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An in vitro mass propagation protocol was developed for Gerbera (Gerbera jamesonii Bolus) var. orange using flower bud explants. The young (7 days) explants were found to be suitable for culture and highest (100%) callus induction was obtained in MS medium supplemented with 5.0 mg/l BAP with 1.0 mg/l NAA. The maximum shoot regeneration (95%) and the number of shoots (10) were observed in MS medium supplemented with 6.0 mg/l BAP with 0.3 mg/l NAA. The highest shoot multiplication frequency (95%) and the number of shoots (60) were obtained in MS medium supplemented with only 2.0 mg/l BAP. The well-developed highest number of roots (5.0) per plant and average root length (5.0 cm) with 100% root induction efficiency without callusing was recorded in the half strength of MS medium devoid of any hormonal supplement. Acclimatized in vitro grown plantlets were successfully transferred into the field condition. Plant Tissue Cult. & Biotech. 32(2): 217-226, 2022 (December)

Tobacco has been widely used as a model plant for stable and non-stable gene function analysis. Successful Agrobacterium-mediated transformation mainly depends on in vitro regeneration of tobacco plant. However, a reliable and standard regeneration protocol of tobacco using multiple explants is limited. In this study, we established a reliable and reproducible regeneration protocol of tobacco using three different explants i.e. cotyledon, hypocotyl and leaf. Preliminary, surface sterilized tobacco seeds were germinated on growth regulator free MS medium. Thereafter, in vitro germinated explants were inoculated into Murashige and Skoog [1] media supplemented with different combination and types of growth regulators for callus induction and subsequent regeneration of plantlets. It was revealed that, regeneration ability of explants is greatly influenced by type and nature of the explant. Among the three explants, higher callus induction (95%) was obtained in MS medium supplemented with 2.0 mg l-1 kinetin + 2.0 mg l-1 IAA from leaf explant. Also, leaf explant exhibited much higher regeneration ability (95%) than hypocotyl (60%) and cotyledon (45%) explants. Significantly highest number of shoots (8.0) were regenerated from leaf explants cultured on MS medium supplemented with 3.0 mg l-1 Kinetin+1.0 mg l-1 IAA compared to the other hormone combinations. Regenerated mature shoots were showed normal root after transferred onto ½ MS medium containing 0.3 mg l-1 IBA. This study will provide valuable information related to in vitro regeneration of tobacco plantlets using cotyledon, hypocotyl and leaf explants and will be used as a standard protocol for Agrobacterium-mediated transformation for gene function analysis.

Novel mutant alleles related to isoflavone content are useful for breeding programs to improve the disease resistance and nutritional content of soybean. However, identification of mutant alleles from high-density mutant libraries is expensive and time-consuming because soybean has a large, complicated genome. Here, we identified the gene responsible for increased genistein-to-daidzein ratio in seed of the mutant line F333ES017D9. For this purpose, we used a time- and cost-effective approach based on selective genotyping of a small number of F2 plants showing the mutant phenotype with nearest-neighboring-nucleotide substitution-high-resolution melting analysis markers, followed by alignment of short reads obtained by next-generation sequencing analysis with the identified locus. In the mutant line, GmCHR5 harbored a single-base deletion that caused a change in the substrate flow in the isoflavone biosynthetic pathway towards genistein. Mutated GmCHR5 was expressed at a lower level during seed development than wild-type GmCHR5. Ectopic overexpression of GmCHR5 increased the production of daidzein derivatives in both the wild-type and mutant plants. The present strategy will be useful for accelerating identification of mutant alleles responsible for traits of interest in agronomically important crops.

Runner tips explants of strawberry give rise to multiple shoots when cultured on MS medium supplemented with different concentrations and combinations of BAP with KIN or NAA or GA3.The highest response of shoot multiplication was obtained on MS containing 2.5 mgl-1 BAP + 0.5 mgl-1 Kin + 0.5 mgl-1 GA3. The maximum frequency of rooting (83%) and highest number of roots (3.49) was produced in medium containing 1.0 mgl-1 IBA. The well grown rooted plantlets were acclimatized and successfully established in autoclaved vermiculate soil and as well as natural condition. Using our established protocol, it is also possible to provide large numbers of micropropagated plantlets of this cultivars to produce high quality strawberry fruit for commercial cultivation practices.

RNA silencing is mediated through RNA interference (RNAi) pathway gene families, i.e., Dicer-Like (DCL), Argonaute (AGO), and RNA-dependent RNA polymerase (RDR) and their cis-acting regulatory elements. The RNAi pathway is also directly connected with the post-transcriptional gene silencing (PTGS) mechanism, and the pathway controls eukaryotic gene regulation during growth, development, and stress response. Nevertheless, genome-wide identification of RNAi pathway gene families such as DCL, AGO, and RDR and their regulatory network analyses related to transcription factors have not been studied in many fruit crop species, including banana (Musa acuminata). In this study, we studied in silico genome-wide identification and characterization of DCL, AGO, and RDR genes in bananas thoroughly via integrated bioinformatics approaches. A genome-wide analysis identified 3 MaDCL, 13 MaAGO, and 5 MaRDR candidate genes based on multiple sequence alignment and phylogenetic tree related to the RNAi pathway in banana genomes. These genes correspond to the Arabidopsis thaliana RNAi silencing genes. The analysis of the conserved domain, motif, and gene structure (exon-intron numbers) for MaDCL, MaAGO, and MaRDR genes showed higher homogeneity within the same gene family. The Gene Ontology (GO) enrichment analysis exhibited that the identified RNAi genes could be involved in RNA silencing and associated metabolic pathways. A number of important transcription factors (TFs), e.g., ERF, Dof, C2H2, TCP, GATA and MIKC_MADS families, were identified by network and sub-network analyses between TFs and candidate RNAi gene families. Furthermore, the cis-acting regulatory elements related to light-responsive (LR), stress-responsive (SR), hormone-responsive (HR), and other activities (OT) functions were identified in candidate MaDCL, MaAGO, and MaRDR genes. These genome-wide analyses of these RNAi gene families provide valuable information related to RNA silencing, which would shed light on further characterization of RNAi genes, their regulatory elements, and functional roles, which might be helpful for banana improvement in the breeding program.

Shoot tip and nodal segment explants from field grown plants were used as experimental materials in this investigation. All explants were cultured on MS medium supplemented with various plant growth regulators. For surface sterilization of explants, HgCl2 (0.1%) for 2 minutes was found to be most effective for complete killing of surface pathogens and getting healthy tissues. Shoot regeneration was observed from both shoot tips and nodal explants for the studied plant. Various concentrations of BAP (0.1, 0.2, 0.3mg/l) and GA3 (0.1, 0.2, 0.3mg/l) were used for shoot multiplication. In case of BAP, the highest length of shoot was recorded 4 cm and the highest percentage of shoot multiplication (73%) was noticed in MS+0.2mg/l BAP. And in case of GA3, the highest response for shoot multiplication (82%) was noticed in MS+ 0.1 mg/l GA3.But among all of the media formulations used in this experiment, the highest response for shoot multiplication (95%) within7-10 days was noticed in hormone free MS media. In case of root regeneration, the highest percentage (96%) of root induction was recorded in MS medium supplemented with no hormone. Asian Australas. J. Biosci. Biotechnol. 2017, 2 (1), 81-88

Novel mutant alleles related to isoflavone content are useful for breeding programs to improve the disease resistance and nutritional content of soybean. However, identification of mutant alleles from high-density mutant libraries is expensive and time-consuming because soybean has a large, complicated genome. Here, we identified the gene responsible for increased genistein-to-daidzein ratio in seed of the mutant line F333ES017D9. For this purpose, we used a time- and cost-effective approach based on selective genotyping of a small number of F2 plants showing the mutant phenotype with nearest-neighboring-nucleotide substitution–high-resolution melting analysis markers, followed by alignment of short reads obtained by next-generation sequencing analysis with the identified locus. In the mutant line, GmCHR5 harbored a single-base deletion that caused a change in the substrate flow in the isoflavone biosynthetic pathway towards genistein. Mutated GmCHR5 was expressed at a lower level during seed development than wild-type GmCHR5. Ectopic overexpression of GmCHR5 increased the production of daidzein derivatives in both the wild-type and mutant plants. The present strategy will be useful for accelerating identification of mutant alleles responsible for traits of interest in agronomically important crops.