Green Plant Regeneration Research Articles

Response to Callus Induction and Regeneration of Newly Released BRRI Rice Varieties

A study was carried out for developing an efficient callus induction and regeneration system for three newly developed BRRI varieties namely BRRI dhan86, BRRI dhan87 and BRRI dhan89. Dehusked seeds were plated onto MS and N6 media with two hormone combinations for callus induction. Calli obtained from each callus induction medium were transferred to four different regeneration media. Callus induction frequency and regeneration ability were significantly influenced by rice varieties, and interactions of variety and media. Among the media compositions, the highest callus (59.44%) were obtained from C1 (MS+2mg/l 2,4-D) followed by C2 ( MS+2 mg/l 2,4-D+0.5 mg/l kinetin) , C3 ( N6+2 mg/l 2,4-D) and C4 (N6+2 mg/l 2,4-D+0.5 mg/l kinetin) medium. The highest regeneration (45.74%) was obtained from R2 (MS+4 mg/ml BAP+1.2 mg/ml kinetin+0.5 mg/ml NAA), followed by R3 (1 mg/ml BAP+1 mg/ml Kinetin+1 mg/ml NAA), R4 (2 mg/ml kinetin+1 mg/ml NAA+300 mg casein hydrolysate) and R1 (2 mg/ml BAP+1 mg/ml kinetin+1 mg/ml NAA). BRRI dhan86 showed the highest regeneration ability (53.06%) than the other two varieties. It is observed that all varieties performed better in C1 medium for callus induction and R2 medium for regeneration. This study also revealed that BRRI dhan86 was more responsive to callus induction and regeneration of green plants than the other two varieties.
 Bangladesh Rice j. 2019, 23(2): 17-25

Impact of Ionic Liquids on Induction of Wheat Microspore Embryogenesis and Plant Regeneration

Ionic liquids are novel compounds with unique chemical and physical properties. They can be received based on synthetic auxins like 2,4-dichlorophenoxyacetic acid or dicamba, which are commonly used hormones in microspore embryogenesis. Nevertheless, ionic liquids have not been adapted in plant in vitro culture thus far. Therefore, we studied the impact of ionic liquids on the ability to undergo microspore embryogenesis in anther cultures of wheat. Two embryogenic and two recalcitrant genotypes were used for this study. Ten combinations of ionic liquids and 2,4-dichlorophenoxyacetic acid were added to the induction medium. In most cases, they stimulated induction of microspore embryogenesis and green plant regeneration more than a control medium supplemented with only 2,4-dichlorophenoxyacetic acid. Two treatments were the most favorable, resulting in over two times greater efficiency of microspore embryogenesis induction in comparison to the control. The effect of breaking down the genotype recalcitrance (manifested by green plant formation) was observed under the influence of 5 ionic liquids treatments. Summing up, ionic liquids had a positive impact on microspore embryogenesis induction and green plant regeneration, increasing the efficiency of these phenomena in both embryogenic and recalcitrant genotypes. Herbicidal ionic liquids can be successfully used in in vitro cultures.

Highly efficient and genotype-independent barley gene editing based on anther culture

Recalcitrance to tissue culture and genetic transformation is the major bottleneck for gene manipulation in crops. In barley, immature embryos of Golden Promise have typically been used as explants for transformation. However, the genotype dependence of this approach limits the genetic modification of commercial varieties. Here, we developed an anther culture-based system that permits the effective creation of transgenic and gene-edited plants from commercial barley varieties. The protocol was tested in Golden Promise and four Australian varieties, which differed in phenology, callus induction, and green plant regeneration responses. Agrobacterium-mediated transformation was performed on microspore-derived callus to target the HvPDS gene, and T0 albinos with targeted mutations were successfully obtained from commercial varieties. Further editing of three targets was achieved with an average mutation rate of 53% in the five varieties. In 51 analyzed T0 individuals, Cas9 induced a large proportion (69%) of single-base indels and two-base deletions in the target sites, with variable mutation rates among targets and varieties. Both on-target and off-target activities were detected in T1 progenies. Compared with immature embryo protocols, this genotype-independent platform can deliver a high editing efficiency and more regenerant plants within a similar time frame. It shows promise for functional genomics and the application of CRISPR technologies for the precise improvement of commercial varieties.

Effect of sucrose on calus induction and green plantlet regeneration in anther culture of Indica x Indica rice

Anther culture is an important biotechnological tool. By rice anther culture homozygous pure lines in the form of doubled haploid (DH) plants can be produced within a year as compared to the long inbreeding method, which might take 8-10 years. Application of anther culture technique in breeding and genetics research is limited due to the very low regeneration frequency of anthers of rice in general, and indica cultivars in particular. Therefore, the successful use of the technique depends on the adequate production of DH plants for selection and field evaluation. Effect of various concentrations of sucrose in callus induction media was investigated on callus induction and regeneration of green plantlets from anther culture of several F1s derived from indica x indica crosses. Cold pretreated anthers of five genotype of anther donor plants (F1s) at 5 °C for 8 days were cultured on N6 callus induction medium containing sucrose at a concentration of 6.0%, 6.5%, and 7.0%. Results revealed that all genotypes showed similar response to callus induction, but a significantly different responses to plant regeneration depended on sucrose level in the callus induction media. Two genotypes, i.e. IR78788 / Inpara 5 and Dendang/Inpari 30 only regenerated albino plantlets. In this experiment, 6.5% sucrose compared to two other sucrose treatments was suitable for inducing high frequency callus induction and high green plant regeneration in all genotypes. The high concentration of sucrose (7.0%) in the culture medium not only resulted in a decrease in the number and percentage of callus formation but also decreased the regeneration of green plantlets.

The Effect of Caffeine and Trifluralin on Chromosome Doubling in Wheat Anther Culture.

Challenges for wheat doubled haploid (DH) production using anther culture include genotype variability in green plant regeneration and spontaneous chromosome doubling. The frequency of chromosome doubling in our program can vary from 14% to 80%. Caffeine or trifluralin was applied at the start of the induction phase to improve early genome doubling. Caffeine treatment at 0.5 mM for 24 h significantly improved green plant production in two of the six spring wheat crosses but had no effect on the other crosses. The improvements were observed in Trojan/Havoc and Lancer/LPB14-0392, where green plant numbers increased by 14% and 27% to 161 and 42 green plants per 30 anthers, respectively. Caffeine had no significant effect on chromosome doubling, despite a higher frequency of doubling in several caffeine treatments in the first experiment (67–68%) compared to the control (56%). In contrast, trifluralin significantly improved doubling following a 48 h treatment, from 38% in the control to 51% and 53% in the 1 µM and 3 µM trifluralin treatments, respectively. However, trifluralin had a significant negative effect on green plant regeneration, declining from 31.8 green plants per 20 anthers (control) to 9–25 green plants per 20 anthers in the trifluralin treatments. Further work is required to identify a treatment regime with caffeine and/or anti-mitotic herbicides that consistently increases chromosome doubling in wheat without reducing green plant regeneration.

Влияние инициальных сред и длительности холодовой предобработки на эффективность андрогенеза в культуре изолированных пыльников озимой пшеницы сорта Иркутская

Haploid technologies are based on the regeneration of gamete cells to form whole plants. The main advantage of haploid technologies is the ability to quickly in one generation obtain homozygous plants with the required parameters. Nevertheless, the use of these technologies today is limited, what is associated with insufficient knowledge of the physiological and biochemical features of the application of this method. In this regard, this work is devoted to the study of the features of androgenesis in vitro in the culture of isolated anthers of winter wheat cultivar Irkutskaya and the determination of the methodological features of obtaining intact plants. In the autumn-winter period, donor plants were grown at the artificial climate station SIPPB SB RAS. In the spring-summer period, donor plants were grown in the field at the experimental plots of the Zalarinsky agroecological station (Tungui village). The plant material was selected at the booting stage. Anthers were isolated from 6-10 spikelets of the middle third of the spike. The features of androgenesis were assessed by the frequency of formation of calli and embryo-like structures to the total number of anthers and the frequency of formation of the total number of seedlings and the number of green seedlings to the number of embryo-like structures and calli. It was found that the optimal medium for the induction of androgenesis in the anther culture of this common wheat cultivar is the modification of medium N6 containing 9% sucrose, with the addition of glycine (2 mg/L), 2.4-D (1 mg/L) and NAA (1 mg/L). The maximum yield of embryoid-like structures was observed in the case of low-temperature pretreatment of donor plants at + 4 °C for 6-12 days. At the same time, the most intensive calli formation in the culture of isolated anthers occurred after 3-6 days of donor plant cold pretreatment. Regeneration of green plants took place on 190-2Cu medium with the addition of growth regulators (2.4-D, 0.5 mg/L; kinetin, 0.5 mg/L) with obligatory cold pretreatment of androclinic structures at +4 °C for 3-7 days. Obtained results indicate that low-temperature exposure at different stages, from pretreatment of donor ears to exposure to androclinic structures, is a key factor for winter wheat that promotes androgenesis in the culture of isolated anthers and the successful formation of intact plants.

Comparison of the Androgenic Response of Spring and Winter Wheat (Triticum aestivum L.)

Androgenesis is potentially the most effective technique for doubled haploid production of wheat. It is not however widely used in breeding programmes due to its main limitation: the genotype dependence. Due to genetic differences between spring and winter wheat, it was assumed that both phenotypes are different in their capacity to conduct androgenesis. And so, the aim of this investigation was to verify the effectiveness of androgenesis induction and plant regeneration of spring and winter wheat genotypes while considering varying amounts of growth hormones in the induction medium. Fifteen genotypes of spring wheat and fifteen of winter wheat were used in the experiment. Six hundred anthers of each of the 30 genotypes were plated and analysed. Previous studies have allowed selection of the best medium for wheat androgenesis and a combination of growth hormones that are the most effective in stimulating microspore proliferation. Therefore, C17 induction media with two combinations of growth hormones were used: I—supplemented only by auxins (2,4-D and dicamba), and II—supplemented by auxin and cytokinin (2,4-D and kinetin). Data was recorded according to the efficiency of androgenic structure formation (ASF), green plant regeneration (GPR), and albino plant regeneration (APR). The results showed that the induction and regeneration of androgenesis in the spring wheat were more efficient than in the winter ones. The spring genotypes formed more androgenic structures and green plants on anthers plated on the medium supplemented only by auxins, in contrast to the winter genotypes which were better induced and regenerated on the medium supplemented by auxin and cytokinin. The study showed that to increase the efficiency of androgenesis, it is necessary to select appropriate factors such as concentration and type of hormones in medium composition, affecting the course of the culturing procedure according to the winter or spring phenotype of donor plants.

Improvement of anther cultures conditions using the Taguchi method in three cereal crops

Background: Plant tissue cultures have the potential to reprogram the development of microspores from normal gametophytic to sporophytic pathway resulting in the formation of androgenic embryos. The efficiency of this process depends on the genotype, media composition and external conditions. However, this process frequently results in the regeneration of albino instead of green plants. Successful regeneration of green plants is affected by the concentration of copper sulfate (CuSO 4 ) and silver nitrate (AgNO 3 ) and the length of induction step. In this study, we aimed at concurrent optimization of these three factors in barley ( Hordeum vulgare L.), wheat ( Triticum aestivum L.), and triticale (x Triticosecale spp. Wittmack ex A. Camus 1927) using the Taguchi method. We evaluated uniform donor plants under varying experimental conditions of in vitro anther culture using the Taguchi approach, and verified the optimized conditions. Results: Optimization of the regeneration conditions resulted in an increase in the number of green regenerants compared with the control. Statistic Taguchi method for optimization of the in vitro tissue culture plant regeneration via anther cultures allowed reduction of the number of experimental designs from 27 needed if full factorial analysis is used to 9. With the increase in the number of green regenerants, the number of spontaneous doubled haploids decreased. Moreover, in barley and triticale, the number of albino regenerants was reduced. Conclusion: The statistic Taguchi approach could be successfully used for various factors (here components of induction media, time of incubation on induction media) at a one time, that may impact on cereals anther cultures to improve the regeneration efficiency. Normal 0 false false false PL X-NONE X-NONE /* Style Definitions */ table.MsoNormalTable {mso-style-name:Tabla normal; mso-tstyle-rowband-size:0; mso-tstyle-colband-size:0; mso-style-noshow:yes; mso-style-priority:99; mso-style-parent:; mso-padding-alt:0cm 5.4pt 0cm 5.4pt; mso-para-margin-top:0cm; mso-para-margin-right:0cm; mso-para-margin-bottom:8.0pt; mso-para-margin-left:0cm; line-height:107%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri,sans-serif; mso-ascii-font-family:Calibri; mso-ascii-theme-font:minor-latin; mso-hansi-font-family:Calibri; mso-hansi-theme-font:minor-latin; mso-bidi-font-family:Times New Roman; mso-bidi-theme-font:minor-bidi; mso-ansi-language:PL;}

Plastid differentiation during microgametogenesis determines green plant regeneration in barley microspore culture

Developing plants from in vitro culture of microspores or immature pollen grains (androgenesis) is a highly genotype-dependent process whose effectiveness in cereals is significantly reduced by occurrence of albino regenerants. Here, we examined a hypothesis that the molecular differentiation of plastids in barley microspores prior to in vitro culture affects the genotype ability to regenerate green plants in culture. At the mid-to-late uninucleate (ML) stage, routinely used to initiate microspore culture, the expression of most genes involved in plastid transcription, translation and starch synthesis was significantly higher in microspores of barley cv. ‘Mercada’ producing 90% albino regenerants, than in cv. ‘Jersey’ that developed 90% green regenerants. The ML microspores of cv. ‘Mercada’ contained a large proportion of amyloplasts filled with starch, while in cv. ‘Jersey’ there were only proplastids. Using additional spring barley genotypes that differed in their ability to regenerate green plants we confirmed the correlation between plastid differentiation prior to culture and albino regeneration in culture. The expression of GBSSI gene (Granule-bound starch synthaseI) in early-mid (EM) microspores was a good marker of a genotype potential to produce green regenerants during androgenesis. Initiating culture from EM microspores that significantly improved regeneration of green plants may overcome the problem of albinism.

Improving the efficiency of wheat microspore culture methodology: evaluation of pretreatments, gradients, and epigenetic chemicals

The objective of the study was to develop efficient widely adaptable microspore culture methodology for spring and winter wheat genotypes. Current wheat microspore culture methods are still inadequate for routine application, especially for winter genotypes. The present study involved increasing the frequency of microspore embryogenesis and improving the conversion of microspore-derived embryos to fertile green plants. A systematic evaluation was conducted testing numerous factors at all phases of the microspore embryogenesis process. Results from pretreatments, maltose gradients, and histone deacetylase inhibitors are presented. A 28 days cold pretreatment of spikes was optimal for winter wheat genotypes whereas a 21 days cold pretreatment was best for spring genotypes. Microspore suspensions containing a high frequency of embryogenic microspores were collected by resuspension in a 23% maltose gradient with subsequent pelleting with a mannitol containing solution. The application of trichostatin A (TSA) enhanced embryogenesis and/or green plant regeneration. Other epigenetic chemicals like scriptaid, BIX-01294, and sodium butyrate were also evaluated. Scriptaid enhanced embryogenesis but regenerated plantlets showed abnormal shoot and root development. The improved methodology was validated using a range of spring and winter wheat genotypes. Enhanced microspore embryogenesis and green plant regeneration in spring and winter wheat was observed with 21 or 28 days cold pretreatments, a 23% maltose gradient, and the application of trichostatin A (TSA).

Efficiency of spring barley haploid production in anther culture in vitro: comparison of basic and improved technologies

Aim. Evaluation of innovative methodological approaches elaborated on model genotypes for usability to increase frequencies of morphogenic structure induction and plant regeneration in anther culture in vitro in spring barley diverse material. Methods. Spikes isolated from F1 and F2 hybrids of four crosses were pretreated using an improved method (4ºC, 28 days), and anthers were inoculated onto nutrient media containing chemically modified starches D5-M and D5a-1 instead of agar. In control cut tillers were emerged in water and pretreated at 4ºC for 5 days. Anthers were cultivated on agar solidified medium. Results. Positive effects of the improved method of cold pretreatment and cultivation of anthers on media solidified with starches were confirmed. The advantage of new gelling agent D5a-1 was proved. Particularly, its usage resulted in a three-fold increase in the frequency of green plant regeneration. Conclusions. In order to increase spring barley androgenic haploid yield, combination of prolonged cold pretreatment with anther cultivation on media solidified with chemically modified starches instead of agar in an integrated technological process is reasonable.
 Keywords: Hordeum vulgare L., anther culture in vitro, cold pretreatment, starch, agar, embryo formation, plant regeneration.

Accelerating hybrid rice breeding through the adoption of doubled haploid technology for R-line development

Hybrid rice holds significant yield advantage over inbred lines, but its adoption rate is very slow in India. Development of cytoplasmic male sterile line (CMS) and fertility restorer line with superior grain quality is the prerequisite in three line hybrid rice breeding programme. Minimum of three to four years are required for development of useful R-line which severely delays the pace of hybrid breeding. Application of doubled haploid (DH) technology will be very useful to develop elite R-lines within short period of time. An elite inbred line LAXMI was crossed with the R-line RPR158 and anthers from the resulting F1 population was used for development of DH plants. Eight hormonal combinations and four different cold pre-treatment conditions were tried in tissue culture. Maximum callusing frequency was observed in N6 medium supplemented with 2.0 mg L−1 2, 4-diclorophenoxyacetic acid (2,4-D), 0.5 mg L−1 napthalene acetic acid (NAA), 0.5 mg L−1 benzyl amino purine(BAP) and 6% maltose with 3 days cold pre-treatment. Highest green plant regeneration frequency was found with Murashige and Skoog medium supplemented with 0.5 mg L−1 NAA, 0.5 mg L−1 kinetin, 2 mg L−1 BAP. A total of 280 DH plants were generated and evaluated under field conditions for agronomic characters and fertility restoration capacity in CMS system. Two distinct DH lines were identified as improved R-lines based on high restoration ability (94%), superior grain quality and medium slender grain type. This study successfully demonstrated that superior R-lines can be developed within two crop seasons through the application of DH technology.

Exploring factors affecting anther culture in rice (Oryza sativa L.)

Anther culture is an important in vitro culture technique for production of double haploids. It is largely species and genotype-specific. Asian cultivated rice (O. sativa, ssp. indica) is recalcitrant to anther culture which limits its practical application in rice breeding. Several researchers tried to optimize the media recipes and culture techniques for callus induction and plantlet regeneration. Negligible response to callus induction and recovery of higher frequency of albino plants are the major hindrance for use of the technique in crop improvement. Shortening the culture period and sexual hybridization of rice sub-groups (japonica/indica) may be adopted to improve green plant regeneration from anther-derived callus in indica rice. However, genetic transformation technology using individual genes related to different aspects of anther culture could be a more direct approach. Besides, the role of genotypes, physiological status of donor plant, developmental stage of pollen, pre-treatment, culture media, phytohormones and culture conditions for successful anther culture have been discussed. Key words: Anther culture, factors for anther culture, double haploids, genetic improvement, rice.

Anther culture in rice: Progress and breeding perspective

Anther culture is an important biotechnological tool for the rapid recovery of fixed breeding lines in rice. The method is largely species- and genotype-specific. Indica rice is known to have a recalcitrant genetic background. Oryza glaberrima responds better to the anther culture than O. sativa while japonica sub-group is more responsive to microspore embryogenesis than Indica types. Media recipes and culture techniques have been standardized for callus induction and green plantlet regeneration using Indica and Japonica varieties as well as their inter se hybrids. Additive effects seem more important than dominant effects of the genes concerned for callus induction while preponderance of dominant effects is reported for regeneration response. Sexual hybridization of rice sub-groups (Japonica x Indica) followed by the selection of favourable QTL for green plant regeneration may be adopted to improve plant regeneration from anther-derived callus in Indica rice. However, genetic transformation technology using individual genes related to different aspects of anther culture could be a more direct approach. This paper reviews the progress and breeding perspective of anther culture in rice for genetic improvement of agronomic- and stress-related traits using double haploid breeding.

Genotypic features of morphogenetic reactions of varieties and hybrids F1 of winter soft wheat during various stages of androgenesis in vitro

Aim. Testing the haploproduction ability of 47 varieties and 143 F1 hybrids of winter bread wheat. Methods. In vitro culture of isolated anthers of wheat. The percentage of callus and regeneration of green plants for each genotype calculated as a percentage of the planted anthers. Results. The differences in the frequency of сallus induction and the ability to regenerate plants in the process of androgenesiss in vitro of winter soft wheat were detected. The range of variation haploproduction activiti was broad. The sign of "the formation of сallus" for varieties was in limited from 0 to 21.2 % and for hybrids – from 0 to 38.8 %; on the sign of "regeneration of green plants" for varieties – from 0 to 9.4 %, and for hybrids from 0 to 9.1 %. The positive effect of 1BL/1RS translocation on signs of haploproduction in anther culture Triticum aestivum L. Conclusions. Genotype-specific of microspores morphogenetic reactions of soft winter wheat in the process of androgenesis in vitro were revealed. It has been found that F1 hybrids have a higher potential production potential in anther culture than varieties. During the period from 2011 to 2017, the 580 lines of winter wheat were transferred to the breeding departments.
 Keywords: soft winter wheat, anther culture in vitro, callus, regeneration.

Genetic Loci Governing Androgenic Capacity in Perennial Ryegrass (Lolium perenne L.).

Immature pollen can be induced to switch developmental pathways from gametogenesis to embryogenesis and subsequently regenerate into homozygous, diploid plants. Such androgenic production of doubled haploids is particularly useful for species where inbreeding is hampered by effective self-incompatibility systems. Therefore, increasing the generally low androgenic capacity of perennial ryegrass (Lolium perenne L.) germplasm would enable the efficient production of homozygous plant material, so that a more effective exploitation of heterosis through hybrid breeding schemes can be realized. Here, we present the results of a genome-wide association study in a heterozygous, multiparental population of perennial ryegrass (n = 391) segregating for androgenic capacity. Genotyping-by-sequencing was used to interrogate gene- dense genomic regions and revealed over 1,100 polymorphic sites. Between one and 10 quantitative trait loci (QTL) were identified for anther response, embryo and total plant production, green and albino plant production and regeneration. Most traits were under polygenic control, although a major QTL on linkage group 5 was associated with green plant regeneration. Distinct genetic factors seem to affect green and albino plant recovery. Two intriguing candidate genes, encoding chromatin binding domains of the developmental phase transition regulator, Polycomb Repressive Complex 2, were identified. Our results shed the first light on the molecular mechanisms behind perennial ryegrass microspore embryogenesis and enable marker-assisted introgression of androgenic capacity into recalcitrant germplasm of this forage crop of global significance.

Embryogenic Callus Induction and Efficient Plant Regeneration in Three Varieties of Soybean (Glycine max)

An efficient callus induction and plant regeneration system has been developed using three local soybean (Glycine max L.) varieties. All the varieties showed good callusing (78.30 ‐ 88.80%) from shoot tip (ST) in MS + 3.0 mg/l 2,4‐D + 1.0 mg/l BAP. Best callus induction and embryo formation were recorded in T5 for BS‐6 as 88.80 and 81.20%, respectively from ST. In case of cotyledonary node (CN) the maximum callus induction (82.40%) and embryo formation (74.20%) were recorded also in T5 for BS‐6. Highest frequency (79.40%) of plant regeneration was recorded where ST were used and cultured in MS supplemented with 2.5 mg/l BAP + 1.0 mg/l NAA as well as 76.30% from CN in BS‐6. The length of shoot was observed 5.32 and 4.62 cm, respectively from ST and CN for BS‐6 with the same medium composition. It was observed that half strength of MS + 2.0 mg/l IBA showed best rooting (9.04). Among the genotypes BS‐6 proved to be best explants than ST that exhibited better performance on callus induction and green plant regeneration for all parameters.Plant Tissue Cult. & Biotech. 27(1): 41-50, 2017 (June)

Influence of different concentrations of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale

In this work, the effect of ascorbic and gibberellic acids and pH of medium on embryogenesis and regeneration in anther culture of spring triticale was studied. The study of androgenesis was conducted on two model samples YaTKh-327-11 and YaTKh-18-11. The research revealed that the addition of the antioxidant ascorbic acid at a concentration of 2–8 mg/L into the culture medium to induce embryogenesis enhanced the formation of androgen structures and regeneration of green plants. The addition of gibberellic acid into the culture medium to induce embryogenesis gave negative effects. The average yield of green plants in the anther culture of spring triticale was 2 pcs/100 anthers. According to the research, 277 green plants were received and spontaneous doubling of chromosomes was recorded in 26.7%.

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.

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.