The effect of 2,4-dichlorophenoxyacetic acid, benzyl amino purin and cupric sulphate on in vitro propagation system from shoot apices of shoot tiller of hybrid Napier grass (Pennisetum purpureum Schum)

An efficient micropropagation method of hybrid Napier grass (Pennisetum purpureumn Schum) for in vitro plant production and material breeding was established from multiple-shoot clumps (MCS) regeneration system. This system was important for forage breeding system. Shoot apices from shoot-tillers produced MSC on Murashige-Skoog (MS) induction medium containing several combinations of BAP and 2,4-D in induction stage. The addition of 5 μM (v/v) and 50 μM (v/v) CuSO4 were added in best medium for inoculation to proliferate the clump in proliferation/multiplication stage. Plant regeneration was achieved by culturing on solid MS with several combination of medium containing NAA and BAP in regeneration stage. The best results for induction were Murashige-Skoog (MS) induction medium containing 2 mgL−1 BAP and 0.1 mgL−1 2,4-D. The proliferation stage on MS medium containing 5 μM CuSÜ4 effective for proliferation (50% multiple shoot formation). The regeneration stage using 0.1 mgL−1 NAA and 2.0 mgL−1 BAP (51.6% number of shoot can regenerate). All plantlets were successfully grown up in an acclimatization stage. Based on the results, the hybrid Napier grass regeneration via MSC was a stable tissue culture system (no albino plats), which could be applied either for further genetic transformation assay or for alternative supply of nursery plant in the future.

In-vitro propagation and phytochemical profiling of a highly medicinal and endemic plant species of the Himalayan region (Saussurea costus)

Efficient protocols for callus induction and micro propagation of Saussurea costus (Falc.) Lipsch were developed and phytochemical diversity of wild and in-vitro propagated material was investigated. Brown and red compact callus was formed with frequency of 80–95%, 78–90%, 70–95% and 65–80% from seeds, leaf, petiole and root explants, respectively. MS media supplemented with BAP (2.0 mgL−1), NAA (1.0 mgL−1) and GA3 (0.25 mgL−1) best suited for multiple shoot buds initiation (82%), while maximum shoot length was formed on media with BAP (1.5 mgL−1), NAA (0.25 mgL−1) and Kinetin (0.5 mgL−1). Full strength media with IAA (0.5 mgL−1) along with IBA (0.5 mgL−1) resulted in early roots initiation. Similarly, maximum rooting (87.57%) and lateral roots formation (up to 6.76) was recorded on full strength media supplemented with BAP (0.5 mgL−1), IAA (0.5 mgL−1) and IBA (0.5 mgL−1). Survival rate of acclimatized plantlets in autoclaved garden soil, farmyard soil, and sand (2:1:1) was 87%. Phytochemical analysis revealed variations in biochemical contents i.e. maximum sugar (808.32 µM/ml), proline (48.14 mg/g), ascorbic acid (373.801 mM/g) and phenolic compounds (642.72 mgL−1) were recorded from callus cultured on different stress media. Nonetheless, highest flavenoids (59.892 mg/g) and anthocyanin contents (32.39 mg/kg) were observed in in-vitro propagated plants. GC–MS analysis of the callus ethyl acetate extracts revealed 24 different phytochemicals. The variability in secondary metabolites of both wild and propagated plants/callus is reported for the first time for this species. This study may provide a baseline for the conservation and sustainable utilization of S. costus with implications for isolation of unique and pharmacologically active compounds from callus or regenerated plantlets.

Multiple shoot induction and regeneration of Vanilla borneensis Rolfe - a critically endangered orchid of Assam, India

In the present investigation, a micropropagation protocol has been developed for Vanilla borneensis Rolfe – a critically endangered orchid through multiple shoot regeneration. Through in vitro multiple shoot regeneration from both nodal and shoot tip explants, maximum (100%) shoot induction was observed. The minimum time required for shoot bud induction was observed from the shoot tip (5–7 days) on medium supplemented with BAP (4.44 mM) + KIN (2.32 mM) as compared to the nodal explants. Maximum multiple shoot regeneration was observed from nodal explants on the medium supplemented with BAP (4.44 mM) + TDZ (6.82 mM). However, maximum shoot length was observed on the medium supplemented with BAP (4.44 mM) + 15% CW and the number of nodes (5.27±0.33) per shoot after 90 days. Maximum (80-100%) of root initiation was observed in almost all the concentrations of NAA. The shortest time of root initiation was found on the medium supplemented with NAA (5.37 mM). Further, acclimatization period was found to be 15 days with 70% acclimatization while 60% of survivability was observed in the field condition. This efficient micropropagation method of V. borneensis could be successfully used for mass propagation as well as conservation of the critically endangered wild orchid.

DEVELOPMENT OF IN VITRO PLANT REGENERATION PROTOCOL OF BANANA (MUSA SP.) CV. GRAND NAINE USING SUCKER EXPLANT

Banana is an important fruit crop belongs to the family Musaceae. This has more demand for it multifarious uses like food, medicinal as well as industrial values. The present study was carried out to develop micropropagation protocol for large scale production of banana cv. Grand naine using sucker explant. Sucker explants were inoculated on Murashige and Skoog’s (1962) (MS) basal medium and MS basal medium supplemented with different types and concentrations and combination of plant growth regulators. Highest mean number of shoots (10.2) per explant having mean shoot length 5.2 cm was observed on MS medium supplemented with 4.0 mg/L BA, 2.0 mg/L Z, 1.0 mg/L NAA, and 3.0 mg/L ADS. For large scale production of shoot, in vitro regenerated shoots were harvested, cut into small pieces and inoculated on the optimum medium for multiple shoot proliferation. In this way, more than thousand numbers of in vitro shoots were regenerated from a single explant at six month of culture. In vitro regenerated shoots were excised and rooted on ½ MS medium supplemented with 1.0 mg/L IBA. Finally in vitro regenerated plants were acclimatized and subsequently transferred to field with zero mortality. This protocol helps to meet the demand of the farmers.

Clonal propagation of Chrysanthemum morifolium ramat using various explants obtained from field grown plants

A reliable and rapid large scale micropropagation method has been established from the node, shoots tip and leaf explant of Chrysanthemum morifolium growing in field condition. Experiments were conducted to standardize the culture media with plant hormone for multiple shoot proliferation and rooting for obtaining plantlets with uniform characteristics like mother plant in terms of growth and habits. Different concentrations and combinations of auxins (IAA) and cytokinins (BAP, Kin) were used in MS for the above purpose. Maximum shoot regeneration was found in MS treated with 2.0 mg/l BAP both in node and shoot tip explants. In the above combination, nodal explants produced 16 initial shoots. Shoot tip explants produced 12 shoots and leaf segment produced 07 shoots. For in vitro rooting, different concentrations of IBA and NAA were used. Higher rooting percentage was recorded on MS fortified with 1.5 mg/l IBA. The rooted plantlets were hardened and successfully established in the soil. About 90% of the regenerated plantlets survived in the natural environment.

Preservation of representatives the genus Drosera L. using biotechnological methods

The peculiarities of obtaining planting material of rare representatives Drosera spatulate L. and Drosera aliciae L. using microclonal propagation in order to preserve and cultivate them in ex vitro conditions were studied. The method of sterilization of D. spatulate and D. aliciae explants with 80-90% obtaining aseptic material has been developed. The influence of different sterilization options on the development of microshoots has been studied. The best mode of sterilization is 0,1% solution of AgNO3 and 12.5% solution of H2O2. The features of organogenesis and regeneration of the whole organism from cultivated tissues and organs of Drosera L. was investigated. The effect of exogenous growth regulators at different stages of plant morphogenesis in vitro is shown. Improved conditions of rhizogenesis in vitro. It was found experimentally that MS nutrient media with the addition of 2 g∙l-1 PVP is optimal at the stage of introduction into culture in vitro D. spatulate and D. aliciae. The regeneration of microshoots of D. spatulate and D. aliciae depending on the type of explant and the composition of nutrient media was studied. Morphogenesis was most effective on nutrient media with the addition of 0.25 mg∙l-1 kinetin and on the hormone free MS media. Such cultivation conditions provided 100% regeneration of plants with a reproduction rate of 1:8. Studying the effect of cytokinins on the microclonal reproduction of D. spatulate and D. aliciae, it was found that the development and induction of multiple shoot formation in vitro is best performed on hormone free MS media. To induce the formation of the root system, it is necessary to add into MS nutrient media 0.5 mg∙l-1 IBA. According to the results of the research, a method of microclonal propagation was developed by cutting stem culture, which made it possible to obtain genetically stable, disease-free regenerating plants of D. spatulate and D. aliciae with an optimally formed root system and vegetative mass. The obtained homogeneous planting material can be used in floriculture, creation of terrariums, for pharmacological purposes and for the purpose of introduction. Keywords: Drosera L., microclonal reproduction, morphogenes.

Effects of Bavistin and Cefotaxime on in vitro Contaminant free Shoot Regeneration of Ruellia tuberosa L.

In general, antimicrobial agents are often used in micropropagation techniques to obtain contaminant free clones. The objective of the present study was to evaluate the effects of bavistin and cefotaxime on producing contaminant free plants of Ruellia tuberosa cultured on MS supplemented with phytohormones. Field grown nodal explants of Ruellia tuberosa was used to regenerate entire plants via direct organogenesis. Among the decontaminants tested, the fungicide bavistin along with higher concentration of BAP (2.0 mg/l) and lower concentration of NAA (1.0 mg/l) was the most effective in regeneration and producing contaminant free shoots from cultured explants. This fungicide at 300 mg/l minimised fungal contamination with survival rate of 54%. While the addition of decontaminant cefotaxime at low concentration (200 mg/l) along with same concentration of BAP and NAA stimulated the bud formation and controlled the bacterial contamination. However, its increasing concentration adversely affected the survival rate of Ruellia tuberosa. These findings clearly showed that low concentrations of bavistin and cefotaxime were not only non-toxic but also facilitated bud regeneration. The results achieved showed the decisive role not only of the use of successful fungicides and antibiotics, but also of their sufficient doses were very important in reducing contamination and helping multiple shoot proliferation. Plant Tissue Cult. & Biotech. 31(1): 1-12, 2021 (June)

A SIMPLE AND EFFICIENT MICROPROPAGATION PROTOCOL FOR DEVELOPING PLANTLETS OF EXACUM BICOLOR ROXB. – AN ENDANGERED, ORNAMENTAL, AND ANTIDIABETIC HERB

Objective: Exacum bicolor Roxb. is an endangered medicinal herb due to overexploitation by humans and its inefficient vegetative reproduction. Here, we report an efficient and simple procedure for the regeneration of E. bicolor Roxb. using leaf as an explant. Methods: The optimal concentrations of the hormones needed for callus induction were determined by full factorial method using DOE (Design expert ver. 8.0). The hormones selected based on literature were kinetin, indole acetic acid, and 6-Benzylaminopurine (BAP). Multiple shoot regeneration was carried out in liquid and solid media with the optimal concentrations of the hormones obtained by DOE. Rooting was initiated using Murashige and Skoog media containing naphthalene acetic acid 0.5 mg/l, indole butyric acid (IBA) 1.0 mg/l, and gibberellic acid 3 0.5 mg/l along with 0.2% of activated charcoal. Results: Analysis of full factorial design run showed that BAP in combination with kinetin was effective for the growth of callus and multiple shoot regeneration was higher in liquid media (81.25%). The rate of rooting was observed to be 88.23% and the average number of roots was 0.26. Plantlets with budding apical region and well-established leaves and roots were observed in 30 days. Conclusion: The protocol reported here can be used for effective production of E. bicolor plants in a shorter duration compared to the conventional approach.