Haploid Development Research Articles

CDNA cloning of four Hsp genes from Agarophyton vermiculophyllum and transcription analysis in different phases

Agarophyton vermiculophyllum is an agarophytic red alga originating from Northeast Asia that successfully spread to Europe and North America in the last 20 years. In this study, three hsp70 genes (hsp70-1, hsp70-2, and hsp70-3) and one hsp90 gene were cloned, and real-time reverse transcription polymerase chain reaction (RT-qPCR) was used to measure their transcriptional levels in three kinds of thalli (tetrasporophytes, and male/female gametophytes) belonging to diploid or haploid phases in the life cycle of A. vermiculophyllum. The results show that the three Hsp70s in A. vermiculophyllum clustered into three different groups, and the locations of the putative Hsp70-1, Hsp70-2, and Hsp70-3 were in the cytoplasm, endoplasmic reticulum and chloroplast, respectively, according to their sequences. Beside on the same research, the putative Hsp90 was supposed to have a cytoplasmic location. The RT-qPCR results show that the three hsp70 genes were highly upregulated in gametophytes as compared to tetrasporophytes but the transcriptional level of hsp90 did not show such a significant increase. The chloroplast hsp70-3 exhibited the highest upregulation and the transcriptional level increased more than 570 fold in female gametophytes, and 17 fold in male gametophytes, compared with tetrasporophytes. Therefore, cpHsp70-3 might act more like a chaperon molecule involved in haploid development under natural condition, while Hsp70-1 and Hsp70-2 were more active in stress resistance.

A novel approach for haploid generation in Lolium

AbstractDevelopment of haploid and/or dihaploid methodologies for Lolium sp. is important to future improvement efforts as the genus includes the majority of the world's cool‐season annual and perennial turf and forage grass species. Microspore or anther culture haploid induction systems can result in the generation of either maternal or paternal haploids that can be effectively used to advance genetic gains through a breeding or selection program. The successful and traditional approach for the production of haploids and eventually dihaploids in Lolium sp. is most successful when microspore culture approaches are applied. Unfortunately, this approach is labor‐intensive, expensive and often restrictive to amendable genotypes. Within the Lolium‐Festuca genome complex, there is a need for modern breeding approaches that can facilitate the rapid development of haploids or dihaploids for the development of improved germplasm or cultivars. It has been suggested that an in vivo approach could facilitate further advances in Lolium sp. genetic studies, molecular breeding and superior cultivar development. The recent development of a two novel annual ryegrass (Lolium perenne L. subsp. multiflorum (Lam.) Husnot [syn. L. multiflorum Lam.]) line that when hybridized to tall fescue (Schedonorus arundinaceus (Schreb.) Dumort., nom. cons.) has been previously demonstrated. This research investigates and reports on the potential generation of haploids and/or dihaploids through this novel ryegrass when hybridized with L. multiflorum or L. perenne and production of the first invivo haploid/dihaploid Lolium sp. approach.

Effect of ploidy level on guard cell length and use of stomata to discard diploids among putative haploids in maize

AbstractGuard cell (GC) length proved to be efficient in sorting diploids from haploids in doubled haploid development in maize (Zea mays L). It compensates for the weakness of widely used R1‐nj marker approach that showed low reliability in haploids identification from tropical genotypes. Guard cell length differs between haploid and diploid plants, and these differences were evaluated in the progeny of three different induction crosses obtained using Krasnodar haploid inducer. Epidermal impressions of the abaxial surface of leaves removed from the second, third, and fourth nodes (from base to apex) were collected and measured using an optical microscope. This was also conducted on the flowering phenotype. Guard cell length varied according to germplasm source, leaf stage, and ploidy level. Mean GC length ranged from 23.67 to 33.82 μm in haploids, and from 36.1 to 41.25 μm in diploids. Based on these differences in GC length at any of the chosen leaf stages (second, third, or fourth), diploid and haploid maize plants were successfully classified. Classification efficiency was found to be more closely related to germplasm source than leaf stage. Comparing GC length according to phenotype (haploid or diploid), GC limits for classification as a diploid plant (threshold points) were estimated and ranged from 29.74 to 34.49 μm, depending on germplasm source. The highest false discovery rate was 2.93% and false negative rate was 15.06%, indicating that classification based on GC length was reliable.

Optimization of Conditions for Callus Induction from BC2F1 Population (Ranbir Basmati x PAU148) through Anther Culture

With different culture conditions and concentrations of growth regulators and supplements, Anther culture technique can be easily employed for the production of haploids under in vitro conditions.
 Aims: The present study was undertaken with the objective to optimize the development of doubled haploids using anthers for in vitro induction of callus on N6 medium.
 Place and Duration of Study: The samples (BC2F1 seeds) were raised previously in Skuast-J. From total degree program of 3 years, this work related to tissue culture technique was done in one year from January 2018 to January 2019.
 Methodology: The effect of levels of 2, 4-dichlorophenoxy acetic acid (2, 4-D) i.e. 0 to 3 mg/L in basal N6 media was observed on callus induction frequency (CIF). The effect of duration of cold pre-treatment was observed on callus induction frequency at 2.5 mg/L of 2, 4-D by giving the cold pre-treatment at 4ºC from 8 to 12 days. Also the effect of different amino acids was checked on callus induction frequency.
 Results: Highest callus induction frequency of 9.39 per cent was observed in N6 medium fortified with 2.5 mg/L 2, 4-D and lowest callus induction frequency of 2.52 per cent at the concentration of 1.0 mg/L. The cold pre-treatment for 10 days gave highest callus induction frequency of 1.44 per cent and lowest callus induction frequency of 0.44 per cent was obtained at cold pre treatment for 8 days. The highest callus induction frequency of 12.55 per cent was observed in case of media supplemented with 25 mg/L tryptophan and 40 mg/L cysteine and lowest callus induction frequency of 7.18 per cent was observed when media was supplemented with 560 mg/L proline.
 Conclusion: The cold pre-treatment of 10 days at 4ºC on media supplemented with 2.5 mg/L of 2, 4-D and combination of 25 mg/L tryptophan and 40 mg/L cysteine proves to provide best androgenesis conditions for anthers from BC2F1 population.

R1-nj expression in parental inbreds as a predictor of amenability of maize hybrids to R1-nj-based doubled haploid development

The current method of doubled haploid (DH) development in maize involves in vivo production of haploids using R1-njbased haploid inducer lines that upon use as male render a small fraction of seed in the pollinated female ears haploid. Identification of haploid seed relies on R1-nj marker expression in the endosperm and embryo, and the degree of its expression determines efficiency of DH development process. In the present study, R1-nj expression in the endosperm was characterized in crosses of CIMMYT’s R1-nj-based haploid inducer TAILP1 with a set comprising 18 early maturity hybrids and their 23 parental inbreds. Kernel colour inhibition was observed only in a small proportion of the hybrids and inbreds. Comparison of R1-nj expression in the hybrids and their parental inbreds revealed a distinct pattern, which may be useful in identifying source populations and/or determining parental constituents for synthesizing source populations with predicted amenability to doubled haploid development using R1-nj-based haploid inducers. However, deviation from the pattern was noted in hybrids involving inbreds with higher degree of colour inhibition, which suggests complex nature of R1-nj phenotype expression and necessitates further investigation involving larger sets of germplasm for dissecting the role of maternal and paternal genetic factors in determining R1-nj phenotype expression. The hybrids found exhibiting complete kernel anthocyanin expression in present study can be used directly as source populations for DH development using R1-nj based haploid inducers. Besides, since the inbreds used in the study have originated from and/or are accessible to CGIAR/NARS maize breeding programmes, the information on their kernel anthocyanin expression can be helpful in selection of source populations or generating new source populations amenable for DH development using R1-nj based haploid inducers.

Optimising protocol for successful development of haploids in marigold (Tagetes spp.) through in vitro androgenesis

For the first time, we have developed a protocol for rapid and successful induction of haploids in Tagetes spp. using in vitro androgenesis techniques. Various factors affecting the in vitro androgenesis response in Tagetes spp. were optimised. At first, a correlation was established between floret size/bud size and stage of microspore development in different genotypes of Tagetes erecta and Tagetes patula. The floret size of 3.5–4.0 mm (French marigold) and 3.0–3.5 mm (African marigold) contained the highest percent of microspores at early-uninucleate to early-binucleate stage. The frequency of shoot formation from anthers ranged from 0 to 78.6% in T. patula cv. Pusa Arpita and 0 to 63.9% in T. erecta cv. Pusa Basanti Gainda. The French marigold cultivars exhibited higher androgenic response over the African marigold cultivars. The effects of basal media, plant growth regulator combinations and sucrose concentration for direct differentiation of shoot buds and haploid induction were studied. Marigold anthers cultured on newly formulated EMS1 basal media (enriched with coconut water, AgNO3, PVP etc.) exhibited a very high androgenic response over the other commercially available media. We have also optimised the cold temperature pre-treatments and culture condition for high frequency androgenesis in marigold. Ten days cold pre-treated anthers along with 20 days dark incubation of cultures proved highly beneficial for haploid induction. Among a total of 424 anther-regenerants, 56 plants were randomly selected for flow cytometry and cytological analysis. The ploidy analysis revealed 14.3% of anther regenerants as dihaploids, 66.1% as tetraploid (similar to donor mother plant) and 19.6% as polyploid. The determination of ploidy level by counting the number of chloroplast in stomatal guard cells of marigold was also established for rapid screening of haploids. The identified haploids were successfully diploidised and are being utilised in the hybrid breeding programme at our institute. The developed protocol will facilitate doubled haploids based breeding in Tagetes spp. This is the first report of successful induction of haploids in Tagetes spp. using in vitro androgenesis techniques. We have studied various factors affecting the in vitro androgenesis response in Tagetes spp. A correlation was established between floret size/ bud size and stage of microspore development in different genotypes of Tagetes erecta and Tagetes patula. Basal media and cultural conditions were optimised for high frequency androgenesis in marigold. Haploids were successfully induced and their ploidy level was assessed through flowcytometry and cytological analysis.

Present status of haploidy research in onion (Allium cepa) – A review

Research on haploid induction and their subsequent utilization in basic and genomic research and breeding is still at its infancy due to paucity of research efforts, difficulties in various critical steps, large genome size and limited publications. Haploid development in onion (Allium cepa L.) is only feasible through in-vitro gynogenesis and is influenced by genotype, geographic origin, genetic constitution, physiological stage, growth conditions and cultural conditions. Pollination with irradiated pollen has been reported to induce haploids in onion. However, more successful methodology is through whole flower bud culture. After haploid induction, chromosome doubling, for doubled haploids (DHs) induction, is another limiting factor. Whole basal plant as an explant and amiprofos-methyl as a chromosome doubling agent has been found to be safe and effective. Potential of DH lines has been explored through the study of hybrids using DH as parents and hybrids have been found uniform and promising compared to conventionally developed hybrids. DH line 'Onion haploid OH-1' has been released by USDA to serve as a responsive control for the extraction of gynogenic haploids. DH's have been successfully utilized in onion genomic research for identification of gene and genomic regions affecting quality traits like colour, restorer of male sterility (Ms) locus, bolting, fructan etc. Future studies need to focus on identification of haploidy inducing gene/QTLs, gynogenic responsive genes, establishment of efficient chromosome doubling techniques, restoration of fecundity of DH genotypes and methodology standardisation to transfer them in less/non-responsive desirable genotypes.

Endogenous phytohormone profile during oat (Avena sativa L.) haploid embryo development

The development of embryos requires interaction of many endogenous hormones. The aim of the study was to determine which endogenous phytohormones are involved in the process of oat (Avena sativa L.) haploidization. Oat haploids were obtained by wide crossing with Zea mays L. The hormonal profiles of the ovaries with (OE) and without developed embryo (OWE) were compared. Phytohormone contents were measured by UHPLC coupled with mass spectrometer. The total content of indole-3-acetic acid (IAA), trans-zeatin (tZ), and kinetin (KN) in OE was significantly higher than in OWE. 4-Chloroindole-3-acetic acid was detected only in OWE. There were no differences between OE and OWE in the content of gibberellins (GA1, GA3, GA4, GA6, GA7) and stress hormones (abscisic, salicylic, jasmonic acids). Content of endogenous KN was highly negatively correlated with the percentage of haploid embryos, germinated haploid embryos, haploid plants on MS (in vitro), haploid plants in soil (ex vitro), and doubled haploid lines. The tZ content negatively correlated with the frequency of haploid embryo formation, germination, and haploid plants obtained in vitro, as opposed to GA1, which correlated positively. A positive correlation was found between IAA and tZ in OE, whereas in OWE it was a negative correlation. The profiles of phytohormones in OE and OWE were determined; however, their mode of action needs to be clarified.

181 Equine androgenic embryos: ability of the equine sperm to develop in a heterospecific ooplasm

Androgenic haploid embryos were originally produced for the study of certain aspects of early embryo development. The generation of androgenic haploid embryos allows us to better understand the complementary parental contribution to embryonic development, and to examine the effects of haploid development on gene expression. Because mare oocytes for research are scarce, the generation of heterospecific androgenic embryos could be useful to study aspects of the biology of early embryo development, or to identify genes and their variations or mutations that are responsible for reproduction-related problems in mares and stallions, which is of interest for the breeding industry. Therefore, the aim of this work was to study the capability of equine sperm to induce embryonic development after injection into an enucleated oocyte from a different species. Porcine cumulus-oocyte complexes (COC) were obtained from abattoir ovaries and placed in 100-µL drops in vitro maturation (IVM) medium for 42h. Cumulus cells were removed with hyaluronidase and vortexing. Then, mature oocytes were subjected to intracytoplasmic sperm injection (ICSI) with stallion frozen-thawed semen (according to Rodriguez et al. 2015). Immediately after the last injection, the zona pellucida of injected oocytes was removed with protease treatment, the oocytes were treated with cytochalasin B, and the metaphase II enucleated with a 20-µm micropipette. Finally, embryos were placed in culture medium (SOF) in plates with the well-of-the-well (WOW) system. As control treatment, non-enucleated pig oocytes were injected with stallion (CE) and boar (CC) semen. At Day 4, embryos were evaluated for cleavage and number of blastomeres, and stained with Hoechst 33342 to verify the presence of DNA in each blastomere under the UV light. Embryos were stored for future PCR studies to validate the presence of equine DNA. Data were analysed by chi-squared test to compare the cleavage of both controls with the androgenic embryos. From a total of 53 androgenic haploid embryos, the cleavage rate was 62% (33/53). Embryos were cleaved in 2 to 4 cells in 72.7%, 5 to 8 cells in 18.2%, and 9+ cells in 9.1% at Day 4. Presence of DNA in all blastomeres was observed in 60.6% (20/33) of the androgenic haploid embryos, while 21.2% (7/33) of the embryos had 10 to 50% of blastomeres with DNA, and 18.6% (6/33) of the embryos did not have DNA in their blastomeres. The ICSI control embryos cleaved in 45.3% (34/75) and 64.9% (98/151) for groups CC and CE, respectively. Cleavage rates in control CE were significantly higher than those in control CC (P<0.004). No statistical difference was observed in the control groups versus androgenic embryos. This preliminary results showed that a heterospecific ooplasm can be successfully used to allow an equine sperm DNA to decondense and to develop, even in absence of the female counterpart. Using this method, copies of a single sperm DNA can be produced to potentially evaluate individual aspects of early embryo development concerning the male contribution. This is the first report of successful androgenic embryos using a heterospecific oocyte to create copies of a horse sperm DNA.

Improving Response in Genomic Selection with a Population-Based Selection Strategy: Optimal Population Value Selection

Genomic selection (GS) identifies individuals for inclusion in breeding programs based on the sum of their estimated marker effects or genomic estimated breeding values (GEBVs). Due to significant correlation between GEBVs and true breeding values, this has resulted in enhanced rates of genetic gain as compared to traditional methods of selection. Three extensions to GS, weighted genomic selection (WGS), optimal haploid value (OHV) selection, and genotype building (GB) selection have been proposed to improve long-term response, and to facilitate the efficient development of doubled haploids. In separate simulation studies, these methods were shown to outperform GS under various assumptions. However, further potential for improvement exists. In this paper, optimal population value (OPV) selection is introduced as selection based on the maximum possible haploid value in a subset of the population. Instead of evaluating the breeding merit of individuals as in GS, WGS, and OHV selection, the proposed method evaluates the breeding merit of a set of individuals as in GB. After testing these selection methods extensively, OPV and GB selection were found to achieve greater responses than GS, WGS, and OHV, with OPV outperforming GB across most percentiles. These results suggest a new paradigm for selection methods in which an individual's value is dependent upon its complementarity with others.

Enhancing genetic gain in the era of molecular breeding.

As one of the important concepts in conventional quantitative genetics and breeding, genetic gain can be defined as the amount of increase in performance that is achieved annually through artificial selection. To develop pro ducts that meet the increasing demand of mankind, especially for food and feed, in addition to various industrial uses, breeders are challenged to enhance the potential of genetic gain continuously, at ever higher rates, while they close the gaps that remain between the yield potential in breeders' demonstration trials and the actual yield in farmers' fields. Factors affecting genetic gain include genetic variation available in breeding materials, heritability for traits of interest, selection intensity, and the time required to complete a breeding cycle. Genetic gain can be improved through enhancing the potential and closing the gaps, which has been evolving and complemented with modern breeding techniques and platforms, mainly driven by molecular and genomic tools, combined with improved agronomic practice. Several key strategies are reviewed in this article. Favorable genetic variation can be unlocked and created through molecular and genomic approaches including mutation, gene mapping and discovery, and transgene and genome editing. Estimation of heritability can be improved by refining field experiments through well-controlled and precisely assayed environmental factors or envirotyping, particularly for understanding and controlling spatial heterogeneity at the field level. Selection intensity can be significantly heightened through improvements in the scale and precision of genotyping and phenotyping. The breeding cycle time can be shortened by accelerating breeding procedures through integrated breeding approaches such as marker-assisted selection and doubled haploid development. All the strategies can be integrated with other widely used conventional approaches in breeding programs to enhance genetic gain. More transdisciplinary approaches, team breeding, will be required to address the challenge of maintaining a plentiful and safe food supply for future generations. New opportunities for enhancing genetic gain, a high efficiency breeding pipeline, and broad-sense genetic gain are also discussed prospectively.

Erratum to: Development of doubled haploids from an elite indica rice hybrid (BS6444G) using anther culture

Erratum to: Development of doubled haploids from an elite indica rice hybrid (BS6444G) using anther culture

Exploration of wild grass Imperata cylindrica for development of doubled haploids in winter x spring wheat (Triticum aestivum L.) hybrids accompanied with combining ability and hybrid potential estimation

Two spring and three winter wheat genotypes were crossed in Line x Tester manner forming six F1’s and three spring and one winter wheat genotypes were also crossed forming two three way F1’s. Combining ability studies indicated sufficient genetic variability in the breeding material. Among lines, HS542 showed good general combining ability for seed yield/plant and among testers KLE/BER/2*FL-8/DONSK-POLL was good combiner for 1000 seed weight. The cross HS542/Zander33 and HD2997/KLE/BER/2*FL-8/DONSK-POLL showed significant specific combining ability effects for 1000 grain weight. Intergeneric hybridization lead to the haploid embryo development from the crosses: HS542/China 84–40022, HS542/KLE/BER/2*FL-8/DONSK-POLL, HS526/ID 80994W/VEE/3/CHEN/3AL X FLW13 and HD2997/KLE/BER/2*FL-8/DONSK-POLL. These crosses also showed maximum values of heterosis for 1000 seed weight or spikelet per spike or seed yield/plant.

Development of doubled haploids from an elite indica rice hybrid (BS6444G) using anther culture

A total of 200 doubled haploids (DHs) were generated from an elite rice hybrid, ‘BS6444G’ for which an androgenic method was developed by manipulating the physical and chemical factors. The spike pretreated at 10 °C for 7–8 days was effective for callusing and green plant regeneration. The maximum callus frequency was achieved when the anthers cultured in N6 medium supplemented with 2.0 mg L−1 2,4-diclorophenoxyacetic acid, 0.5 mg L−1 6-benzylaminopurine and 3% maltose. Calli induced in N6 media also showed significant green shoot regeneration in MS medium supplemented with 0.5 mg L−1 1-napthalene acetic acid, 0.5 mg L−1 kinetin, 1.5 mg L−1 benzylaminopurine and 3% sucrose producing 210 green plants. Assessment of the ploidy status showed 95.71% fertile diploids and 4.2% polyploids; no haploids were observed. A total of 38 sequence-tagged microsatellite (STMS) markers proved able to discriminate a heterozygote from all the 200 DHs. The DHs grown in the field showed significant variation for their agronomic traits. Comparison of traits with control indicates homogeneity within each DH line and significant variance of traits between DH lines. Nine DH lines produce higher grain yield than the hybrid parent which suggests the possibility of exploiting hybrid vigor in indica rice through the development of DH lines of high yielding hybrids.

Optimizing protocol for efficient microspore embryogenesis and doubled haploid development in different maturity groups of cauliflower (B. oleracea var. botrytis L.) in India

Microspore culture technique is widely used in Brassicas for development of haploids and doubled haploids (DH) in breeding programmes and genetic studies. Indian cauliflower, a group of vegetable Brassica; with tolerance to higher temperature and moisture, has been selected in India for last 200 years. In India, cauliflowers are grown throughout the year in different climatic zones based on their temperature adaptation. For the first time, we report microspore embryogenesis in different maturity groups of Indian cauliflowers. The main objective of our study was to optimize protocol for efficient microspore embryogenesis in different types of cauliflower cultivated in India. We studied different factors like bud size, developmental stage of the microspores, culture density and heat shock treatments for efficient microspore embryogenesis in four different genotypes representing four different maturity groups. Among different factors, the bud size and microspore developmental stage was found to be more critical for successful microspore embryogenesis. The optimum bud size for high frequency microspore embryogenesis for the two early maturity groups (September–October maturity) was 4.0–4.5 mm, whereas, it was 4.5–5.0 mm for the cultivars grown for November onwards harvesting. These optimized bud sizes contained the highest percent of viable microspores and maximum number of microspores at late uninucleate to early binucleate stage. The microspore density of 6–8 × 104 cells per mL was found most suitable for efficient microspore embryogenesis. A post-culture incubation of microspores at 30.0 °C/32.5 °C for 24 h followed by maintenance at 25 °C was found optimum for induction of microspore embryos in different maturity group of Indian cauliflower. The flow cytometry analysis of 646 microspore derived plants revealed that more than 60 % plants were spontaneous diploids and less than 15 % were haploids. However, the genotypes from different groups showed varied response in different culture density and temperature treatments. Thus, it is necessary to optimize genotype specific protocol for efficient microspore embryogenesis before its routine application. The protocol developed in this study will facilitate doubled haploids based breeding and genetic studies of all types of cauliflower grown in India.

Behind the scenes of microspore-based double haploid development in Brassica napus: A review

Abstract Double haploids are extremely valuable for generating completely homozygous genotypes and have been used in plant breeding program of a number of crop species. This method is a much faster way of developing genetically pure

Dihaploid induction in tetraploid durum wheat (Triticum durum L.) using pollen of Imperata cylindrica

Doubled haploidy breeding protocols have revolutionized the varietal development programmes in bread wheat, however, the protocols have not proved much efficient in durum wheat. Presently, the durum wheat × maize system is being widely followed for haploid induction but the frequency of haploid development is very poor which has limited its application in durum wheat improvement programmes. In order to formulate an efficient wide hybridization-mediated approach for haploid induction in durum wheat, different genotypes of durum wheat were subjected to hybridization with Imperata cylindrica, a wild perennial grass for the first time in this laboratory. The investigation was carried out for two seasons. During the 1st year, the investigation was carried out on one genotype only, Langdon, in order to notice the development of haploid embryos and the factors that influence the haploid induction frequency. The most important factor influencing the embryo formation frequency was found to be the concentration of 2,4-dichlorophenoxyacetic acid (2,4-D). Upon pollinating the emasculated spikes of durum wheat genotypes, various concentrations of 2,4-D were injected into the uppermost internode of wheat culm for three consecutive days after pollination to find out the most responding concentration for haploid induction. During the next year, the protocol was applied to eight durum wheat genotypes. The frequency of haploid induction parameters varied with the durum wheat genotypes as well as 2,4-D concentration used. The mean pseudoseed and embryo formation frequency over all the genotypes ranged from 30.2 to 56.3% and 1.2 to 18.4%, respectively. The average embryo formation frequency over all the genotypes was found to be highest (18.4%) at 250 mg/l 2,4-D whereas it was superior for the genotypes WH 896 and Langdon over all the 2,4-D concentrations. At the most responding 2,4-D concentration (250 mg/l), the genotype A-9-30-1 exhibited the highest embryo formation frequency (32.1%). The ploidy status of the developed embryos was identified using cytological analysis carried out on the rootlets of the tissue culture generated plantlets.

The D-genome plays a critical role in the formation of haploid Aegilops tauschii through Imperata cylindrica mediated uniparental chromosome elimination

Doubled haploid (DH) breeding eventually offers unique advantages for rapid genetic improvement of wheat in a single generation by achieving absolute homozygosity and enhanced selection efficiency. A new perennial invasive weed i.e., Imperata cylindrica has already proved its importance in haploid wheat induction through Imperata-mediated chromosome elimination process. Whereas, cytological evidences and the actual mechanism of haploid induction through chromosome elimination in wheat x I. cylindrica hybrids are well known, but which genome(s) of wheat (AA/BB/DD) are actually involved in such haploid formation has remained elusive. Therefore, in order to identify the genome-specific triggering of Imperata-mediated chromosome elimination in development of wheat haploids, 11 wheat genotypes including Ae. tauschii and D-genome chromosome substitution lines of the durum wheat variety ‘Langdon’ were crossed with I. cylindrica and examined with the prime aim of producing haploids of Ae. tauschii. The crosses suggest that only bread wheat, Ae. tauschii and Triticale (containing D genome) were successful to develop haploid plants through chromosome elimination process. Higher frequencies of embryo formation were obtained primarily in substitution lines with chromosomes 7D. The detailed anatomical and cytological analyses suggest that : (i) genome/genotypic specificity plays a key role in haploid induction through Imperata-mediated chromosome elimination process, (ii) D-genome triggers the chromosome elimination and haploid production in wheat x Imperata, (iii) some D-genome chromosomes (primarily 7D) substituted lines in wheat genetic background may enhance crossability with Imperata in the formation of haploid wheat. To the best of our knowledge, this is the first report of haploid induction in Ae. tauschii.

Induction of Gynogenetic and Androgenetic Haploid and Doubled Haploid Development in the Brown Trout (Salmo trutta Linnaeus 1758)

Gynogenetic and androgenetic brown trout (Salmo trutta Linnaeus 1758) haploids (Hs) and doubled haploids (DHs) were produced in the present research. Haploid development was induced by radiation-induced genetic inactivation of spermatozoa (gynogenesis) or eggs (androgenesis) before insemination. To provide DHs, gynogenetic and androgenetic haploid zygotes were subjected to the high pressure shock to suppress the first mitotic cleavage. Among haploids, gynogenetic embryos were showing lower mortality when compared to the androgenetic embryos; however, most of them die before the first feeding stage. Gynogenetic doubled haploids provided in the course of the brown trout eggs activation performed by homologous and heterologous sperm (rainbow trout) were developing equally showing hatching rates of 14.76 ± 2.4% and 16.14 ± 2.90% and the survival rates at the first feeding stage of 10.48 ± 3.48% and 12.78 ± 2.18%, respectively. Significantly, lower survival rate was observed among androgenetic progenies from the diploid groups with only few specimens that survived to the first feeding stage. Cytogenetic survey showed that among embryos from the diploid variants of the research, only gynogenetic individuals possessed doubled sets of chromosomes. Thus, it is reasonable to assume that radiation employed for the genetic inactivation of the brown trout eggs misaligned mechanism responsible for the cell divisions and might have delayed or even arrested the first mitotic cleavage in the androgenetic brown trout zygotes. Moreover, protocol for the radiation-induced inactivation of the paternal and maternal genome should be adjusted as some of the cytogenetically surveyed gynogenetic and androgenetic embryos exhibited fragments of the irradiated chromosomes.

Development of Maize Haploid Inducer Lines and Doubled Haploid Lines in Pakistan

The work on maize doubled haploid development has started at the Maize Research Station, Ayub Agricultural Research Institute, Faisalabad, Pakistan during 2010 in collaboration with University of Agriculture F aisalabad, Pakistan. The aim of current study was to develop locally adopted maize haploid inducer lines utilizing cheap and easy source of Stock6 and indeterminate gametophyte mutant ig1 gene lines imported from Maize genetic Coop Stock Centre. The lines were later utilized to produce doubled haploid inbred lines. The environmental conditions of Faisalabad are extreme in nature. In spring crop during pollination period temperature may reach up to 45°C. Therefore, locally adopted haploid inducers are needed . Best performing local inbred lines were screened having branched heavy tassel, bold seed, good pollen shedding ability and vigor. These lines were utilized as female donor parent while imported lines as recurrent pollen parent in back cross