In vitro regeneration of soybean (a review)

This is an overview of contemporary published works dedicated to the ability of soybean plants to regenerate in vitro and the techniques to achieve high regeneration rates, which is a necessary condition for the inclusion of soybean genotypes in genome editing programs. The main factors that determine the regenerative capacity of explants from various soybean accessions are considered. The greatest effect on the efficiency of regeneration is exerted by the conditions of in vitro culture initiation, type of explant, composition of the nutrient medium, shelf life of seeds, and genotypic characteristics of soybean accessions.

A bioprocess perspective on the production of secondary metabolites by Streptomyces in submerged co-cultures

Filamentous microorganisms are potent sources of bioactive secondary metabolites, the molecules formed in response to complex environmental signals. The chemical diversity encoded in microbial genomes is only partially revealed by following the standard microbiological approaches. Mimicking the natural stimuli through laboratory co-cultivation is one of the most effective methods of awakening the formation of high-value metabolic products. Whereas the biosynthetic outcomes of co-cultures are reviewed extensively, the bioprocess aspects of such efforts are often overlooked. The aim of the present review is to discuss the submerged co-cultivation strategies used for triggering and enhancing secondary metabolites production in Streptomyces, a heavily investigated bacterial genus exhibiting an impressive repertoire of secondary metabolites, including a vast array of antibiotics. The previously published studies on influencing the biosynthetic capabilities of Streptomyces through co-cultivation are comparatively analyzed in the bioprocess perspective, mainly with the focus on the approaches of co-culture initiation, the experimental setup, the design of experimental controls and the ways of influencing the outcomes of co-cultivation processes. These topics are discussed in the general context of secondary metabolites production in submerged microbial co-cultures by referring to the Streptomyces-related studies as illustrative examples.

In vitro Micropropagation of Pomegranate (Punica granatum L.) Derived from Cotyledon

A high frequency in vitro plant regeneration of pomegranate was established on MS medium supplemented with different concentrations and combinations of plant growth regulators. As explant cotyledons were employed for this study. Ninety percent of the cultured explants responded to form shoots from 30 days old in vitro raised seedlings after 90 days of culture initiation in MS containing 1.0 mg/l IBA + 0.1 mg/l NAA. The average number of shoots per explant was 10.0 ± 2.20, shoot length of 12.0 ± 2.40 cm, node per regenerated shoot was 9.0 ± 1.60 and the leaf number was14.0 ± 1.40. Well developed shoots were cultured on half strength of MS medium supplemented with 0.5 mg/l IBA, in which 90% shoot induced roots implanted after one month. The average number of root per shoot was 8.0 ± 0.90 and the average root length of 6.5 ± 0.40 cm was observed in this medium. Eighty percent plantlets were survived in the outdoor condition during the acclimatization period of seven days. Plant Tissue Cult. & Biotech. 31(1): 61-69, 2021 (June)

Growth Evaluation of In-Vitro Propagated Embryo of Morinda Citrifolia L. Seeds

The dormant nature of Morinda citrifolia seeds is a limitation to its efficient in-vitro plantlet multiplication. Hence, the use of embryo culture for successful in-vitro culture initiation. Matured embryo of freshly collected noni seeds were cultured on Murashige and Skoog basal medium supplemented with kinetin (Kn) and Benzyl amino purine (BAP) in the range of A: control (no addition); B: 0.5 mg/l Kn+1.0 mg/l BAP; C: 1.0 mg/l Kn+2.0 mg/l Bap; D: 1.5 mg/l Kn+3.0 mg/l BAP and E: 2.0 mg/l Kn+4.0 mg/l BAP. The results at 4 weeks after inoculation (WAI) showed that germination was faster from medium A without hormone whereas highest percentage germination was obtained from both medium D and E with 80 %. Medium B and C had 65 % each while medium A gave the least (40%). The development of the plantlets showed that longest shoot (3.9 cm) from medium A was closely related to 3.58 cm from Medium B while root lengths (2.28 cm) and number of adventitious roots (26) from medium A were significantly higher than other media at 12 WAI. Highest number of nodes (2.25) obtained from medium D was comparable to Media C and B while medium A had the least at 12 WAI. Number of leaves obtained was similar between the media at 12 WAI. These results indicated that using embryo is reliable for fast in-vitro propagation and shoot development of noni plant with optimum cytokinins (0.5/1.0 mg/l Kn/BAP) application. Keywords: Culture initiation, Cytokinins, Embryo culture, Plantlet, Shoot development

In vitro propagation of plume cultivars selected in Amur Region, Russia

Results are presented on clonal micropropagation for three cultivars selected in Amur Region: 'Blagoveshchenskii chernosliv', 'Lyudmila', and 'Oranzhevaya rannyaya'. Tips of growing young shoots are preferable explants for the tissue culture production as compared to lateral buds of the same shoots. The Quoirin-Lepoivre (QL) agar medium supplemented with sorbitol (20 g/L) and 6-benzylaminopurine (BA, 3 mg/L) was used for the establishment of explants and tissue culture initiation. Shoot proliferation was conducted on a QL agar medium with a modified microelement and vitamin composition and supplemented with sucrose (30 g/L), BA (0.2, 0.5, or 2.0 mg/L) and indole-3-butyric acid (IBA, 0.04 mg/L). Optimal shoot proliferation was noted to be achieved by alternate cultivation cycles on the medium with various BA concentration: 2 mg/L for an increased proliferation rate and 0.2–0.5 mg/L for an increased microshoot length. Higher values of shoot proliferation (the proliferation rate and microshoot length) were found for cv. 'Blagoveshchenskij chernosliv' (1.6 and 1.9 respectively) as compared to cv. 'Lyudmila' (1.3 and 1.5) and cv. 'Oranzhevaya rannyaya' (1.4 and 1.2). In vitro rooting was achieved after a preliminary incubation of microshoots in the aqueous solution of IBA (15 mg/L) with the subsequent cultivation on the growth regulator-­free QL agar medium. The cultivar 'Blagoveshchenskij chernosliv' had higher values of rooting (5.8 roots per shoot and 11.6 cm of total root length) as compared to cv. 'Lyudmila' (4.6 and 6.6 respectively) and cv. 'Oranzhevaya rannyaya' (5.3 and 7.4). The protocol was used for production of own­rooted plants of the plum cultivars.

Problems Encountered during In vitro Culture Establishment in Terminalia arjuna

The main aim of present study was to overcome the problems associated with the in vitro culture initiation in Terminalia arjuna. The micropropagation of tree species is not easy as shrubs and herbs. Many problems encountered from explant collection to in vitro culture establishment. The problems that have been occurred during T. arjuna micropropagation were culture contamination, phenolic exudation, bud growth inhibition, shoots yellowing and leaf fall. All these problems have been solved by applying certain treatments prior to explant collection and inoculation. The mother tree was lopped in November months (six months prior to explant collection) to remove any inhibitory substance and release bud growth. Different sterilizing agents were used to minimize the bacterial and fungal contamination. Some modification in culture media (use of different concentration of NH4NO3 and KNO3 salts and adenine sulphate) was done. Surface sterilization of nodal explants collected from lopped branches with 0.1% HgCl2 for 8 min., treatment with chilled antioxidant solution (Ascorbic acid, Citric acid and PVP) and half strength of NH4NO3 and KNO3 salts of MS medium supported 100% bud break response with proliferation of green and healthy in vitro shoots. Removing these hurdles already in the initial stage of micropropagation is very important and maximize mass in vitro propagation of this medicinally important Arjun tree.

Rhizome Buds Disinfection for Preparation of Red Ginger (Zingiber officinale Roxb. var. rubrum Rosc.) In Vitro Culture

The success of culture initiation depends on explant surface sterilization techniques. Suitable concentration, combinations, and duration of exposure of sterilizing agents are important to raise in vitro culture successfully. The aim of this work is to obtain the suitable sterilization method for explant buds of red ginger rhizome to get the axenic culture. Four sterilizing agents, fungicide, bactericide, Cefotaxime antibiotic, and NaOCl were tested for sterilization by various concentration and duration of exposure. The results showed that sterilizing agents 200 mg/L Cefotaxime and 100 mg/L Benomyl combined with NaOCl decreased the contamination of explants, and achieved 20% axenic culture.

Genotype-specific requirements for in vitro culture initiation and multiplication of Magnolia taxa

The influence of basal media composition, concentration of plant growth regulators (PGRs), and the developmental stage of primary explants (dormancy, stage of bud opening and fruit ripening) on the initiation phase of nine Magnolia genotypes, including M. stellata /Sieb. & Zucc./Maxim., M. × soulangeana ‘Rustica Rubra’, M. denudata Desr., M. × soulangeana ‘Alexandrina’, M. liliiflora Desr., M. officinalis var. biloba Rehd. & Wils., M. salicifolia Maxim., M. × soulangeana ‘Lennei’, and M. kobus DC, was evaluated. The highest efficiency of primary culture initiation of seven Magnolia genotypes (except for M. liliiflora and M. salicifolia) was achieved from primary explants collected in the bud opening stage. A high positive correlation was found between total tannins and efficiency of the primary culture initiation at the fruit ripening stage (r = 0.833). Standardi and Catalano medium (S2) with 0.5 mg l−1 of 6-benzylaminopurine (BAP) was the most appropriate for multiplication of M. × soulangeana ‘Alexandrina’, whereas tissue cultures of M. × soulangeana ‘Lennei’ proliferated and grew better on S2 medium with 1.0 mg l−1 of BAP and 1.0 g l−1 of polyvinylpyrrolidone. The requirements for the composition of basal media and concentration of PGRs in the initiation and multiplication stages of micropropagation of various Magnolia species and cultivars are genotype-specific.

Effect of Rhustox on Micropropagation of Scoparia dulcis L. through Leaf Explants Culture

Aim: To investigate the effect of Rhus toxicodendron (30CH) along with different compositions of phytohormones (Auxin and Cytokinin) on the basis of growth and multiplication of explants under optimum temperature under in-vitro conditions. Study Design: To establish and design the standard protocol for the in-vitro propagation through leaf explant of Scoparia dulcis under stress of phytohormones and homeopathic medicine Rhus toxicodendron (30CH). Place and Duration of Study: The plant materials were procured from the Herbal Botanical Garden Patna Science College, Department of Botany, Patna University, Patna, Bihar. The experimental part was carried out in Plant Tissue Culture Laboratory, between December 2017 to August 2018 in Department of Botany P.U. Patna. Methodlogy: The sterilized leaf explants were inoculated into MS media fortified with different phytohormones (Auxin and Cytokinin) and Rhus tox(30CH) under aseptic environmental conditions for the growth and development of callus, embryoids etc. Result: The explants in MS medium supplemented with auxins phytohormones and Rhus tox(30CH) exhibited that IAA (0.10 to 2.0 mg/l) and BAP (0.10 to 2.5 mg/l) induces green and compact calli. Whereas at 0.30mg/l of IAA and 0.50 mg/l BAP induced brown friable calli. 2,4-D (1.5 mg/l) and Kinetin (1.5-6.5mg/l) concentrations induced brown and friable calli. Rhus tox(30CH) (100 µl/100 ml) enhances proliferation with 2,4-D and Kinetin (1.5/1.5 mg/l.). Conclusion: After 42 days of culture initiation and establishment the callus was 520.0±1.12 mg in the mixture of 2,4-D and Kinetin (1.5 mg/l) in Rhus tox free medium. Whereas weight of callus were found to be 1092±0.74 mg after 42 days in the same medium of 2, 4-D and Kinetin (1.5/5.5 mg/l) supplemented with Rhus tox (100 µl/100 ml). Hence, the investigation proponded that the Rhus tox (CH30) has increased the rate of callus development and plantlet regeneration.