Optimization of the Androgenesis Method in the In Vitro Culture of Anteneson the Basis ofT. AestivumHybrids

This study aimed to examine the regularities of the regenerationprocesses of haploid plants, the dependence of in vitro microspore morphogenesis in anther culture on optimization factors, and their efficiency in F1 hybrids of T. aestivumof different ecological and geographicorigin. It was found that heterosis contributed to an increased yield of haploid chlorophyll-bearing regenerants from hybrids obtained from the crossing of parental forms with different responsiveness to androclinia. Results were obtained for the complex optimization of the androgenesis method for the in vitro anther culture of T. aestivum, in order to create diploidized haploid lines (DHL) regardless of the influence of the genotype. The agroecological properties for a complex of economically useful traits were also assessed. DHLs were created that combined high yield (5.1-6.8 t / ha) with lodging resistance (straw height – 60-80 cm) and consistently high grain quality; these were characterized by increased resistance to major leaf diseases in comparison with the standard variety in the conditions of the Central Economic Region of the Non-Black Earth Zone of the Russian Federation. Keywords: spring soft wheat, androgenesis, embyroidogenesis, callusogenesis, diploidized haploids, in vitro, yield and quality

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.

Aromatic Rice with Multiple Disease Resistance Developed through Anther Culture

The experiments were conducted during kharif (June to September) 2018 and 2019 to screen 7 aromatic rice double haploid (DH) lines developed against Bacterial Leaf Bligh (BLB) and blast disease. Three genes Xa21, xa13, xa5 for BLB resistance and two genes Pi1, Pi2 for blast resistance was introgressed into a local aromatic rice variety Dubraj from Chhattisgarh State of India. Dubraj is extremely popular aromatic short medium grain variety in Chhattisgarh and Madhya Pradesh region and hence used in this study. RPBIO-226 and PR122 rice variety is used as donor for BLB and blast resistance respectively. 7 DH lines (L-1 to L-7) were developed from the crosses (Dubraj X RPBIO-226 X PR122) in the year 2017. Phenotypic evaluation for BLB diseases resistance has shown that L-1, L-6 and L-7 (score 1) has a similar resistance as the control check RPBIO-226(score-1) and 3 lines with moderate resistance (L-3 score 2) (L-4 score 3) (L-5 score 3). For blast disease, three lines (L-4, L-6 and L-7) were identified as resistance with the disease score of 1, whereas three were grouped under the section moderately resistant (L-1, L-3 and L- 5) with a disease score of 3. Sensory test (1.7% KOH) was conducted for the estimation of presence / absence of aroma. Moderately strong aroma was present in four lines (L-1, L-3, L-6 and L-7). On the basis of yield and presence of aroma in grain, L-3 has been selected with moderately resistant to BLB and blast for further advancement and subsequently evaluated under State Initial Evaluation Trial (SIET) during kharif 2020. Within 3 years of development of DH line, L-3 is under varietal developmental trail, which itself indicate the efficacy of anther culture in fixing homozygosity and speedy development of desired variety within short period of time.

Genetic variation of doubled haploids derived from anther culture of M1 red rice plants

Nurmansyah, Setyadi AH, Fatumi NC, Fatmawati Y, Wulandari RA, Purwantoro A. 2021. Genetic variation of doubled haploids derived from anther culture of M1 red rice plants. Biodiversitas 22: 4923-4929. The doubled haploid (DH) technology when integrated with induced mutation could accelerate development of local rice cultivars with several desirable traits. Anthers from the first generation of mutant (M1) plants were utilized for DH production. However, the effectiveness of inducing and fixing mutation requires detailed evaluation with the help of molecular techniques for its accuracy and reliability than that of morphological or biochemical assessments. The objective of this research was to develop and detect genetic variation of DH plants derived from anther culture of M1 plants. Seeds of local red rice cultivar, Cempo Abang, were treated with four gamma-ray doses (0, 100, 200, 300 Gy) and planted to produce the M1 plants. Panicles at the booting stage of the M1 plants were collected and used as donor anthers. The present study suggested that gamma irradiation treatments indirectly could increase callus formation. However, it also reduced the number of callus producing plantlets due to higher frequency of brown calli. Eleven spontaneous DH plants obtained in the study along with parental plants were assessed to determine the occurrence of genetic variation using six RAPD primers. The RAPD primers generated 51 bands, of which 34 alleles or 66.7% were polymorphic with an average of 5.6 polymorphic alleles per primer. The genetic similarity among parent and 11 DH lines based on Jaccard’s similarity index ranged from 0.622 to 0.902. The DNA polymorphism among the DH plants demonstrated the effect of gamma irradiation to create genetic variation. Therefore, this method could be used as an alternative for rice breeding programs especially to develop preferred traits in the local rice cultivars.

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.

Effect of sowing dates of donor plants and concentration of 2,4-D on the purity of productive anther formation of common barley (Hordeum vulgare L.) in anther culture in vitro.

Creating dihaploid lines of agricultural plants is a labour-intensive but essential step in variety production in modern plant breeding. This stage allows significantly accelerate the process of creating new varieties of common barley and other crops. Barley digaploids are produced mainly by anther culture and microspore culture. The authors preferred anther culture in vitro. In the present study, the influence of climatic factors in the cultivation of donor plants on the yield of productive anthers at different sowing dates was established. The authors also identified the more stable culti- vars with a high anther production regardless of sowing date (Signal, Laureate and Eifel). Varieties showed the highest number of embryo-like structures formation at the first and third sowing dates (Zu Suren, Zu Zaza); and sorts with a high rate of productive anther formation at the second sowing date (Acha, Exploer) were identified. Different concentrations of 2,4-D in N6 medium on the frequency of embryogenesis and yield of productive anthers were studied. As a result of this study, the authors found that different concentrations of 2,4-D (1 mg/l and two mg/l) had no significant effect on the for- mation frequency of productive anthers in all the varieties studied. When the embryogenesis capacity of the cultivars was reviewed, all the samples were found to be positive in anther culture. However, the array Zu Suren had a significantly lower effective anthers yield than the samples Signal and Acha. As a result of correlation analysis, the authors found a close relationship between the length of the ear tube of donor plants and the frequency of formation of productive anthers (r = -0.69). A close relationship with the development of optimal microspore phase for the induction of androgenesis in anthers extracted from the ear tube with an average length of 6 cm was determined. This information can significantly speed up the selection of donor plants, but it is recommended to confirm the stage of microspore development microscopically for each new cultivar used.

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.