Green Plant Regeneration Research Articles

Androgenesis in vitro in anther culture of bread winter wheat

Aim. Study of the effectiveness of stages of in vitro androgenesis in anther culture for the creation of dihaploid lines of bread winter wheat. Methods. In vitro culture of isolated wheat anthers. For each genotype, the following parameters of androgenesis in vitro were determined: the percentage of caluses and the percentage of regeneration of green plants from the number of planted anthers; the percentage of acclimatized plants from regenerated green plantlets, as well as the percentage of fertile (doubled haploid) plants. Results. Androgenesis in vitro in the anther culture of 105 genotypes of soft winter wheat of different genetic origin was studied. Differences regarding the frequency of callusogenesis induction (from 0.17 to 21.25 percent of planted anthers) and the ability to regenerate plants (from 0 to 4.94 percent of planted anthers) were found. The dihaploid lines in anther culturefor almost 50 % of the studied genotypes were obtained. Spontaneous diploidization averaged 29.41 % of the obtained regenerants. Conclusions. Genotype-specific morphogenetic reactions of bread winter wheat microspores in the process of in vitro androgenesis were revealed. 122 dihaploid lines of bread winter wheat were obtained.

β-glucans, SAM, and GSH fluctuations in barley anther tissue culture conditions affect regenerants’ DNA methylation and GPRE

BackgroundMicrospore embryogenesis is a process that produces doubled haploids in tissue culture environments and is widely used in cereal plants. The efficient production of green regenerants requires stresses that could be sensed at the level of glycolysis, followed by the Krebs cycle and electron transfer chain. The latter can be affected by Cu(II) ion concentration in the induction media acting as cofactors of biochemical reactions, indirectly influencing the production of glutathione (GSH) and S-adenosyl-L-methionine (SAM) and thereby affecting epigenetic mechanisms involving DNA methylation (demethylation—DM, de novo methylation—DNM). The conclusions mentioned were acquired from research on triticale regenerants, but there is no similar research on barley. In this way, the study looks at how DNM, DM, Cu(II), SAM, GSH, and β-glucan affect the ability of green plant regeneration efficiency (GPRE).ResultsThe experiment involved spring barley regenerants obtained through anther culture. Nine variants (trials) of induction media were created by adding copper (CuSO4: 0.1; 5; 10 µM) and silver salts (AgNO3: 0; 10; 60 µM), with varying incubation times for the anthers (21, 28, and 35 days). Changes in DNA methylation were estimated using the DArTseqMet molecular marker method, which also detects cytosine methylation.Phenotype variability in β-glucans, SAM and GSH induced by the nutrient treatments was assessed using tentative assignments based on the Attenuated Total Reflectance-Fourier Transform Infrared (ATR-FTIR) spectroscopy. The effectiveness of green plant regeneration ranged from 0.1 to 2.91 plants per 100 plated anthers. The level of demethylation ranged from 7.61 to 32.29, while de novo methylation reached values ranging from 6.83 to 32.27. The paper demonstrates that the samples from specific in vitro conditions (trials) formed tight groups linked to the factors contributing to the two main components responsible for 55.05% of the variance (to the first component DNM, DM, to the second component GSH, β-glucans, Cu(II), GPRE).ConclusionsWe can conclude that in vitro tissue culture conditions affect biochemical levels, DNA methylation changes, and GPRE. Increasing Cu(II) concentration in the IM impacts the metabolism and DNA methylation, elevating GPRE. Thus, changing Cu(II) concentration in the IM is fair to expect to boost GPRE.

An insight into tissue culture-induced variation origin shared between anther culture-derived triticale regenerants

BackgroundThe development of the plant in vitro techniques has brought about the variation identified in regenerants known as somaclonal or tissue culture-induced variation (TCIV). S-adenosyl-L-methionine (SAM), glutathione (GSH), low methylated pectins (LMP), and Cu(II) ions may be implicated in green plant regeneration efficiency (GPRE) and TCIV, according to studies in barley (Hordeum vulgare L.) and partially in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927). Using structural equation models (SEM), these metabolites have been connected to the metabolic pathways (Krebs and Yang cycles, glycolysis, transsulfuration), but not for triticale. Using metabolomic and (epi)genetic data, the study sought to develop a triticale regeneration efficiency statistical model. The culture’s induction medium was supplemented with various quantities of Cu(II) and Ag(I) ions for regeneration. The period of plant regeneration has also changed. The donor plant, anther-derived regenerants, and metAFLP were utilized to analyze TCIV concerning DNA in symmetric (CG, CHG) and asymmetric (CHH) sequence contexts. Attenuated Total Reflectance–Fourier Transfer Infrared (ATR-FTIR) spectroscopy was used to gather the metabolomic information on LMP, SAM, and GSH. To frame the data, a structural equation model was employed.ResultsAccording to metAFLP analysis, the average sequence change in the CHH context was 8.65%, and 0.58% was de novo methylation. Absorbances of FTIR spectra in regions specific for LMP, SAM, and GSH were used as variables values introduced to the SEM model. The average number of green regenerants per 100 plated anthers was 2.55.ConclusionsThe amounts of pectin demethylation, SAM, de novo methylation, and GSH are connected in the model to explain GPRE. By altering the concentration of Cu(II) ions in the medium, which influences the amount of pectin, triticale’s GPRE can be increased.

Obtaining dihaploid lines of soft winter wheat with complex resistance to rust and hard smut by anther culture in vitro

Aim. Obtaining homozygous linear material of winter soft wheat of different genetic origin (complex hybrids), which are resistant to different types of rust by androgenesis in vitro. 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. Methods of phytopathological assessment of resistance to complex of various diseases. Results. The differences in the frequency of сallus induction (from 0.99 to 16.26 % of planted anthers) and the ability to regenerate plants (from 0 to 3.05 % of planted anthers) in the process of androgenesiss in vitro of winter soft wheat were detected. Fertile regenerants in the anthers culture 5 out of 9 studied hybrids have been obtained. The effectiveness of the last process was different: from 12.5 to 60.0 % (an average of 29.41 %) from the received regenerants. The high degree (8–9 points) of the resistance of the obtained dihaploids to a complex of diseases was shown. Conclusions. Genotype-specific of microspores morphogenetic reactions of soft winter wheat in the process of androgenesis in vitro were revealed. The highest level of callus formation were shown for samples 120/20 and 132/20. 15 dihaploid lines with complex rust resistance and hard smut were obtained.

Metabolomic Changes as Key Factors of Green Plant Regeneration Efficiency of Triticale In Vitro Anther Culture

Green plant regeneration efficiency (GPRE) via in vitro anther culture results from biochemical pathways and cycle dysfunctions that may affect DNA and histone methylation, with gene expression influencing whole cell functioning. The reprogramming from gametophytic to sporophytic fate is part of the phenomenon. While DNA methylation and sequence changes related to the GPRE have been described, little attention was paid to the biochemical aspects of the phenomenon. Furthermore, only a few theoretical models that describe the complex relationships between biochemical aspects of GPRE and the role of Cu(II) ions in the induction medium and as cofactors of enzymatic reactions have been developed. Still, none of these models are devoted directly to the biochemical level. Fourier transform infrared (FTIR) spectroscopy was used in the current study to analyze triticale regenerants derived under various in vitro tissue culture conditions, including different Cu(II) and Ag(I) ion concentrations in the induction medium and anther culture times. The FTIR spectra of S-adenosyl-L-methionine (SAM), glutathione, and pectins in parallel with the Cu(II) ions, as well as the evaluated GPRE values, were put into the structural equation model (SEM). The data demonstrate the relationships between SAM, glutathione, pectins, and Cu(II) in the induction medium and how they affect GPRE. The SEM reflects the cell functioning under in vitro conditions and varying Cu(II) concentrations. In the presented model, the players are the Krebs and Yang cycles, the transsulfuration pathway controlled by Cu(II) ions acting as cofactors of enzymatic reactions, and the pectins of the primary cell wall.

S-Adenosyl-L-Methionine and Cu(II) Impact Green Plant Regeneration Efficiency.

The biological improvement of triticale, a cereal of increasing importance in agriculture, may be accelerated via the production of doubled haploid lines using in vitro culture. Among the relevant factors affecting the culture efficiency are Cu(II) or Ag(I) acting, e.g., as cofactors of enzymes. The copper ions are known to positively affect green plant regeneration efficiency. However, the biochemical basis, mainly its role in the generation of in vitro-induced genetic and epigenetic variation and green plant regeneration efficiency, is not well understood. Here, we employed structural equation modeling to evaluate the relationship between de novo DNA methylation affecting the asymmetric context of CHH sequences, the methylation-sensitive Amplified Fragment Length Polymorphism related sequence variation, and the concentration of Cu(II) and Ag(I) ions in induction media, as well as their effect on S-adenosyl-L-methionine perturbations, observed using FTIR spectroscopy, and the green plant regeneration efficiency. Our results allowed the construction of a theory-based model reflecting the biological phenomena associated with green plant regeneration efficiency. Furthermore, it is shown that Cu(II) ions in induction media affect plant regeneration, and by manipulating their concentration, the regeneration efficiency can be altered. Additionally, S-adenosyl-L-methionine is involved in the efficiency of green plant regeneration through methylation of the asymmetric CHH sequence related to de novo methylation. This shows that the Yang cycle may impact the production of green regenerants.

Triticale doubled haploid plant regeneration factors linked by structural equation modeling

Triticale regeneration via anther culture faces many difficulties, e.g., a low percentage of regenerated plants and the presence of albinos. Plant regeneration may be affected by abiotic stresses and by ingredients added to the induction medium. The latter influences biochemical pathways and plant regeneration efficiency. Among such ingredients, copper and silver ions acting as cofactors for enzymatic reactions are of interest. However, their role in plant tissue cultures and relationships with biochemical pathways has not been studied yet.The study evaluated relationships between DNA methylation, changes in DNA sequence variation, and green plant regeneration efficiency influenced by copper and silver ions during triticale plant regeneration. For this purpose, a biological model based on donor plants and their regenerants, a methylation-sensitive amplified fragment length polymorphism, and structural equation modeling were employed.The green plant regeneration efficiency varied from 0.71 to 6.06 green plants per 100 plated anthers. The values for the components of tissue culture-induced variation related to cytosine methylation in a CHH sequence context (where H is A, C, or T) were 8.65% for sequence variation, 0.76% for DNA demethylation, and 0.58% for de novo methylation. The proposed model states that copper ions affect the regeneration efficiency through cytosine methylation and may induce mutations through, e.g., oxidative processes, which may interfere with the green plant regeneration efficiency. The linear regression confirms that the plant regeneration efficiency rises with increasing copper ion concentration in the absence of Ag ions in the induction medium. The least absolute shrinkage and selection operator regression shows that de novo methylation, demethylation, and copper ions may be involved in the green plant regeneration efficiency. According to structural equation modeling, copper ions play a central role in the model determining the regeneration efficiency.

Получение растений-регенерантов озимой мягкой пшеницы с использованием метода культуры пыльников

Abstract. The purpose of the current study was to evaluate the effect of cultivation conditions and the genotype of winter bread wheat samples on the formation of embryogenic callus and regenerative capacity, to identify regenerated plants in order to develop new initial material for breeding new varieties. Scientific novelty. There has been estimated the share of influence of induction nutrient media with different composition and genotype on the amount of regenerated plants of winter wheat in anther culture; there have been identified the new samples with high regeneration potential. Methods. The objects of the study were the donor plants of winter bread wheat (Triticum aestivum L.) of F3 generation from breeding nurseries of the winter wheat department of the FSBSI “ARC “Donskoy”, namely 13 hybrid combinations developed in the laboratory for breeding and seed production of winter bread wheat of half-intensive type. There has been used a methodology of obtaining haploids in anther culture in vitro including such methods as sterilization during work with in vitro; preparation of nutrient media; selection and pretreatment of plant explants; determination of the stage of microspores’ development; isolation and planting of anthers on an induction nutrient medium; regeneration of plants from callus tissues. Results. There has been found the most favorable nutrient media for the induction of and rogenesis in vitro. There has been identified a maximum percentage of new formations from the anthers of winter bread wheat in the sample Vol’nitsa × Gerda. There have been developed the largest number of green haploid regenerated plants from pollen callus from the samples Vol’nitsa × Gerda, Kapitan × Vol’nyy Don. There has been revealed that the contribution of the genotype and nutrient medium into the number of new formations and the regeneration of green plants was statistically significant.

Glutathione and copper ions as critical factors of green plant regeneration efficiency of triticale in vitro anther culture.

Plant tissue culture techniques are handy tools for obtaining unique plant materials that are difficult to propagate or important for agriculture. Homozygous materials derived through in vitro cultures are invaluable and significantly accelerate the evaluation of new varieties, e.g., cereals. The induction of somatic embryogenesis/androgenesis and the regeneration and its efficiency can be influenced by the external conditions of tissue culture, such as the ingredients present in the induction or regeneration media. We have developed an approach based on biological system, molecular markers, Fourier Transform Infrared spectroscopy, and structural equation modeling technique to establish links between changes in sequence and DNA methylation at specific symmetric (CG, CHG) and asymmetric (CHH) sequences, glutathione, and green plant regeneration efficiency in the presence of variable supplementation of induction medium with copper ions. The methylation-sensitive Amplified Fragment Length Polymorphism was used to assess tissue culture-induced variation, Fourier Transform Infrared spectroscopy to describe the glutathione spectrum, and a structural equation model to develop the relationship between sequence variation, de novo DNA methylation within asymmetric sequence contexts, and copper ions in the induction medium, as well as, glutathione, and green plant efficiency. An essential aspect of the study is demonstrating the contribution of glutathione to green plant regeneration efficiency and indicating the critical role of copper ions in influencing tissue culture-induced variation, glutathione, and obtaining green regenerants. The model presented here also has practical implications, showing that manipulating the concentration of copper ions in the induction medium may influence cell function and increases green plant regeneration efficiency.

High throughput anther culture response in an upland rice cross ‘Khandagiri x Dular’

Aim: The experiment was conducted to maximize anther culture response in an upland indica rice cross amenable for drought tolerance breeding. Methodology: The cold pre-treated anthers of a cross Khandagiri (drought sensitive) x Dular (drought tolerant) were cultured on N6, SK3, MS and CIM media at varying concentration of hormones (2,4-D, Kn and NAA) to assess callusing response. Embryogenic calli were placed on regeneration medium (RM) with varying concentration of Kn, BAP and NAA for plant regeneration. The plantlets were acclimatized in half-strength MS basal liquid medium for one week before transferring to pot mixture (peat moss: perlite 2:1, v/v) for plant establishment in glasshouse under partial shade. Finally, the plantlets were checked for doubled haploid status by cytological study of root tips. Results: Anther culture response was found to be media and genotype-specific. F1-progenies responded better than either of the parents. A modified MS callus induction medium (CIM) with 1.5mg/l 2,4-D + 0.5mg/l Kn resulted impressively higher callusing response (30.2%) with nodular calli than SK3>MS>N6. High frequency (12.8%) of albino-free green plant regeneration with well developed rooting was achieved in RM medium (a modified MS) containing 2mg/l BAP and 0.5mg/l NAA. A total 129 doubled haploid (DH) plants (each with 12 pairs of chromosomes) were recovered which maintained normal growth, set seeds and resulted in true breeding DH lines. Interpretation: The high throughput regeneration system is amenable for doubled haploid production from indica crosses. Besides, the present doubled haploid stock can serve as an ideal mapping population and as such targeted for marker aided selection for early development of drought tolerant rice varieties.

Triticale Green Plant Regeneration Is Due to DNA Methylation and Sequence Changes Affecting Distinct Sequence Contexts in the Presence of Copper Ions in Induction Medium

Metal ions in the induction medium are essential ingredients allowing green plant regeneration. For instance, Cu(II) and Ag(I) ions may affect the mitochondrial electron transport chain, influencing the Yang cycle and synthesis of S-adenosyl-L-methionine, the prominent donor of the methylation group for all cellular compounds, including cytosines. If the ion concentrations are not balanced, they can interfere with the proper flow of electrons in the respiratory chain and ATP production. Under oxidative stress, methylated cytosines might be subjected to mutations impacting green plant regeneration efficiency. Varying Cu(II) and Ag(I) concentrations in the induction medium and time of anther culture, nine trials of anther culture-derived regenerants of triticale were derived. The methylation-sensitive AFLP approach quantitative characteristics of tissue culture-induced variation, including sequence variation, DNA demethylation, and DNA de novo methylation for all symmetric-CG, CHG, and asymmetric-CHH sequence contexts, were evaluated for all trials. In addition, the implementation of mediation analysis allowed evaluating relationships between factors influencing green plant regeneration efficiency. It was demonstrated that Cu(II) ions mediated relationships between: (1) de novo methylation in the CHH context and sequence variation in the CHH, (2) sequence variation in CHH and green plant regeneration efficiency, (3) de novo methylation in CHH sequences and green plant regeneration, (4) between sequence variation in the CHG context, and green plant regeneration efficiency. Cu(II) ions were not a mediator between de novo methylation in the CG context and green plant regeneration. The latter relationship was mediated by sequence variation in the CG context. On the other hand, we failed to identify any mediating action of Ag(I) ions or the moderating role of time. Furthermore, demethylation in any sequence context seems not to participate in any relationships leading to green plant regeneration, sequence variation, and the involvement of Cu(II) or Ag(I) as mediators.

Development of haploids in the winter bread wheat anthers

In the modern world, the use of isolated anther cultivation technology is currently an integral part of the wheat breeding process. The development of haploids in the winter bread wheat anthers will allow obtaining new forms of wheat in the shortest possible time and without large areas. The purpose of the current study was to estimate the F3 winter bread wheat hybrids according to the anthers’ sensitivity to androgenesis and plant regeneration in vitro and to identify the factors affecting the yield of haploid production. There has been studied the ability to androgenesis in vitro in the anthers of four winter bread wheat hybrids of intensive and semi-intensive type of the FSBSI “ARC “Donskoy”. There has been assessed the role of the mineral composition of three induction nutrient media N6, W14 and NPB-99. There has been established a correlation between the main stages of development of haploids and a genotype. The highest regeneration rate of green plants was obtained in the sample F3 623 of intensive type (3.3%). The most suitable medium for androgenesis of the winter bread wheat anthers in vitro is NPB-99. Since the genotype F3 623 of intensive type demonstrated high values of haploid production capacity, it could be successfully used in breeding programs for the rapid production of homozygous wheat anther lines in vitro. Using two-way analysis of variance, there has been identified a correlation between the effects of a genotype, nutrient medium and their interaction with the main parameters of haploid formation in winter wheat. The formation of embryogenic structures is mainly associated with the effect of a genotype (46.52%). The proportion of the nutrient composition of the medium was low (1.82%), and the correlation factor was 2.1%. The genotype had the greatest effect on the indicator of the regenerants’ number. The nutrient medium had little effect. Regarding the regeneration of green plants, which is the main indicator of the haploid production, the share of a genotype effect was the largest (47.32%). The contribution of the medium and the correlation of factors were less important, but statistically significant.

The Effect of Daminozide, Dark/Light Schedule and Copper Sulphate in Tissue Culture of Triticum timopheevii.

Triticum timopheevii Zhuk. is a tetraploid wheat that is utilized worldwide as a valuable breeding source for wheat improvement. Gene-based biotechnologies can contribute to this field; however, T. timopheevii exhibits recalcitrance and albinism in tissue cultures, making this species of little use for manipulation through genetic engineering and genome editing. This study tested various approaches to increasing in vitro somatic embryogenesis and plant regeneration, while reducing the portion of albinos in cultures derived from immature embryos (IEs) of T. timopheevii. They included (i) adjusting the balance between 2,4-D and daminozide in callus induction medium; (ii) cultivation using various darkness/illumination schedules; and (iii) inclusion of additional concentrations of copper ions in the tissue culture medium. We achieved a 2.5-fold increase in somatic embryogenesis (up to 80%) when 50 mg L−1 daminozide was included in the callus induction medium together with 3 mg L−1 2,4-D. It was found that the dark cultivation for 20–30 days was superior in terms of achieving maximum culture efficiency; moreover, switching to light in under 2 weeks from culture initiation significantly increased the number of albino plants, suppressed somatic embryogenesis, and decreased the regeneration of green plants. Media containing higher levels of copper ions did not have a positive effect on the regeneration of green plants; contrarily, the elevated concentrations caused albinism in plantlets. The results and relevant conclusions of the present study might be valuable for establishing an improved protocol for the regeneration of green plants in tissue cultures of T. timopheevii.

Proteins, Small Peptides and Other Signaling Molecules Identified as Inconspicuous but Possibly Important Players in Microspores Reprogramming Toward Embryogenesis

In this review, we describe and integrate the latest knowledge on the signaling role of proteins and peptides in the stress-induced microspore embryogenesis (ME) in some crop plants with agricultural importance (i.e., oilseed rape, tobacco, barley, wheat, rice, triticale, rye). Based on the results received from the most advanced omix analyses, we have selected some inconspicuous but possibly important players in microspores reprogramming toward embryogenic development. We provide an overview of the roles and downstream effect of stress-related proteins (e.g., β-1,3-glucanases, chitinases) and small signaling peptides, especially cysteine—(e.g., glutathione, γ-thionins, rapid alkalinization factor, lipid transfer, phytosulfokine) and glycine-rich peptides and other proteins (e.g., fasciclin-like arabinogalactan protein) on acclimation ability of microspores and the cell wall reconstruction in a context of ME induction and haploids/doubled haploids (DHs) production. Application of these molecules, stimulating the induction and proper development of embryo-like structures and green plant regeneration, brings significant improvement of the effectiveness of DHs procedures and could result in its wider incorporation on a commercial scale. Recent advances in the design and construction of synthetic peptides–mainly cysteine-rich peptides and their derivatives–have accelerated the development of new DNA-free genome-editing techniques. These new systems are evolving incredibly fast and soon will find application in many areas of plant science and breeding.

Structural Equation Modeling (SEM) Analysis of Sequence Variation and Green Plant Regeneration via Anther Culture in Barley.

The process of anther culture involves numerous abiotic stresses required for cellular reprogramming, microspore developmental switch, and plant regeneration. These stresses affect DNA methylation patterns, sequence variation, and the number of green plants regenerated. Recently, in barley (Hordeum vulgare L.), mediation analysis linked DNA methylation changes, copper (Cu2+) and silver (Ag+) ion concentrations, sequence variation, β-glucans, green plants, and duration of anther culture (Time). Although several models were used to explain particular aspects of the relationships between these factors, a generalized complex model employing all these types of data was not established. In this study, we combined the previously described partial models into a single complex model using the structural equation modeling approach. Based on the evaluated model, we demonstrated that stress conditions (such as starvation and darkness) influence β-glucans employed by cells for glycolysis and the tricarboxylic acid cycle. Additionally, Cu2+ and Ag+ ions affect DNA methylation and induce sequence variation. Moreover, these ions link DNA methylation with green plants. The structural equation model also showed the role of time in relationships between parameters included in the model and influencing plant regeneration via anther culture. Utilization of structural equation modeling may have both scientific and practical implications, as it demonstrates links between biological phenomena (e.g., culture-induced variation, green plant regeneration and biochemical pathways), and provides opportunities for regulating these phenomena for particular biotechnological purposes.

Stabilization of soft wheat selection material which is the result of crossing with wild relatives

Aim. Testing the haploproduction ability of 30 hybrids of winter soft 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 callus induction and the ability to regenerate plants in the process of androgenesiss in vitro of winter soft wheat were detected. The microspores of 17 of 30 hybrids formed callus by in vitro anther culture were shown. The intensity of one process was different: more than half of the genotypes (18 pcs.) were characterized by a low percentage of callus (from 0.10 to 1.0%), 6 genotypes - medium (from 1.0 to 3.0%), and three - high (4.36%; 15.11% and 15.81%, respectively). Conclusions. Genotype-specific of microspores morphogenetic reactions of soft winter wheat in the process of androgenesis in vitro were revealed Samples P26 and P27 showed the highest level of callus formation. The 10 green regenerating plants were obtained.
 Keywords: hybrids, soft winter wheat, anther culture in vitro, callus, regeneration.

Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes

Abstract. Carsono N, Juwendah E, Liberty, Sari S, Damayanti F, Rachmadi M. 2021. Optimize 2,4-D concentration and callus induction time enhance callus proliferation and plant regeneration of three rice genotypes. Biodiversitas 22: 2555-2560. The development of callus in the course of transgenic rice avoids the somaclonal variants. To obtain a high number of normal phenotypes and a low number of somaclonal variants requires an appropriate 2,4-D concentration. In this study, we obtained the best callus induction time and a high number of green plant regeneration for three responsive rice genotypes on different 2,4-D concentrations in NB5 medium. The mature seeds of rice embryos were used as explants. A completely randomized factorial design was applied with four levels of 2,4-D concentrations (0, 1, 3, and 5 ppm), two levels of induction time (one and two weeks), and three rice genotypes (cv. Fatmawati, Nipponbare, and Kitaake). The study revealed that there was no interaction effect among genotype, 2,4-D concentration, and callus induction time. Three rice genotypes performed best in callus proliferation and regeneration. One-week callus induction time showed higher callus growth capacity (CGC) as compared to two-week callus induction time. Shoot regeneration capacity (SRC) was independently affected by genotype as well as by callus induction time. The interaction effect between 2,4-D concentration and callus induction time was observed on plant regeneration capacity (PRC). Without the addition of 2,4-D and 1 ppm of 2,4-D, the green plant regeneration capacity (GRC) was comparatively higher. Addition of 2,4-D showed a significant effect, especially at the plant regeneration stage. We found that one-week callus induction was the best treatment for callus proliferation and plant regeneration. We recommend the use of one-week callus induction and 1 ppm of 2,4-D for rice callus proliferation (sub-culture) and subsequent plant regeneration.

Improved protocol for efficacious in vitro androgenesis and development of doubled haploids in temperate japonica rice.

DH (Doubled haploid) is the immortal mapping population and an outcome of single meiotic cycle, contributed from male partner. An improved procedure was developed for high frequency androgenesis in japonica genotypes, K-332 and GS-88 and their F1s. A total of 207 fertile, green, di-haploid plants were generated from K-332 × GS-88 hybrids using the improved anther culture protocol. The investigation was carried out to evaluate callus induction potential and regeneration response for the genotypes and the derived F1s on N6 media and modified N6 media (N6M). Whereas, N6 failed to induce callusing, agarose solidified N6M media supplemented with 4% maltose, growth regulators; NAA (2 mg/l), 2, 4-D (0.5 mg/l), Kinetin (0.5 mg/l), and silver nitrate induced high calli percentage of 27.6% in F1s, 9.5% and 6.7% in GS-88 and K-332 respectively. Murashige and Skoog (MS) media supplemented with 3% sucrose, and the hormonal combination BAP (2 mg/l), Kinetin (1 mg/l) and NAA (1 mg/l) induced high green shoot regeneration rates (0–60.0%). The effect of cold pre-treatment at 4°C and the stage of anther collection and their interaction was studied. The effect of cold pre-treatment (CP) of collected boots at 4°C (for CP2: 2, CP4: 4, CP6: 6 and CP8: 8 days) at different stages of panicle emergence (BES4-6: 4–6, BES7-10: 7–10, BES11-13: 11–13, BES>13: more than 13 inches was worked out in relation to the effect on response of calli induction, albino regeneration, green plant regeneration and number of shoots/green calli. CP referred to the number of days for which the collected boots were incubated before they were inoculated. BES was the length (inches) between flag leaf and penultimate leaf at the time of boot collection. We concluded that CP6 and BES7-10 showed better response to callus proliferation and regeneration of plantlets across genotypes. The appropriate pre-treatment, stage of anther collection and favourable media composition resulted in high calli induction and green plant regeneration rates in recalcitrant japonica genotypes. The modified N6 media resulted into efficient callus induction and is expected to be useful for studies which aim at rapid generation of mapping populations for genetic studies.

The testing of haploproduction ability of soft winter wheat different hybrids in anther culture in vitro

Aim. Study the the efficiency of androgenesis in vitro in anther culture of different genotyps winter wheat. Methods. Obtaining of soft winter wheat double haploid lines by anther culture in vitro. The statistical methods. Results The ability to androgenesis in an in vitro anthers culture of 30 soft winter wheat genotypes was tested. The differences in the frequency of callus 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 (from % of implanted anthers) was broad. The sign of "the formation of callus" was in limited from 0 to 13.2 % and on the sign of "regeneration of green plants" – from 0 to 1.8 %. The 126 green plants-regenerants were received. Conclusions. Genotype-specific of microspores morphogenetic reactions of soft winter wheat in the process of androgenesis in vitro were revealed. It is advisable to use the anthers from the F2 hybrid population plant as a donor material to create an effective technology for producing double haploids of soft winter wheat was shown.
 Keywords: wheat hybrid, anther culture in vitro, callus, regeneration.

Grain barley starch as a gelling agent of nutrient media for cultivation of anthers and isolated embryos of H. vulgare L.

Aim. Determination the possibility of agar replacement with normal and waxy type starches produced from barley seeds in the nutrient media for spring barley haploid production. Methods. Anthers of spring barley line DH00-126 were inoculated on inductive media containing N6 macro-, MS micronutrients, organic supplements, maltose (9.0 %) and differed in solidifying agents (agar, starch from barley seeds or chemically modified starch D5a-1). Trophic capacities of barley starch were determined both in anther and embryo culture of barley. Results. It was determined that barley starch of waxy type was not suitable for solidifying of nutrient medium. Use of normal type barley starch (6.5 %) resulted in sufficient decrease in the frequency of green plant regeneration. At the same time, this modification had no effect on the number of morphogenic anthers. Barley starch was inferior to pea starch in the ability to support seedling growth in embryo culture in vitro. Conclusions. Barley starch of normal type has worse trophic capacities in comparison to pea starch in embryo culture. It was appeared barley starch to be a less suitable gelling agent of medium for spring barley haploid production in anther culture in vitro than agar and chemically modified starch D5a-1.
 Keywords: Hordeum vulgare L., anther culture in vitro, embryo culture in vitro, agar, barley starch.