeopardizes its large scale adoption. One of the approaches to limit the variation in DH plant production and increase the efficiency of the method is manipulation of embryogenesis-related genes. Here we set up a study to map BABY BOOM in a bread wheat genome. The gene is one of the morphogenic regulators of somatic embryogenesis in plants. To achieve this task, we used amino acid sequences of Zea mays BBM-like proteins. TaBBM homoeologs were mapped to chromosomes 6AL, 6BL and 6DL. Amino acid sequence analysis revealed the presence of two AP2 domains and bbm-1 motif in the A and D copies and only one AP2 domain and bbm-1 motif in the B copy. This, along with the absence of both gene expression and predictable TATA-box, suggests that TaBBM-gB is a pseudogene. The expression pattern of the identified A and D homoeologs was similar to that for the BBM-like genes in other species and presence of the transcript was detected in an embryogenic microspore population. Identification of the TaBBM homolog can have application in elevating the efficiency of DH production, tissue culture, plant transformation and genome editing for wheat improvement.

Single nucleus sequencing reveals spermatid chromosome fragmentation as a possible cause of maize haploid induction

Production of maternal haploids using a conspecific haploid inducer is routine and highly efficient in maize. However, the underlying mechanism of haploid induction (HI) is unclear. We develop a method to isolate three nuclei from a pollen grain and four microspores from a tetrad for whole-genome sequencing. A high rate of aneuploidy is observed at the three-nucleus stage (6/22 pollens) rather than at the tetrad stage (1/72 microspores) in one HI line CAU5. Frequent aneuploidy is also observed in another two inducer lines, but not in two regular lines, which implies that HI may be associated with pollen aneuploidy. We further sequence the individual embryos and endosperms of 88 maize kernels crossing between regular and inducer lines. Genome-wide elimination of the CAU5-derived chromosome is identified in eight of 81 embryos. Together, these results suggest that continuous chromosome fragmentation occurring post meiosis in the gametophyte may cause haploidy of the embryo.

Doubled haploid wheat lines with high molecular weight glutenin alleles derived from microspore cultures

ABSTRACTA doubled haploid (DH) production system for wheat using a microspore culture method produced seeds and subsequently regenerated haploid wheat plants derived from the F1 plants of two wheat populations (Irena × Ksu102 and Klasic × Ksu105). 68 DH were obtained from both populations. Field trials of these DH lines were conducted over two seasons. Genotypic differences were highly significant for all agronomic traits recorded. Sets of five gene- or allele-specific PCR markers were used for identification of genes encoding high molecular weight (HMW) glutenin subunits Ax1 and Ax2* at the Glu-A1 locus, Bx7, Bx7*, By8 and By17 at the Glu-B1 locus and Dx5, Dy10 and Dy12 at the Glu-D1 locus in wheat inbred lines. The method developed for providing DH lines and subsequent molecular analysis and quality could be useful for improving bread-making quality and developing new cultivars.Abbreviations: DH: doubled haploid; HMW: high molecular weight; HMW-GS: high molecular weight glutenins subunits; MAB: marker-assisted breeding

Gynogenesis and doubled haploid production from unpollinated ovary culture of cucumber (Cucumis sativus L.)

The effects of the genotypes of donor plants and three steps of culture media (induction, differentiation, and regeneration media) were evaluated in unpollinated ovary culture of cucumber (Cucumis ...

Production of doubled haploids in sugar beet (Beta vulgaris): an efficient method by a multivariate experiment

The present paper describes a detailed study of a highly efficient protocol to multiply the number of haploids in sugar beet production and subsequent chromosome doubling. The protocol involves an experiment investigating factorial interactions between cold pretreatment, seven genotypes of sugar beet, and kinetin to improve haploid embryo induction. In addition, the effects of color of ovules and flower bud position on haploid embryo induction were investigated. After subjecting the data to analysis of variance or Student’s t test (P < .05), the effect sizes of the independent variables were also estimated. Cold pretreatment was effective in stimulating the ovules. The haploid embryo induction rate for 1-week cold pretreated ovules (9.01%) was higher than that of freshly cultured ones (6.15%). In comparison with hormone-free medium (5.16%), the gynogenesis rate for the media supplemented with 0.05 or 0.5 mg L−l kinetin increased to 7.58 and 10.05%, respectively. The genotype responses were significantly different. Interactions of kinetin × cold pretreatment, genotype × hormonal treatment, genotype × cold pretreatment, and the three-way interaction were statistically significant. Moreover, the main effects of flower bud position, ovule color, and comma-form ovule on gynogenic response were significant. After investigating the effect of 5 g L−l colchicine for 3, 5, or 7 min on one genotype’s (SG2) specimens, all the haploid plantlets from the other genotypes were treated for 5 min as the best treatment. The paper discusses interactions of the factors, which may be interesting for others aiming to breed doubled haploid sugar beet or possibly other related plant species.

Arabinogalactan proteins improve plant regeneration in barley (Hordeum vulgare L.) anther culture

Androgenesis-based methods of doubled haploid (DH) production show considerable variation in efficiency in different barley genotypes. Arabinogalactan proteins (AGPs) have been shown to play a key role in several developmental processes, including embryogenesis, in different plant species. In this study we investigated the effect of exogenous AGPs from gum arabic on androgenesis and the regeneration efficiency in barley anther culture. Supplementation of the induction medium with 10 mg l−1 gum arabic increased the total plant regeneration rate up to 2.8 times; when exposure to GA was extended to also include the pretreatment step, the regeneration rate was up to 6.6-times higher than in control. The effect of gum arabic was reversed by the Yariv reagent, an AGPs antagonist. This suggests a direct involvement of AGPs in androgenic development from barely microspores. Addition of gum arabic reduced cell mortality, increased the frequency of mitotic divisions of microspores and the number of multicellular structures (MCSs) when compared to control. The positive effect of gum arabic also included reduction in time required for the androgenic induction and substantially improved the quality of formed embryos. Observations made in this study imply a complex role of AGPs during androgenic development and confirmed the usefulness of gum arabic in production of barley androgenic plants.

Maternal doubled haploid production in interploidy hybridization between Brassica napus and Brassica allooctaploids.

We found a new in vivo route to produce maternal doubled haploid of Brassica napus . The pollen donor, an allooctaploid rapeseed, acts as a DH inducer. Inbred line has a powerful advantage in cultivar breeding and genetic analysis. Compared to the traditional breeding methods, doubled haploid production can save years off the breeding process. Though genotype-dependent tissue culture methods are widely used in the Brassica crops, seed-based in vivo doubled haploid developing systems are rare in nature and in the laboratory. As interspecific cross and interploid hybridization play an important role in genome evolution and plant speciation, we created a new Brassica artificial hybrid, a Brassica allooctaploid (AAAACCCC, 2n=8×=76), by interspecific crossing and genome doubling. A homozygous line was observed at the third self-generation of a synthesized Brassica allohexaploid (AAAACC, 2n=6×=58). Crosses between B. napus as female and Brassica allooctaploid as pollen donor were conducted, which yielded maternal doubled haploid B. napus that were identified based on phenotype, ploidy, and molecular analysis. The Brassica octaploid acted as a maternal doubled haploid inducer and had a relatively high induction rate. Our research provides a new insight for generation of homozygous lines in vivo using a single-step approach, as well as promotes the understanding in breeding programs and genetic studies involving the Brassicas.

Trichostatin A increases embryo and green plant regeneration in wheat.

Chemical agents such as trichostatin A (TSA) can assist in optimization of doubled haploidy for rapid improvements in wheat germplasm and addressing recalcitrance issues in cell culture responses. In wheat, plant regeneration through microspore culture is an integral part of doubled haploid (DH) production. However, low response to tissue culture and genotype specificity are two major constraints in the broad deployment of this breeding tool. Recently, the structure of chromatin was shown to be linked with cell transitions during tissue culture. Specifically, repression of genes that are required for cell morphogenesis, through acetylation of histones, may play an important role in this process. Reduction of histone acetylation by chemical inhibition may increase tissue culture efficiency. Here, the role of trichostatin A (TSA) in inducing microspore-derived embryos was investigated in wheat. The optimal dose of TSA was determined for wheat cultivars and subsequently validated in F1 hybrids. A significant increase in the efficiency of DH production was observed in both cultivated varieties and F1 hybrids. Thus, the inclusion of TSA in DH protocols for wheat breeding programs is advocated.

Pollen Preservation Potential of Imperata cylindrica \u2014 an Efficient Source for Doubled Haploid Production in Wheat

Pollen of two I. cylindrica genotypes, one having spike with brown anthers (Ic-Pbr) and other with yellow anthers (Ic-Pye) was collected from three collection environments (early, mid and late flowering) and preserved in sealed Petri dishes under three preservation regimes, P1 (4 °C temperature and 60% RH), P2 (–20 °C and 65% RH) and P3 (–80 °C temperature and 45% RH). The pollen viability and germination tests of the preserved pollen were conducted after every 15 days till complete loss of viability. The study revealed that Ic-Pbr was superior to Ic-Pye in terms of the mean absolute pollen viability (APV) under preservation conditions. Among the collection environments, pollen collected just after first anthesis, that is, early flowering was superior in terms of the preservation potential. The preservation regime, P2 was found to be best among the three for better preservation of I. cylindrica pollen. By utilizing the one month old preserved pollen for pollination of wheat spikes, pollen from P2 regime induced haploid embryos to the tune of 25.53%. The study concluded that by preserving I. cylindrica pollen at –20 °C, the period of DH production in wheat can be extended by at least one month. The preserved pollen will also open avenues for exploring the possibilities of inducing haploids in other cereals like rice and maize whose flowering does not coincide with I. cylindrica naturally.

Isolation and Culture of Pollen Tetrad Protoplasts from Solanum tuberosum

Pollen protoplasts provide a sexual and haploid system for haploid production, cell fusion and mutation studies used in plant improvement. Due to the multiploidy, heterozygosity, and often self-incompatibility in tetraploid genotypes, haploid potatoes are desirable for breeding schemes via ploidy manipulations. In this study, two tetraploid varieties and two dihaploid lines of potato were used for pollen tetrad protoplast isolation and culture. The meiotic tetrad buds were first pre-treated at 5 °C for 0–12 days, then the tetrads were transferred into enzyme solutions containing different concentrations of snailase (0.5–1.5%), 0.3 M osmolites (sucrose, mannitol, glucose or sorbitol), 1.0% Cellulose, 0.5% Hemicellulase, 0.5% Pectolyase, 0.3% Sucrose, 3 mM 2-(N-Morpholino) ethane sulfonic acid, 1% polyvinyl pyrrolidone, 0.01% casein hydrolysate and K3 medium compositions. Among the four donor materials, tetraploid cv. Gannongshu No. 3 (‘GNS No.3’) showed the greatest protoplast yield (74.6 ± 2.4%). In this variety, most of the tetrad protoplasts regenerated a cell wall and continued cell divisions were observed when they were inoculated in K3 basic medium supplemented with (0.5–1.0) mg/L 2,4-D + (0.1–0.5) mg/L KT + 0.4 mg/L 6-BA +800 mg/L glutamine +100 mg/L serine. ‘GNS No.3’ also showed the greatest first division frequency (21.6 ± 1.5%) and sustained division to form multicellular structures. The study findings suggested that cultured tetrad pollen protoplasts could reverse the gametophytic developmental pattern programmed in vivo to a sporophytic pathway leading to multicellular microspore-derived colonies.

Gametocidal Factor Transferred from Aegilops geniculata Roth Can Be Adapted for Large-Scale Chromosome Manipulations in Cereals.

Segregation distorters are curious, evolutionarily selfish genetic elements, which distort Mendelian segregation in their favor at the expense of others. Those agents include gametocidal factors (Gc), which ensure their preferential transmission by triggering damages in cells lacking them via chromosome break induction. Hence, we hypothesized that the gametocidal system can be adapted for chromosome manipulations between Triticum and Secale chromosomes in hexaploid triticale (×Triticosecale Wittmack). In this work we studied the little-known gametocidal action of a Gc factor located on Aegilops geniculata Roth chromosome 4Mg. Our results indicate that the initiation of the gametocidal action takes place at anaphase II of meiosis of pollen mother cells. Hence, we induced androgenesis at postmeiotic pollen divisions (via anther cultures) in monosomic 4Mg addition plants of hexaploid triticale (AABBRR) followed by production of doubled haploids, to maintain the chromosome aberrations caused by the gametocidal action. This approach enabled us to obtain a large number of plants with two copies of particular chromosome translocations, which were identified by the use of cytomolecular methods. We obtained 41 doubled haploid triticale lines and 17 of them carried chromosome aberrations that included plants with the following chromosome sets: 40T+Dt2RS+Dt2RL (5 lines), 40T+N2R (1), 38T+D4RS.4BL (3), 38T+D5BS-5BL.5RL (5), and 38T+D7RS.3AL (3). The results show that the application of the Gc mechanism in combination with production of doubled haploid lines provides a sufficiently large population of homozygous doubled haploid individuals with two identical copies of translocation chromosomes. In our opinion, this approach will be a valuable tool for the production of novel plant material, which could be used for gene tracking studies, genetic mapping, and finally to enhance the diversity of cereals.

The effect of copper on plant regeneration in barley microspore culture

Isolated microspore culture is an excellent system for the production of doubled haploids in many crops, including barley. In a more traditional barley anther culture method copper sulphate is known to enhance plant regeneration. Here we report that one hundred times higher concentration of copper sulphate in the isolated microspore culture of two spring barley genotypes compared to the standard content in the induction medium resulted in a 34% increase of total plant regeneration. Detailed analysis of plant regeneration showed that additional supplementation of copper sulphate increased not only the regeneration of green plants but also proportionately that of albino plants. Hence, the results from two studied genotypes do not support an assumption that the addition of copper reduces albinism in barley microspore culture.

Reduced ascorbate and reduced glutathione improve embryogenesis in broccoli microspore culture

Isolated microspore culture (IMC) is an effective alternative technique for the production of doubled haploid (DH) homozygous lines in plant breeding. However, death of most of the cultured cells during culture is a critical obstacle that affects embryo production in the practical application of IMC technology. In this present paper, we evaluated the effects of two antioxidants of both reduced ascorbate (ASC) and reduced glutathione (GSH) on microspore viability and the induction of embryogenesis in the IMC of broccoli (Brassica oleracea L. var. italica). The results showed that cell death occurred soon after stress treatment (at 32.5°C for 24h), and the number of dead cells reached about 80% after 2d. The addition of 10mgl−1 ASC and 20mgl−1 GSH significantly decreased microspore mortality, and had a strong effect on the number of embryos produced. In the genotype ‘B415’, embryo yields increased approximately 1.2-fold and 2.5-fold after the addition of the 10mgl−1 ASC and 20mgl−1 GSH, respectively. Moreover, the addition of ASC and GSH also influenced positively the rate of plant regeneration, which respectively increased by 4.2% and 9.7% for ‘B415’. Taken together, our results show that the induction of embryogenesis in broccoli is strongly affected by the use of ASC or GSH. The effects on embryogenesis induction could confirm the importance of the redox environmental regulation in IMC, specifically at the early phase of IMC. The obtained knowledge may accelerate and guide the application of microspore-derived DH plants as a breeding tool in broccoli.

MATRILINEAL, a sperm-specific phospholipase, triggers maize haploid induction.

Sexual reproduction in flowering plants involves double fertilization, the union of two sperm from pollen with two sex cells in the female embryo sac. Modern plant breeders increasingly seek to circumvent this process to produce doubled haploid individuals, which derive from the chromosome-doubled cells of the haploid gametophyte. Doubled haploid production fixes recombinant haploid genomes in inbred lines, shaving years off the breeding process. Costly, genotype-dependent tissue culture methods are used in many crops, while seed-based in vivo doubled haploid systems are rare in nature and difficult to manage in breeding programmes. The multi-billion-dollar maize hybrid seed business, however, is supported by industrial doubled haploid pipelines using intraspecific crosses to in vivo haploid inducer males derived from Stock 6, first reported in 1959 (ref. 5), followed by colchicine treatment. Despite decades of use, the mode of action remains controversial. Here we establish, through fine mapping, genome sequencing, genetic complementation, and gene editing, that haploid induction in maize (Zea mays) is triggered by a frame-shift mutation in MATRILINEAL (MTL), a pollen-specific phospholipase, and that novel edits in MTL lead to a 6.7% haploid induction rate (the percentage of haploid progeny versus total progeny). Wild-type MTL protein localizes exclusively to sperm cytoplasm, and pollen RNA-sequence profiling identifies a suite of pollen-specific genes overexpressed during haploid induction, some of which may mediate the formation of haploid seed. These findings highlight the importance of male gamete cytoplasmic components to reproductive success and male genome transmittance. Given the conservation of MTL in the cereals, this discovery may enable development of in vivo haploid induction systems to accelerate breeding in crop plants.

Improved production of doubled haploids of winter and spring triticale hybrids via combination of colchicine treatments on anthers and regenerated plants

Double haploids (DH), obtained during androgenesis in vitro or by genome diploidisation in regenerated haploids, are one type of basic materials used in triticale breeding programmes. The aim of this study was to improve DH production by a combination of colchicine treatment methods on a sample of five winter and five spring triticale hybrids. Colchicine was applied in vitro either in the C17 medium to induce embryo-like structures (ELS) or in the 190-2 medium for green plant (GP) development. Regenerants which remained haploid were immersed in a colchicine solution either when placed on the medium prior to transferring to soil or when growing in pots, followed by the application or absence of cooling. Colchicine treatment during anther culture affected neither ELS nor GP development, but significantly increased the number of DH plants in comparison to spontaneous chromosome doubling. The highest efficiency was recorded when colchicine was applied in the induction medium (55%) versus the regeneration medium (44.5%) or no colchicine treatment (30%). The effectiveness of chromosome duplication in haploid plants ranged from 32 to 64.5% and it was the highest for the treatment on the medium followed by cooling. Individual hybrids differed regarding their capability of regeneration and chromosome doubling, which were consistent only to a low or moderate extent. However, taken together, winter and spring hybrids did not differ significantly. Combined colchicine application resulted in a high yield of DH production, 82.6% for all triticale hybrids, and can provide a considerable number of fertile DH lines for triticale breeding programmes.

Florogenesis and Female Gametophyte Development in &amp;lt;i&amp;gt;Allium cepa&amp;lt;/i&amp;gt; L. cv. Krishnapuram

Florogenesis is one of the most complicated and interesting processes in the nature. This process involves developmental, physiological and molecular events leading to transformation from vegetative to reproductive phase for optimal seed production and the continuation of species. The basic knowledge about flowering processes, male and female sexual systems support basic and applied research and breeding programs. Most of the onion varieties from India are short day varieties, more diverse than other exotic germplasm and useful as a source of new alleles for supporting breeding programs. The present investigation was focused to study for the first time florogenesis process by scanning electron study for the first time florogenesis process by scanning electron microscopy (SEM) and development of female gametophytes by light microscopy in order to acquire basic knowledge useful for optimizing in vitro process to produce gynogenic haploid to support and speed breeding program in short-day onion Allium cepa L. cv. Krishnapuram (KP) or Bangalore Rose. This study revealed that shoot primordium differentiated into inflorescence meristem in the month of December, while seeds were planted in the field in the September. The individual florets are preceded by a variying number of floral initials. The female gametophyte developed from chalazal side megaspore. The embryo sac development is a bisporic Allium type showing short-lived antipodals. The histological study suggests that the use of big or preanthesis flower buds with embryo sac for production of gynogenic haploids to support breeding program in onion cv. Krishnapuram (KP). However further studies are needed for confirmation of this observation.