Doubled Haploid Production In Wheat Research Articles

Improving detached tiller culture for doubled haploid production in wheat (T. aestivum L.) by post‐pollination fungicides application and UV radiation treatment

AbstractThis work aims at improving the proven protocol for doubled haploid production in wheat. This procedure has some deficits, for example, microbial infection of ears as well as of the nutrient culture medium. In the present study, the potential effect of sterilization by detached tiller culture fungicides of ears as well as of nutrient medium to control microbial infection, and its effect on the formation of caryopses and embryos (CFF, EFF) was studied. In a further experiment, the UV‐light was used as physical sterilant for the nutrient medium. All of the fungicides and their concentrations studied were found to be effective to control infection on ears except the concentration 0.05% tilt. 0.1% tilt gave the highest CFF (79.5%) followed by 0.1% Bayleton (78.0%). The lowest CFF was recorded in control treatment (48.8%). Among Bavistin treatments, the 0.2% Bavistin gave maximum CFF (68.5%) as well as EFF (29.1%) followed by 0.1% Bavistin (64.9%, 23.7%). The higher concentration of tilt (0.2%) showed detrimental effects on EFF (17.5%). Bavistin in tiller culture nutrient medium reduced infection in medium and was shown to improve CFF and EFF (67.4%, 29.8%).

Anther Culture Protocols for Barley and Wheat.

Doubled haploid (DH) techniques remain valuable tools for wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) genetic improvement, and DH populations are used extensively in breeding and research endeavors. Several techniques are available for DH production in wheat and barley. Here, we describe two simple, robust anther culture methods used to produce more than 15,000 DH wheat and barley lines annually in Australia.

In vitro kultura antera kao efikasno primenjena tehnika za proizvodnju dvostrukih haploida pšenice (Triticum aestivum L.)

In vitro anther culture is the most commonly-used technology for doubled haploid production in wheat (Triticum aestivum L.) and various cereals. The technology application allows the genetic attainment of homozygous pure lines from heterozygous breeding material in a single generation, and has become a rapid alternative to the conventional breeding methods. This technology also contributes to more accurately assess QTL × environmental interactions and has been applied in: genetic researches for marker-trait association study, genomics and as a target for transformation, genetic engineering, gene mapping, and mapping of quantitative trait loci (QTLs). This paper reviews the most important factors that limit the efficiency of winter wheat (Triticum aestivum L.) anther culture, such as genotype dependency and albinism, in addition to other affecting factors including the collection time of tillers and physiological growth environments of plants. Factors that improve the efficiency of wheat anther culture in mitigating both genotypic dependency and albinism have been included in this review, too, for example, genetic improvements by following a strategy to realize this purpose through using responsive breeding material for crossing, application of convenient stress pre-treatments (cold pre-treatments, heat shock treatment, colchicine, hormones, and chemicals) to induce androgenesis, and improvement of the composition of anther culture media and culture conditions. We have also highlighted the methods of chromosome doubling for the haploid wheat produced by anther culture androgenesis, such as spontaneous chromosome doubling or use of colchicine and anti-mitotic chemicals at an early stage of anther culture or as root immersion treatment.

New protocol for colchicine induced efficient doubled haploidy in haploid regenerants of tetraploid and hexaploid wheats at In vitro level

An investigation to standardize the protocol for in vitro application of colchicine for enhancing the doubled haploid production in wheat was done. Two tetraploid (PDW-314 and A-9-30-1); and two hexaploid (DH-40 and C-306) wheat genotypes were used as maternal parents, whereas, the pollen sources involved Zea mays (cv. Bajaura Makka) and Imperata cylindrica. During the rabi seasons of years 2013–14 and 2014–15, wheat × maize and wheat × I. cylindrica hybridization was carried out followed by treatment of their haploids produced as a result of elimination of chromosomes of maize and I. cylindrica respectively, with varied doses of colchicine for different durations The various doses of colchicine were categorized into two groups: lower doses for longer durations (0.01, 0.025, 0.05% each for 5, 7, 9, 11 hrs) and higher doses for shorter durations (0.05, 0.075, 0.10, 0.15, 0.20, 0.25% each for 5, 4, 3, 2 hrs). The response of different concentrations of colchicine applied for varied durations revealed significant differences for various doubled haploidy parameters viz., per cent survived plants, per cent doubled haploid formation and per cent doubled haploid seed formation. In hexaploid and tetraploid wheats, colchicine doses of 0.075% for 4 hrs and 0.15% for 4 hrs, respectively were established as optimum for enhanced doubled haploid production.

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.

Bcl-2△21 and Ac-DEVD-CHO Inhibit Death of Wheat Microspores

Microspore cell death and low green plant production efficiency are an integral obstacle in the development of doubled haploid production in wheat. The aim of the current study was to determine the effect of anti-apoptotic recombinant human B-cell lymphoma-2 (Bcl-2△21) and caspase-3-inhibitor (Ac-DEVD-CHO) in microspore cell death in bread wheat cultivars AC Fielder and AC Andrew. Induction medium containing Bcl-2△21 and Ac-DEVD-CHO yielded a significantly higher number of viable microspores, embryo-like structures and total green plants in wheat cultivars AC Fielder and AC Andrew. Total peroxidase activity was lower in Bcl-2△21 treated microspore cultures at 96 h of treatment compared to control and Ac-DEVD-CHO. Electron paramagnetic resonance study of total microspore protein showed a different scavenging activity for Bcl-2△21 and Ac-DEVD-CHO. Bcl-2△21 scavenged approximately 50% hydroxyl radical (HO•) formed, whereas Ac-DEVD-CHO scavenged approximately 20% of HO•. Conversely, reduced caspase-3-like activities were detected in the presence of Bcl-2△21 and Ac-DEVD-CHO, supporting the involvement of Bcl-2△21 and Ac-DEVD-CHO in increasing microspore viability by reducing oxidative stress and caspase-3-like activity. Our results indicate that Bcl-2△21 and Ac-DEVD-CHO protects cells from cell death following different pathways. Bcl-2△21 prevents cell damage by detoxifying HO• and suppressing caspase-3-like activity, while Ac-DEVD-CHO inhibits the cell death pathways by modulating caspase-like activity.

Production of wheat doubled haploids by in vitro anther culture

The in vitro culture response of anthers, originating from high yielding and resistance to lodging F1 soft wheat hybrid plants selected under drought conditions, was investigated. The spikes were collected when the microspores were at the mid-uninucleate stage and after 3 days at 40 C for cold pre-treatment, the anthers were cultured in N6 and Gamborg- Eveleg B5 (B5) solid medium. Progeny from 17 different F1 families were used and 9 of them responded to in vitro anther culture and 7 of them produced fertile green plants. Doubled haploid green plants were fertile and produced seeds. Induction of aploid development depends on genotype, stage of male gametophyte development, composition of nutrient media and conditions of cultivation. The effect of the composition of media and different genotypes on wheat anther culture was investigated. From the genotypes produced regenerants, the high yielding genotypes from local breeding collection of wheat produced more albino plants (35.2%) compared to high yielding genotypes from CYMMIT collection (24.92%) ones. Independent of culture medium composition a ratio of doubled haploid/haploid was 4.57:1 and a ratio of haploid/aneuploid was 18.5:1, in particular 81.28 % of green plants were doubled haploid, 17.76% were haploid and the rest (0.96%) were aneuploid

Enhanced induction of microspore embryogenesis after n-butanol treatment in wheat (Triticum aestivum L.) anther culture

The aim of this study was the improvement of embryo production in wheat anther culture. Three butanol alcohols, n-butanol, sec-butanol and tert-butanol, were evaluated for their effect on microspore embryogenesis in two spring cultivars of wheat, Pavon and Caramba. Application of n-butanol, at 0.1 and 0.2% (v/v) in the induction media for 5 h, highly improved embryo production in both cultivars. Sec- and tert-butanol performed similarly to control plates. Regeneration ability was unaffected by any butyl-alcohol treatment. As a consequence of the higher embryo production after n-butanol treatment, the number of green regenerated plants increased up to five times in cultivar Pavon and up to three times in cultivar Caramba. The percentage of green plants was improved or unaffected by the treatment. Doubled haploid plant production was between 2 and 4 times higher after n-butanol treatment than in control plates. Therefore, n-butanol was successfully applied in the production of wheat doubled haploids. This primary alcohol is known as an activator of phospholipase D and has been previously reported to disrupt cortical microtubules and detach them from the plasma membrane in plants. Its effects on androgenetic induction could confirm the importance of microtubule regulation in plant cell fate, specifically in microspore development. A possible implication of phospholipase D is discussed.

Production of wheat doubled haploids by pollination with Job's tears (Coix lachryma-jobi. L.).

Wheat (Triticum aestivum L.) haploids were produced by crossing with Job's tears (Coix lachryma-jobi L.) as the pollen parent. Pollination was followed by 2,4-D treatment, detached tiller culture, and embryo culture, as described for maize pollination. The frequency of embryo formation was similar to that obtained by crossing wheat with maize pollen. Job's tears is a perennial plant which forms several stalks and its pollen can be collected throughout the year when the plant is maintained in a controlled environment. Our results indicate that Job's tears can be used as the pollen parent for wheat crosses for haploid production without requiring synchronization of flowering dates.

Production of wheat doubled haploids via wide crosses in New Zealand wheat

Doubled haploid (DH) technology allows the production of homozygous cereal lines in a single generation. The integration of DH technology into New Zealand wheat (Triticum aestivum) breeding and genetics programmes has the potential to reduce the breeding time of new cultivars and improve our understanding of agronomic ally important genetic traits. Wheat × maize (Zea mays) crosses are now widely used in the production of wheat DHs. The aim of this study was to develop a wheat × maize method of DH production using New Zealand germplasm. A number of New Zealand wheat cultivars were crossed with different maize genotypes. A successful method for producing wheat DHs was achieved with all genotypes. Several factors were identified as being important in the efficiency of wheat DH production. These included: seasonal constraints, time of embryo excision, method of auxin application, and culture media composition. Of these factors, seasonal constraints were the major limitation in the use of the wheat × maize method for producing DHs in New Zealand.

Agronomic performance of lines derived from anther culture, maize pollination and single-seed descent in a spring wheat cross

Anther culture and maize hybridization are two frequently used techniques for doubled haploid production in wheat (Triticum aestivum L.). Information on the field performance of lines derived from these techniques is limited. This study was conducted to compare the performance of F(4:6) lines obtained by single-seed descent with lines obtained by anther culture and maize (Zea mays L.) pollination from the same cross of spring wheat, 'Chris'/MN 7529. Thirty-three lines derived from each of those techniques were evaluated in six environments for grain yield, protein content, test weight, heading date, kernel weight and plant height. Mean performance of the single-seed descent lines exceeded performance of the anther culture lines for grain yield, kernel weight and plant height with no apparent differences for grain protein content, test weight and heading date. No differences between trait means for the single-seed descent and maize pollination lines were found except for plant height. The best 5 lines from each method for grain yield, protein content and test weight were similar in performance except that the protein content was higher for the maize pollination lines than for the single-seed descent lines. Acceptable levels of agronomic performance could be found among lines from each method. Wide acceptance of the doubled haploid technique for pure line production in breeding programs may, however, be limited by the often poor efficiency of doubled haploid line production, resulting in smaller population sizes for selection of desirable traits in comparison to the single-seed descent method.

The Effects of Temperature and Light Intensity on Embryo Numbers in Wheat Doubled Haploid Production through Wheat×Maize Crosses

Triticum aestivum×Zea mayscrosses are now widely used in the production of wheat doubled haploids to produce homozygous lines. Seasonal effects are known to influence the number of haploid embryos produced through wheat×maize crosses, but the effects of temperature and light have not been quantified. This study investigated the effect of temperature and light intensity on haploid embryo production. New Zealand wheat cultivars were grown in a glasshouse until booting when they were transferred to growth cabinets at three temperatures (day/night; 17/12, 22/17 or 27/22 °C at an irradiance of 250 μmol m−2s−1PAR). In another experiment, wheat lines were transferred to a growth cabinet at one of three light intensities (300, 500 or 1000 μmol m−2s−1PAR at 22/17 °C day/night, with a photoperiod of 16 h). The temperature and light intensity at which pollinations were made and subsequent fertilisation and embryo development occurred, significantly (P<0.01) influenced the frequency of haploid embryo production. The optimal temperature for embryo recovery was 22/17 °C. The greatest number of embryos was produced at a light intensity of 1000 μmol m−2s−1. These findings will result in improvements in the overall efficiency of the wheat×maize system for wheat doubled haploid production.

Nuclear and Cytoplasmic Control of Anther Culture Response in Wheat: III. Common Wheat Crosses

Anther culture represents a potentially important technology for production of wheat doubled haploids for plant breeding and genetics research; however, the yields of haploids from culture must be increased to achieve competitive efficiencies for the method. Genetic differences for culturability are now well known, but the relative roles of nuclear and cytoplasmic genetic factors are still unclear. The purpose of this study was to determine the significance of cytoplasm genetic variation for anther culture responses among common T. aestivum wheats. Plants were grown in a greenhouse and potato‐4 and 190‐2 media were used as callus induction and plant regeneration media, respectively. Large genotype differences in an ther culture response were observed. Among the 18 pairs of reciprocal crosses studied, 9 showed reciprocal differences for green plant regeneration, 4 showed significant differences for callus induction, and 3 were different for plant regeneration. The occurrence of reciprocal differences for all components of anther culture response, especially for green plant regeneration, indicates the the cytoplasm of microspores or maternally inherited factors interact with nuclear genes to control the response of wheat genotypes to anther culture. The results provide substantial new supporting evidence that nuclear, cytoplasm, and nuclear ✕ cytoplasm genetic interactions control the responses of common wheats to anther culture.