Fertile Doubled Haploid Plants Research Articles

Factors inducing regeneration response in oat (Avena sativa L.) anther culture

The efficiency of embryogenesis of anther culture was compared using four cultivars of oat (Avena sativa L.): ‘Akt’, ‘Bingo’, ‘Bajka’, and ‘Chwat’. Despite the high resistance of oat to the process of androgenesis, all tested cultivars produced embryo-like structures and only two of them, ‘Akt’ and ‘Chwat’, produced fertile doubled haploid plants. A strong cultivar dependency was observed during induction of androgenesis. Further, cold pretreatment together with high temperature shock enhanced the efficiency of this technique. The highest number of embryo-like structures and haploid plants was obtained from cv. ‘Chwat’ (3.6% and 0.8%, respectively). Embryo-like structure formation also depended on the distance from the base of the flag leaf to the penultimate leaf of the panicle. Most of them were observed on anthers harvested from panicles of which the distance from the base of the flag leaf to the penultimate leaf was less than 4 cm. The presence of the induction medium supplemented with different plant growth regulators was essential for the induction of embryo-like structures but did not increase the production of haploid plants and doubled haploid lines. The highest number of embryo-like structures and plants was obtained on W14 medium with the addition of 2.0 mg/dm3 2,4-dichlorophenoxyacetic acid and 0.5 mg/dm3 kinetin (2.7%). The low haploid plant regeneration rate (from 0.03 to 0.05%) still limits the practical application of anther culture for the production of doubled haploid lines in oat.

Isolated microspore culture of oat (Avena sativa L.) for the production of doubled haploids: effect of pre-culture and post-culture conditions

The production of doubled haploid (DH) plants from microspores is an important technique used in plant breeding programs and basic research. Although doubled haploidy efficiencies in wheat and barley are sufficient for breeding purposes, oat (Avena sativa L.) is considered recalcitrant. The objective of this project was to develop a protocol for the production of microspore-derived embryos of oat and further develop these embryos into fertile DH plants. A number of experiments were conducted evaluating the factors influencing microspore embryogenesis, i.e. donor plant conditions, pretreatments, media composition, and culture conditions. The initial studies yielded little response, and it was not until high microspore densities (106 microspores/mL and greater) were used that embryogenesis was achieved. Depending on the treatment, yields of over 5,000 embryos/106 microspores were obtained for breeding line 2000QiON43. The doubled haploidy protocol includes: a 0.3 M mannitol pretreatment of the tillers for 7 days, culture in W14 basal medium with a pH of 6.5–7.5, a microspore density of 106 microspores/mL, and continuous incubation at 28 °C incubation. The resulting embryos observed after 28 days were plated onto solidified W14 medium with 0.8 or 1.0 g/L activated charcoal. A colchicine treatment of 0.2 % colchicine for 4 h resulted in conversion of 80 % of the plants from haploid to DH. This protocol was successful for the production of oat microspore-derived embryos and DH green plants with minimal albinism. DH seed was produced and planted for evaluation in a field nursery.

Improvements in the production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this study was to produce durum wheat doubled haploid (DH) plants through the induction of microspore embryogenesis. The microspore culture technique was improved to maximize production of green plants per spike using three commercial cultivars. Studies on factors such as induction media composition, induction media support and the stage and growth of donor plants were carried out in order to develop an efficient protocol to regenerate green and fertile DH plants. Microspores were plated on a C(17) induction culture medium with ovary co-culture and a supplement of glutathione plus glutamine; 300 g/l Ficoll Type-400 was incorporated to the induction medium support. Donor plants were fertilized with a combination of macro and microelements. With the cultivars 'Ciccio' and 'Claudio' an average of 36.5 and 148.5 fertile plants were produced, respectively, from 1,000 anthers inoculated. This technique was then used to produce fertile DH plants of potential agronomic interest from a collection of ten F(1) crosses involving cultivars of high breeding value. From these crosses 849 green plants were obtained and seed was harvested from 702 plants indicating that 83% of green plants were fertile and therefore were spontaneously DHs. No aneuploid plant was obtained. The 702 plants yielded enough seeds to be field tested. One of the DH lines obtained by microspore embryogenesis, named 'Lanuza', has been sent to the Spanish Plant Variety Office for Registration by the Batlle Seed Company. This protocol can be used instead of the labor-intensive inter-generic crossing with maize as an economically feasible method to obtain DHs for most crosses involving the durum wheat cultivars grown in Spain.

Production of doubled haploids in durum wheat (Triticum durum Desf.) through culture of unpollinated ovaries

The objective of this work was to produce doubled haploid plants from durum wheat through gynogenesis using unpollinated ovary culture of three local Tunisian genotypes (Jenah Khotifa, Hmira, Azizi) and three improved cultivars (Karim, Khiar, Razzek). A total of 12,000 unpollinated ovaries were cultured in this study. Spikes were either pretreated at 4°C for 14 days or at 4°C in a mannitol solution (0.3 M) for 7 days. Induction was performed using two media. We showed that ovary development, callus and plantlet regeneration was influenced significantly by genotype and growth conditions. The highest regeneration frequency was obtained when the microspore population was in the late mononucleate to binucleate stage. Our results suggested that the cold pretreatment for 14 days was more efficient than the cold treatment in a mannitol solution. Furthermore, the addition of 2,4-D, vitamins and glutamine, and the use of maltose as sugar source in media improved the ovary culture. When the unpollinated ovaries were cultured under the conditions found to be optimal in the present study, a total of 84 plants were produced, all green and haploid. The best levels for regenerated plants were obtained with the cultivars Khiar (3.5%), Hmira (3.1%) and Karim (1.5%). Fertile doubled haploid plants were obtained by colchicine treatment. This result represents a modern tool for breeders to produce durum wheat homozygous lines in a few months.

Complementary strategies for ploidy manipulations in gynogenic onion ( Allium cepa L.)

Successful application of doubled-haploid (DH) technology in onion ( Allium cepa L.) breeding programs requires production of large numbers of fertile DH plants. Only a small percentage of the onion plants recovered via gynogenesis are spontaneous DH, so induction of chromosome doubling is required. The chromosome doubling step is currently one of the major limitations in onion DH programs. To address this problem, three complementary strategies for increasing the frequency of DH onion plants obtained via gynogenesis were investigated. First, the doubling efficiencies of three anti-mitotic agents (amiprofos methyl (APM), colchicine, and oryzalin) on whole basal explants from in vitro haploid plant were tested. APM at 100 and 150 μM and colchicine at 750 and 1000 μM gave similar regeneration of explants (70–80%) and recovery of diploid plants (25–32%). Colchicine is highly toxic to mammalian cells, so the comparable efficacy of APM at much lower concentrations offers a safer approach. Some gynogenic plants remain haploid even after exposure to anti-mitotic agents. The second strategy recovered diploid plants from such haploid plants via spontaneous chromosome doubling in somatic shoots regenerated from haploid flowers cultured in vitro. About 60% of the somatic shoots formed on flowers from haploid plants were diploid. Addition of colchicine (12.5–50 μM) to the shoot regeneration medium did not increase the frequency of diploid plants recovered but raised the frequency of tetraploid somatic plants. The third strategy used a second cycle of gynogenesis to recover diploid plants from in vivo tetraploid and mixoploid plant materials. The diploid gynogenic plants recovered produced bulbs comparable to those of diploid control plants, averaging ca. 200 g. Use of the three strategies in combination can maximize the recovery of fecund DH plants in gynogenesis-derived populations for use in onion breeding programs.

In vitro microspore selection in maize anther culture with oxidative-stress stimulators

In order to produce doubled-haploid maize plants tolerant of oxidative stress, in vitro microspore selection was carried out in anther culture with reactive oxygen species (ROS) progenitors such as paraquat, menadione, tert-butylhydroperoxide (t-BHP), and methionine combined with riboflavin. All the ROS progenitors reduced the anther induction, the formation of microspore-derived structures, and their regeneration potential. Abnormal cell divisions and progeny cell degradation could be observed during the development of microspores treated with ROS progenitors. Menadione and t-BHP influenced the microspore developmental pathway, as menadione induced the formation of embryoids, while t-BHP increased the proportion of calli in the microspore-derived structures. As the result of in vitro selection, 15, 10, 10, and 3 fertile doubled-haploid plants were obtained in cultures treated with paraquat, t-BHP, methionine combined with riboflavin, and menadione, respectively.

Efficiency of Microspore Culture for Doubled Haploid Production in the Breeding Project "Czech Winter Rape"

A selected set of twenty inter-line F<sub>1</sub> hybrids of Brassica napus L. was evaluated for their capacity to produce microspore-derived doubled haploid (DH) plants subsequently utilized in the breeding project “Czech Winter Rape”. All tested genotypes were able to produce microspore-derived embryos based on the standardized protocol previously adopted for the Czech winter rape varieties/breeding lines; nevertheless, significant differences were found in the frequency of microspore-derived embryo production as related to particular genotypes. However, the fertile DH regenerants were obtained only in 12 hybrid lines of this set. The other 8 lines failed to produce fertile DH plants for several reasons, mainly due to altered morphology of microspore embryos connected with limited germination/conversion potential and due to unsuccessful colchicine treatment resulting in haploid or mixoploid (chimeric) regenerants exhibiting partial or full sterility. In general, colchicine treatment induced diploid (doubled haploid), polyploid (triploid, tetraploid) and mixoploid (containing both euploid and aneuploid nuclei) plants. The fully aneuploid plants were recorded only sporadically. Some modifications of culture protocol (removal of embryo cotyledons before transfer to a germination medium; production of more plantlets from a single embryo by rooting multiple shoots; repeated colchicine treatment of unsuccessfully doubled plants in a later phase of growth) could enhance the final yield of DH fertile regenerants in our experiments. In total, 590 microspore-derived regenerants were produced in a series of experiments, 236 (40%) of them represented fully fertile DH plants. The R2 seeds (progeny of DH R1-generation) of the most promising hybrid-lines were included in the tests of agronomic performance in field conditions.  

Production of doubled haploids in durum wheat (Triticum turgidum L.) through isolated microspore culture

The objective of this work was to produce doubled haploid plants from durum wheat through the induction of androgenesis. A microspore culture technique was developed and used to produce fertile doubled haploid plants of agronomic interest. Five cultivars, one selected line, plus a collection of 20 F(1) crosses between different genotypes of high breeding value were used. Studies on several factors such as pre-treatments and media components were carried out in order to develop a protocol to regenerate green haploid plantlets. Anthers were pre-treated in 0.7 M mannitol. Microspores, from anther maceration, were plated on a C(17) induction culture medium with ovary co-culture. The optimum regeneration medium J25-8 was used. From 35 microspore isolations, 407 green plantlets were obtained. With this technique mature embryos were obtained. Green plants were regenerated from all genotypes used and approximately 67% of them were spontaneously doubled haploids. Some haploids and a very few polyploids plants were obtained. From the 407 plants, 275 were completely fertile and gave enough seeds to be assayed in the field. This protocol could be used complementary to or instead of the intergeneric crossing with maize as an economically feasible method to obtain doubled haploids from most durum wheat genotypes.

Green haploid plants from unpollinated ovary culture in tetraploid wheat (Triticum durum Defs.)

In Triticum durum Defs., gynogenesis was investigated on six genotypes, Cocorit, Isly, Jori, Oued Zenati, Sarif, issued from Morocco,and Cham1 from Syria. The experiments described here were made during two seasons (spring and summer) in which batches of 1,036 and 3,750 unpollinated ovaries were used. A 4 °C pretreatment was applied, during 7, 11 or 15 days and the A (modified Jahne's et al.,1991) and B (modifed San Nœum's, 1976) induction media were compared. The spikes were harvested when microspores were at bi- or trinucleate stage. After the 4 °C cold pretreatment, they were sterilized, and the excised ovaries underwent the following phases and sequence of successive media. The A or B ‘induction’ media, induced cell divisions in the female gametophytes, and after 4 to 9 weeks in dark conditions, the swelling ovaries burst. The growing calluses were excised and transferred to 16 h. light onto a differentiation medium (dif); there generated shoots were then placed on a development (dev), followed by a rooting medium (r). The comparison of the two experiments showed that summer was the best season, for in vitro unpollinated ovary culture; this period corresponds to a growth of mother-plants during spring, while those developing in winter gave rise to lower scores. In Experiment 1 the best level of regenerated plants per 100 ovaries were obtained for Isly with 14.3% and for Oued Zenati with 10.1%. In Experiment 2, the best values were those of Isly with 21.8%, after a 7 day cold pretreatment and use of induction medium B, and of Cocorit with 18.1%. Jori, exhibited good results, in three define situations, with 17.1%, 16%, and 9.3%.Only Cham1 and Sarif showed very low scores or no plants. Jori remaining aside, for each genotype, the best values rose up from a specific treatment combination, as a very strong interaction was expressed between genotype, induction medium and cold pretreatment duration. After this ‘first phase’ of regeneration by ovary culture 150 haploid plants were obtained. When combining these results with other attempts, a total number of 191 green plants was regenerated, 90 for Isly, 74 for Jori, 19 for Cocorit, 7 for Oued Zenati and 1 for Cham1. In a ‘second phase’, permanent regenerating callus lines were obtained through subculture on dif medium. Depending on genotype, 2 to 9 transfers (up to one year for Jori) were performed, giving 507 regenerated plants. From direct regeneration and subcultures, a total of 698 plants were produced, all green and mostly haploid. Fertile doubled haploid plants were obtained, either spontaneously or by colchicine treatment, for all these genotypes. These results are of interest for breeders, who need pure lines for genotypes evaluation and for the creation of improved homozygous varieties.

Regeneration of fertile doubled haploid plants from colchicine‐supplemented media in wheat anther culture

AbstractThe effect of colchicine added to induction medium for the production of fertile doubled haploid plants after in‐vitro anther culture was studied in wheat, Triticum aestivum L. For this, one winter and two spring wheat varieties were used. Anther cultures of the three genotypes were treated with 0.03% colchicine for 3 days at the beginning of microspore induction. Colchicine had no significant effect on anther response and embryoid production of the genotypes examined. However, in the winter wheat genotype ‘Mv Szigma’, colchicine caused a significant reduction in microspore‐derived structures. A significant decrease was also observed in plant regeneration ability of two genotypes (‘Vergina’ and ‘Acheloos’) after colchicine treatment. In addition, a significant reduction of the albinos produced was observed in all genotypes after olchicine treatment. In contrast, the regenerants obtained from the colchicine‐supplemented induction media produced significantly higher percentages of fertile plants in all genotypes. However, the level of fertility, was significantly different among the fertile plants obtained. This, together with the observation that in the case of the winter wheat variety the colchicine treatment resulted in 100% completely fertile plants with a high seed‐setting ability indicate that there is space for further improvement of the method when it is applied to spring cultivars. Finally, the increased number of seeds per 100 plated anthers obtained from all three genotypes after colchicine treatment, clearly demonstrates that the addition of colchicine to induction medium was superior to the conventional anther culture method and it could therefore be introduced into wheat breeding programmes.

Heritable variation in the ability to produce haploid embryos via pollination with maize and embryo rescue in durum wheat

Pollination with maize is commonly used to produce fertile doubled haploid plants in durum wheat. However, the genetic basis for the ability of parental lines to produce haploid seedlings is not well documented. A maximum likelihood procedure was used to analyse the response of 15 F1, from a half diallel design involving six inbred lines, and to document rigorously the existence of heritable variation for haploid embryo production. Cross-effects and "general combining abilities" (GCA) were computed to estimate the ability to produce swollen ovaries and viable embryos. On average, the ovary swelling production was 56% and 11% of emasculated flowers produced a viable embryo. For both variables, cross-effect was significant. Based on GCA estimations, the 6 parental lines could be distributed in 3 different groups. The GCA values explained respectively 69% and 79% of the cross-effect for each variable. On the whole, ovary swelling was a poor predictor of embryo formation. No significant correlation was found between the per se values of the 6 parental lines and their GCA.Key words: Triticum durum, haploid, general combining ability.

Application of trifluralin to embryogenic microspore cultures to generate doubled haploid plants in Brassica napus

Microspore or anther culture has been used to produce desirable meiotic recombinants in numerous species. However, the utilization of these recombinants relies on inefficient genome doubling procedures to obtain fertile doubled haploid plants. This study presents a simple and rapid procedure to generate fertile doubled haploids in Brassica napus cv. Topas using trifluralin (α,α,α‐trifluoro‐2,6‐dinitro‐N,N‐dipropyl‐p‐toluidine), a plant specific microtubule inhibitor. The effects of trifluralin on microtubule depolymerization and chromosome doubling in embryogenic microspore cultures of B. napus were examined and compared with those of colchicine. Indirect immunofluorescence labeling of isolated microspores indicated that microtubules were depolymerized within 30 min of trifluralin treatment and after 3–8 h of colchicine treatment. The direct application of these microtubule inhibitors to microspore cultures resulted in the recovery of fertile doubled haploid plants. Continuous culture in the presence of colchicine, was more effective than 18‐h treatments for fertile plant production but resulted in abnormal embryo formation and recalcitrant plant regeneration. The application of 1 or 10 μM trifluralin during the first 18 h of microspore culture was found to be the superior method for doubled haploid production. The embryos generated after trifluralin treatment developed normally, germinated readily and of the plants produced, close to 60% were fertile. The use of trifluralin to double chromosomes very early in microspore cultures is a simple process requiring minimal manipulation and should be very useful for genetic studies and breeding programs of B. napus and possibly other species.

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

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

Comparison of Anther Culture and the Hordeum bulbosum Method for the Production of Doubled Haploids in Winter Barley

AbstractThe comparison, between the efficiency of anther culture and the Hordeum bulbosum method in barley was extended to the chromosome number distributions of all planes derived from the two techniques and the proportions of fertile doubled haploid plants which survived until maturity.The frequencies of haploid and spontaneously doubled haploid plants which were useful for practical breeding purposes were found to be around 90 % for both techniques. The remainder consisted of polyploid, mixoploid and aneuploid variants in the case of microspore‐derived plants and diploid interspecific hybrids in the progeny of the H. bulbosum method. The ploidy level distributions of the microspore‐ and H. bulbosum‐derived plants appeared to be independent of the genotype of the donor.There were no significant differences between techniques regarding the proportions, of plants which survived a severe winter and the production of fertile doubled haploid plants. Both techniques can therefore complement each other in a breeding programme and their relative merits are discussed.Possible ways of improving doubled haploid production in barley are suggested for increasing its use in breeding schemes.