N6 Medium Research Articles

Genome-Wide Association Analysis Identifies Candidate Loci for Callus Induction in Rice (Oryza sativa L.).

Callus induction (CI) is a critical trait for transforming desirable genes in plants. A genome-wide association study (GWAS) analysis was conducted on the rice germplasms of 110 Indica rice accessions, in which three tissue culture media, B5, MS, and N6, were used for the CI of those rice panels' mature seeds. Seven quantitative trait loci (QTLs) on rice chromosomes 2, 6, 7, and 11 affected the CI percentage in the three media. For the B5 medium, one QTL (qCI-B5-Chr6) was identified on rice chromosome 6; for the MS medium, two QTLs were identified on rice chromosomes 2 and 6 (qCI-MS-Chr2 and qCI-MS-Chr6, respectively); for the N6 medium, four QTLs were identified on rice chromosomes 6, 7, and 11 (qCI-N6-Chr6.1 and qCI-N6-Chr6.2, qCI-N6-Chr7, and qCI-N6-Chr11, respectively). Fifty-five genes were identified within the haplotype blocks corresponding to these QTLs, thirty-one of which showed haplotypes associated with different CI percentages in those media. qCI-B5-Chr6 was located in the same region as qCI-N6-Chr6.2, and the Caleosin-related family protein was also identified in this region. Analysis of the gene-based haplotype revealed the association of this gene with different CI percentages in both B5 and N6 media, suggesting that the gene may play a critical role in the CI mechanism. Moreover, several genes, including those that encode the beta-tubulin protein, zinc finger protein, RNP-1 domain-containing protein, and lysophosphatidic acid acyltransferase, were associated with different CI percentages in the N6 medium. The results of this study provide insights into the potential QTLs and candidate genes for callus induction in rice that contribute to our understanding of the physiological and biochemical processes involved in callus formation, which is an essential tool in the molecular breeding of rice.

Potential utilization of filaments and ovules as explant sources in in vitro propagation of Lilium sp.

Lilies are important cut and pot flowers locally and globally; however, qualified planting materials from in vitro culture are still limited. The culture generally uses scales and leaves, while filaments and ovules are rarely utilized. Filaments and ovules of EXO CF/E, RDF CG/E, and DZ CG/E genotypes of Lilium sp. were explored for their potential to establish in vitro propagation protocol using different Chu N6 medium and their modifications. The 9.9 shoots/explant from 1.5 regenerative explants/replication of RDF CG/E filaments was proven on Chu N6 medium containing 6.79 µM 2,4-dichlorophenoxyacetic acid (2,4-D), 2.27 µM thidiazuron (TDZ), and 90 g L-1 sucrose; 3.3 regenerative explants/replication and 8.4 shoots/explant of EXO CF/E ovules were determined on Chu N6 medium with 2.26 µM 2,4-D, 6.81 µM TDZ and 90 g L-1 sucrose; and slightly improved on Chu N6 macro elements, full vitamins of MS basal medium with 3.39 µM 2,4-D, 3.41 µM TDZ, 150 ml L-1 coconut water (CW) and 60 g L-1 sucrose. The shoots were proliferated on the established media. The shoots of EXO CF/E, RDF CG/E and DZ CG/E genotypes produced varied leaves/shoot, leaf length, leaf width, roots/shoot, root length and bulb diameter on Chu N6 medium hormone free with 1.5% AC, followed by hardening them in a glass house for one month. The hardened-plantlets were acclimatized in plastic pots containing charcoal husk and bamboo compost (1:1) with 39.8-63.6% survivability. The protocol has the potential to be applied to other genotypes or lilies by specific improvements at the acclimatization stage.

Improvement of Rice Blast Resistance in TGMS Line HD9802S through Optimized Anther Culture and Molecular Marker-Assisted Selection.

Rice blast caused by Magnaporthe oryzae is one of the most serious rice diseases worldwide. The early indica rice thermosensitive genic male sterile (TGMS) line HD9802S has the characteristics of stable fertility, reproducibility, a high outcrossing rate, excellent rice quality, and strong combining ability. However, this line exhibits poor blast resistance and is highly susceptible to leaf and neck blasts. In this study, backcross introduction, molecular marker-assisted selection, gene chipping, anther culture, and resistance identification in the field were used to introduce the broad-spectrum blast-resistance gene R6 into HD9802S to improve its rice blast resistance. Six induction media were prepared by varying the content of each component in the culture medium. Murashige and Skoog's medium with 3 mg/L 2,4-dichlorophenoxyacetic acid, 2 mg/L 1-naphthaleneacetic acid, and 1 mg/L kinetin and N6 medium with 800 mg/L casein hydrolysate, 600 mg/L proline, and 500 mg/L glutamine could improve the callus induction rate and have a higher green seedling rate and a lower white seedling rate. Compared to HD9802S, two doubled haploid lines containing R6 with stable fertility showed significantly enhanced resistance to rice blast and no significant difference in spikelet number per panicle, 1000-grain weight, or grain shape. Our findings highlight a rapid and effective method for improving rice blast resistance in TGMS lines.

In Vitro Induction of Interspecific Hybrid and Polyploidy Derived from Oryza officinalis Wall.

Oryza officinalis Wall is a potential genetic resource for rice breeding; however, its distant genome limits its crossing ability with cultivated rice. The interspecific hybridization of O. officinalis and cultivated rice, establishment of its tissue culture, and induction of polyploidy are ways to improve O. officinalis's poor crossability. We developed an interspecific hybrid and studied its reproductive pollen development process in this work, and the results showed that abortive pollens (81.94%) and embryo sac abnormalities (91.04%) were the key causes of its high sterility. In order to induce callus formation in interspecific hybrid explants, two different culture media, namely Chu's N-6 medium (N6) and 1/2 Murashig and Skoog medium (1/2 MS), were employed. Additionally, two plant growth regulators (PGRs), namely 2,4-dichlorophenoxyacetic acid (2,4-D) and 6-benzylaminopurine (BA), along with L-proline (Pro) and acid hydrolyzed casein, were utilized in the experiment. The optimal N6 medium, supplemented with 2.0 mg·L-1 2,4-D, produced the highest induction rate (80.56 ± 5.44)%. For callus differentiation and proliferation, the MS medium supplemented with 2.0 mg·L-1 BA + 0.2 mg·L-1 NAA produced the highest differentiation rate (75.00 ± 4.97)% and seedling emergence ratio (28.97 ± 4.67)%. The optimal combination for seedling rooting was the 1/2 MS medium supplemented with 2.0 mg L-1 NAA and 0.2 mg L-1 BA, which produced an average of 13.95 roots per plant. For polyploidy induction in the interspecific hybrid, the concentration of colchicine treatment at 400 mg·L-1 for three days is an ideal protocol. We devised tissue culture and interspecific hybrid polyploidy induction to overcome O. officinalis' poor crossability and introduce its favorable features into cultivated rice.

Introducing sorghum DREB2 gene in maize (Zea mays L.) to improve drought and salinity tolerance.

Salinity and drought are significant abiotic stresses causing a considerable loss of seed and biomass yield in most commercial crops. Some of the most critical players in the abscisic acid pathway are drought responsive element binding (DREB) proteins that are a part of AP2/ethylene response factor transcription factors that bind to promoters of some family genes needed to be expressed under abiotic stresses. In this study, salt- and drought-tolerant maize plants were produced from immature maize embryos bombarded by the sorghum (Sorghum bicolor L.) DREB2 gene that is linked to hygromycin resistance (hpt) genes. The putative transgenic calli were transferred to an N6 medium containing 1mg/L benzylaminopurine and 50mg/L hygromycin. Regeneration was completed after 4 weeks on selective media under a 16/8h light/dark condition at 25°C. Polymerase chain reaction (PCR) and reverse transcription-PCR approved the existence of upstream promoter (rd29a), hpt gene, and the expression of the DREB2 in transgenes up to the third generation (T2). It was found that the K+/Na+ ratio and the amount of proline as a screening indicator were higher in transgenic plants compared to their wild types. This result is a promising model to enhance maize tolerance to abiotic stressors.

In Vitro Doubled Haploid Production of Bacterial Blight Resistant Plants from BC2F1 Plants (Ranbir Basmati X Pau148) Through Anther Culture

Doubled haploid plants are very important for the development of complete homozygous plants from heterozygous parents in one generation as they possess duplicate copy of haploid chromosome. Haploid production is easily obtained from in vitro anther culture. The present study was undertaken with the objective to develop doubled haploids using anthers for in vitro induction of callus on N6 medium supplemented with various combinations and concentrations of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5-2.5 mg/L), Kinetin (0.5-1.0 mg/L) and Naphthalene acetic acid (NAA) (2.0 mg/L) as callus induction medium (CIM). The highest callus induction frequency was obtained when N6 medium fortified with 2,4-D (2.5 mg/L), Kinetin (0.5 mg/L) and NAA (2 mg/L) of 10.07 per cent. The induced callus was sub cultured for shoot regeneration on Murashige and Skoog medium (MS) supplemented with growth regulators: Kinetin and NAA (0.5 mg/L each) in combination with BAP (0.0 - 2.5 mg/L). MS medium supplemented with NAA (0.5 mg/L), Kinetin (0.5 mg/L) and BAP (1.5 mg/L) was most responsive exhibiting regeneration frequency of 28.1 per cent which resulted in maximum regeneration of green plantlets and only 5.21 per cent of albinos. Individual plantlets were separated and immersed in liquid MS medium augmented with NAA (0.5-1.0 mg/L) and BAP (0.5-1.0 mg/L). Maximum rooting was observed in MS medium with NAA (0.5 mg/L) and BAP (1.0 mg/L). The survival rate of in-vitro raised plants was 51.51 per cent. Of these surviving plants, 21 plants were observed to have the sterility percentage above 50 percent and hence can be considered as the doubled haploid plants. Plant DH8 is susceptible and DH20 is heterozygous for gene Xa21. Two plants are susceptible for gene xa13

Expression of cytokinin responsive and ethylene biosynthesis genes in rice callus with different regeneration rates

This study aimed to investigate the correlation between callus regeneration rate and the expression of several genes responsible for cytokinin response and ethylene biosynthesis in the Ciherang, Mentik Wangi Susu, Hwayoung and Tarabas rice varieties. The callus regeneration rate of each rice variety was in vitro tested using N6 media, while the gene expression during the callus regeneration stages was examined using quantitative real‐time PCR (qRT‐PCR). Our results showed that the callus of Ciherang and Mentik Wangi Susu showed earlier green spot formation that then turned brown at a later stage, resulting in a low regeneration rate. While Hwayoung and Tarabas showed late green spot formation, high shoot regeneration was observed in both calluses. Gene expression analysis of regeneration media‐grown calluses showed that two cytokinin‐responsive genes, OsRR2 and OsRR6, were highly expressed in the Ciherang and Hwayoung callus, respectively. We also observed that ethylene biosynthesis genes such as OsACS1 and OsACO1 were highly expressed in the Mentik Wangi Susu and Hwayoung callus, respectively. Moreover, the expression of OsBBM1 was high in Hwayoung and Tarabas. Thus, the positive correlation between the expression of cytokinin‐responsive and ethylene biosynthesis genes with somatic embryogenesis activity likely depends on the induction level of OsBBM1.

Androgenesis in indica rice: A comparative competency in development of doubled haploids.

Rice is critical to global food security which demands immediate attention to meet the ever-growing population. Development of improved variety is the major focus area of research, in which doubled haploid (DH) technology plays a vital role. Since, androgenesis shows its potential in DH production, this method was not capitalized specially in indica rice due to due to its recalcitrant nature to tissue culture. Success of androgenesis is governed by many important factors such as stage of anther, pre-treatment conditions, accurate concentrations of media, and plant growth regulators. Though reports of androgenesis are abundant in rice, most of them either used japonica or a specific cultivar of indica rice ecotypes. In this study, a media combination was established which is successful in producing doubled haploids from F1s of Savitri x Pokkali, IR20 x Mahulata along with the popular indica hybrids of Arize 8433DT, Arize 6453, Arize Bold, and Swift Gold. Out of 12 different media combinations tested, and 5 different durations of cold-treatments studied, N6 media with 2,4-D (2.0 mg/l) and BAP (0.5 mg/l) with 7th day cold pre-treatment was found to be most effective in all of the F1s for callus induction. Among all the F1s, rice hybrid, Arize 8433DT showed highest of 52% callus induction. In case of green shoot regeneration, MS media with NAA (0.5 mg/l), BAP (2.0 mg/l) and Kn (1.0 mg/l) (MS+C4) was found to be the most efficient of six treatments studied with highest of 58.25% regeneration in Arize 8433DT. Further, MS+C4 in combination with proline (5.0 mg/l) increased the regeneration rate to 85.99%. Besides, MS media with NAA (1.0 mg/l), Kn (0.1 mg/l) and 50 g/l sucrose was found to be most efficient for supporting root induction in all F1s. This study claims the establishment of genotype independent androgenic protocol for indica rice which could be capitalized in indica rice improvement.

Leaf pigments and concentrations of phenolic compound in Begonia grandis plantlets obtained from the floral explants

The objective of this study was to design a protocol of the successful establishment of plants of Begonia grandis dry. in culture in vitro, and to evaluate the concentrations of chlorophyll a and b, carotenoids, anthocyanins and phenolic compounds, as well as the ratios of chlorophyll a/b, total chlorophyll/carotenoids and total chlorophyll/anthocyanins in the in vitro plantlets in comparison to the same parameters assessed in the leaves of greenhouse plants. A significant increase in the percentage of shoot formation (22.0 ± 1.5%) was recorded on the modified N6 medium supplemented with plant growth regulators in comparison with the standard breeding technology. Begonia grandis plantlets possessed the elevated contents of all photosynthetic pigments per unit mass of plant compared to the greenhouse plants. The increase in chlorophyll b was most substantial. The average anthocyanin concentrations in the plantlets and greenhouse plants were approximately equal, but the essential variation within B. grandis microclones was observed. Phenolic profiles of in vitro plantlets and greenhouse plants was rather similar. Concentrations per unit mass for the majority of components (especially organic and phenolic acids) in the plantlets increased in comparison to those in greenhouse plants (47.23 and 38.22 mg g−1 total phenolics, respectively). Overall data suggest that this protocol can be used for the production of a large number of plants of B. grandis in a good physiological state and with high phenolic content. Graphical abstract

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.

Efficient callus induction and regeneration in proso millet

AbstractProso millet (Panicum miliaceum L.) is an important drought‐tolerant crop, and recently it has become a research focus because of its health‐care and nutrition significance. A study was carried out to investigate callus induction and differentiation and plantlet rooting in Shaanmi 1 and Shaanmi 2 with young leaves of the two varieties. Its result showed that in proso millet, the age of aseptic seedlings could influence on inducing calli from young leaves with the highest callus induction frequency at the seedling age of 7 d. N6 medium performed better in inducing calli from young leaves. There did appear to be no calli on MS medium without 2,4‐D, but calli were induced on N6 medium without 2,4‐D in Shaanmi 2, with its induction frequency standing 6.45% and no calli were induced on the medium without 2,4‐D in Shaanmi 1. With 2,4‐D added, there appeared the highest callus induction frequency at a 2,4‐D concentration of 3.0 mg L–1. The optimal medium to induce calli from young leaves in proso millet was N6 medium plus 2,4‐D (3.0 mg L–1) and KT (0.5 mg L–1), with its callus induction frequency at 64.33% (Shaanmi 1) and 68.67% (Shaanmi 2), respectively. The optimal callus differentiation medium was MS medium plus 1.0 mg L–1 6‐BA and 0.5 mg L–1NAA, with its differentiation rate at 87.78% (Shaanmi 1) and 96.29% (Shaanmi 2), respectively. Proline adding at 600 mg L–1 was conducive to callus differentiation. The optimal rooting medium was 1/2MS medium plus NAA (0.1 mg L–1), with its rooting rate at 100%.

Improved Anther Culture Media for Enhanced Callus Formation and Plant Regeneration in Rice (Oryza sativa L.).

Anther culture technique is the most viable and efficient method of producing homozygous doubled haploid plants within a short period. However, the practical application of this technology in rice improvement is still limited by various factors that influence culture efficiency. The present study was conducted to determine the effects of two improved anther culture media, Ali-1 (A1) and Ali-2 (A2), a modified N6 medium, to enhance the callus formation and plant regeneration of japonica, indica, and hybrids of indica and japonica cross. The current study demonstrated that genotype and media had a significant impact (p < 0.001) on both callus induction frequency and green plantlet regeneration efficiency. The use of the A1 and A2 medium significantly enhanced callus induction frequency of japonica rice type, Nipponbare, and the hybrids of indica × japonica cross (CXY6, CXY24, and Y2) but not the indica rice type, NSIC Rc480. However, the A1 medium is found superior to the N6 medium as it significantly improved the green plantlet regeneration efficiency of CXY6, CXY24, and Y2 by almost 36%, 118%, and 277%, respectively. Furthermore, it substantially reduced the albino plantlet regeneration of the induced callus in two hybrids (CXY6 and Y2). Therefore, the improved anther culture medium A1 can produce doubled haploid rice plants for indica × japonica, which can be useful in different breeding programs that will enable the speedy development of rice varieties for resource-poor farmers.

The Effect of Plant Growth Regulators (PGRs) on Efficient Regeneration of 12 Recalcitrant Indica Rice (Oryza Sativa L.) Genotypes

Biotechnological approaches provide an efficient and expeditious system for developing agronomic features of rice at the cellular and molecular level compared to traditional breeding methods. The in-vitro regeneration method optimization of recalcitrant indica rice genotypes is a critical step for the biotechnological approaches for further development as the optimum concentrations of Plant Growth Regulators (PGRs) for in-vitro regeneration vary from genotype to genotype. Here, 12 important rice genotypes, including local cultivars and varieties, were collected from different regions of Bangladesh and evaluated for their in-vitro regeneration potentiality. Mature dehusked sterilized rice seeds were inoculated on different basal media (MS, N6, B5) supplemented with different concentrations of 2,4-Dichlorophenoxyaceitic acid (2,4-D) for callus induction. Induced calli were transferred to N6 media supplemented with different combinations of 6-Benzylaminopurine (BA) and 1-Napthaleneacetic Acid (NAA) for shoot induction. Regenerated shoots were rooted on half-strength MS media without supplementation of any hormones. Treatments of different concentrations and combinations of plant growth regulators showed varying results. Maximum callus induction frequencies were observed with 2.5-3.0 mg/l supplementation of 2,4-D on N6 media where Saat, Jamaibabu and Nimikhanai showed the highest callus induction frequencies (84.44%; 78.89 and 80% respectively) and maximum shoot initiation was observed at 2.5 mg/l BA +1.0 mg/l NAA and 3.0 mg/l 6-BA +1.5 mg/l NAA supplemented media where rice genotypes BRRI Dhan71, Nayapaijam, Nimikhanai and BRRI Dhan28 showed the highest shoot induction frequencies-86.67, 85.00, 86.67 and 88.33% respectively. Nearly all of the induced shoots showed root formation on half-strength MS media. This study demonstrates variations in regeneration response of the rice genotypes where cultivars Jamaibabu, Nayapaijam and Nimikhanai showed the highest in-vitro regeneration potential.

Peach palm plantlet growth in different culture media in a temporary immersion system

ABSTRACT: Peach palm is a domesticated palm commercially important for the production of fruits and hearts of palm. Somatic embryogenesis, an effective technique for mass propagation, was successfully established for this species. Furthermore, a temporary immersion system improved plant regeneration. However, production can be further improved by understanding the peach palm’s growth dynamic and modifications of culture media. The aims of this study were to evaluate the growth of plantlets cultured in different culture media in a temporary immersion system and to correlate the results with nutrient uptake during the growth period. Somatic embryo-derived young plantlets approximately 1 cm in length were cultivated for 12 weeks in a twin flask system containing MS, Y3 or N6 salts, Morel and Wetmore vitamins and 3% sucrose, with a monthly medium refreshment. Growth was measured and mineral analysis of the plantlets was carried out after 12 weeks of culture. The Y3 and MS salts were the most appropriate for the plant growth. Number of roots was 52.52% higher and the root size was 40.42% between the N6 and MS medium and the root number in Y3 medium was 37.74% greater than in MS medium, which is important for post acclimatization survival. K and Na are important elements for peach palm. N is not required at such a high concentration as in Murashige and Skoog formulation. The Chu (N6) medium did not generate high quality plantlets, possibly due to the absence of some micronutrients, like Mo, Cu and Co.

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.

Anther Culture Efficiency in Quality Hybrid Rice: A Comparison between Hybrid Rice and Its Ratooned Plants.

An immense increase in human population along with diminished lands necessitates the increase of rice production since, it serves the human population as a staple food. Though rice hybrids (RH) are showing considerable yield enhancement over inbreds in terms of both quality and quantity, farmers’ adoption of hybrid rice technology has been much slower than expected because of several constraints such as seed cost and quality. Doubled haploid (DH) technology was considered useful for the development of inbred lines from rice hybrids in a single generation. Androgenesis shows its significance in development of DHs in rice which requires an efficient method to establish the production of large population. To start the anther culture, anthers are the main component of androgenesis to be isolated from unopened spikes. However, the duration of spikes availability for anther culture coupled with the segregation of rice hybrids in the next generation requires the main crop be ratooned to reduce the cost of cultivation. Therefore, the efficiency of the androgenic method was tested in main crop using a quality indica rice hybrid, 27P63 and its ratooned ones. The effects of various factors such as cold temperature pre-treatment of boots, treatment duration, and different combination of plant growth regulators (PGR) on callus response along with shoot regeneration were tested for development of DHs from both ratooned and non-ratooned plants. The N6 medium supplemented with 2.0 mg/L 2,4-D (2,4-dichlrophenoxy acetic acid), 0.5 mg/L BAP (6-benzylamino purine), and 30 g/L maltose was found to be most effective for callusing as compared to MS (Murashige and Skoog) medium. The N6 media inducted calli showed maximum response rate for green shoot regeneration in MS media supplemented with 0.5 mg/L NAA (1-napthaleneacetic acid), 0.5 mg/L Kn (Kinetin; 6-furfurylaminopurine), 1.5 mg/L BAP and 30 g/L sucrose after 2 weeks of culture. The pre-treatment of spikes at 10 °C for 2 d followed by a 7th and 8th d were found to be most effective for callusing as well as for regeneration, producing a total of 343 green plants from ratooned and main rice hybrid, 27P63. Morpho-agronomic trait-based assessment of ploidy status revealed 94.46% diploids, 3.49% polyploids, 0.58% mixploids, and 1.45% haploids. Microsatellite markers could authenticate all 324 fertile diploids as true DHs. Though this study shows a reduction in generation of DHs from ratooned plants as compared to the main crop, manipulation of chemical factors could optimize the method to enhance the production of considerable number of DHs. Utilization of ratooned of hybrid rice in androgenesis would save time and cost of cultivation.

Vigna caracalla L. Verdc. Bitkisinde In Vitro Klonal Mikroçoğaltım

Vigna caracalla L. Verdc. is an ornamental plant, having with its beautiful odorous remarkable flowers, named as "İzmir's ivy". In this study, it was aimed to develop an effective protocol for in vitro clonal micropropagation in Vigna caracalla L. Verdc. plant. The seeds were cultured in MS medium after exposed to different sterilization methods. The highest percentage of sterilization (93.33%) were achieved when the seeds were soaked in 70% ethyl alcohol for 1 min, 0.1% HgCl2 for 4 min and then kept sterilized distilled water for 7 hours. The highest percentage of germination (95.24%) was observed in 250 mL Erlenmeyer flasks containing N6 medium. The highest shoot length (5.05 cm) was obtained from shoot tip explants cultured in MS medium containing 1 mg/L IBA + 0.5 mg/L BAP. In the experiments carried out to determine the effect of gelling agent, light intensity and explant type on shoot regeneration, the highest multiplication coefficient (1.71) and leaf length (0.82 cm) was achieved in node explants cultured in N6 medium solidified with Duchefa agar and exposed to 4200 lux light intensity. Within the scope of this study, it was determined that 16 clones that did not show much hyperhydricity and had higher multiplication rate gave appropriate responses for micropropagation experiments. The highest root regeneration (85.71%) was achieved from shoot tip explants cultured in N6 medium solidified with Fluka agar and exposed to 4200 lux light intensity. The rooted plantlets were acclimatized with 70% success.

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.&#x0D; Bangladesh Rice j. 2019, 23(2): 17-25

Selection of purification and formation of double haploid Toraja endemic black rice through anther culture

To maximize the potential of Ko’bo, plant breeding was involved to produce the identic color of rice grains, which is full black. Anther culture is a method that is considered effective in plant breeding. This study aimed to find the most suitable combination of growth regulators in the calli induction of Ko’bo rice anther and observe the calli growth response in a regeneration medium. Calli induction experiment used N6 medium with a combination of auxin (2,4-D 1-3 mg L−1) and cytokinin (kinetin 0.5-1 mg L−1 and BAP 0.5-1 mg L−1). Calli regenerated in MS medium, added with 0.5 mg NAA L−1 + kinetin 0.5 mg L−1+ BAP 1.5 mg L−1. The experiment was arranged in a Completely Randomized Design. The experimental unit was a petri dish, containing ± 250 anthers. The highest calli induction was 13.33%, obtained from a combination of 2,4-D 3.0 mg L−1 + kinetin 1.0 mg L−1. All regenerated plantlets were albino.

Optimization of Conditions for Callus Induction from BC2F1 Population (Ranbir Basmati x PAU148) through Anther Culture

With different culture conditions and concentrations of growth regulators and supplements, Anther culture technique can be easily employed for the production of haploids under in vitro conditions.&#x0D; Aims: The present study was undertaken with the objective to optimize the development of doubled haploids using anthers for in vitro induction of callus on N6 medium.&#x0D; Place and Duration of Study: The samples (BC2F1 seeds) were raised previously in Skuast-J. From total degree program of 3 years, this work related to tissue culture technique was done in one year from January 2018 to January 2019.&#x0D; Methodology: The effect of levels of 2, 4-dichlorophenoxy acetic acid (2, 4-D) i.e. 0 to 3 mg/L in basal N6 media was observed on callus induction frequency (CIF). The effect of duration of cold pre-treatment was observed on callus induction frequency at 2.5 mg/L of 2, 4-D by giving the cold pre-treatment at 4ºC from 8 to 12 days. Also the effect of different amino acids was checked on callus induction frequency.&#x0D; Results: Highest callus induction frequency of 9.39 per cent was observed in N6 medium fortified with 2.5 mg/L 2, 4-D and lowest callus induction frequency of 2.52 per cent at the concentration of 1.0 mg/L. The cold pre-treatment for 10 days gave highest callus induction frequency of 1.44 per cent and lowest callus induction frequency of 0.44 per cent was obtained at cold pre treatment for 8 days. The highest callus induction frequency of 12.55 per cent was observed in case of media supplemented with 25 mg/L tryptophan and 40 mg/L cysteine and lowest callus induction frequency of 7.18 per cent was observed when media was supplemented with 560 mg/L proline.&#x0D; Conclusion: The cold pre-treatment of 10 days at 4ºC on media supplemented with 2.5 mg/L of 2, 4-D and combination of 25 mg/L tryptophan and 40 mg/L cysteine proves to provide best androgenesis conditions for anthers from BC2F1 population.