Multiple Shoot Regeneration Research Articles

In-vitro Regeneration of An Endangered Medicinal Plant Anodendron paniculatum (Apocynaceae)

Objectives: The present study aims to establish a standardized and efficient regeneration protocol for Anodendron paniculatum A. DC. (Apocynaceae) by optimizing the effect of various plant growth regulators as it is categorized globally as an endangered medicinal plant. Methods: MS medium fortified with 3% sucrose (w/v) and different concentrations of cytokinins 6-benzylamino purine (BAP), kinetin, thidiazuron either alone or in combination with auxins, indole 3-acetic acid (IAA), naphthalene acetic acid (NAA) and GA3. For the in vitro root induction, individual and healthy shoots (3-4 cm in length) were excised in the 7th week and were cultured onto basal and half-strength MS medium supplemented with various concentrations of auxins and incubated in the culture room. Findings: MS medium supplemented with thidiazuron (2.5 𝜇M) in combination with indole 3-acetic acid (2.5 𝜇M) and GA3 (1.25 𝜇M) was most effective, providing multiple shoot regeneration for 79.33% of cotyledonary leaf explants associated with a high number of shoots per explant (20.00 ± 1.15) and length of shoot (2.66 ± 0.08 cm). Whereas, the frequency of response, mean number of shoots, and respective lengths were varied with different concentrations of 6-benzylamino purine when used in combination with IAA or NAA and BAP (5.0 𝜇M) along with IAA (2.5 𝜇M) at the frequency of 47.66%. Whereas, the addition of GA3 (1.25 𝜇M) in combination with BAP (5.0 𝜇M) and IAA (2.5 𝜇M) has shown a maximum number of multiple shoot formation/explant (12.33) with length of shoot (1.53 ± 0.08 cm). MS medium supplemented with 10 𝜇M 6-benzyl amino purine and 2.5 𝜇M naphthalene acetic acid recorded the highest response (53.66%) with more number of the shoot (9.66) and length of the shoot (1.83 ± 0.03 cm) per explant. The maximum percentage of response (84.33%) was recorded at 1 2 strength MS medium with 5.0 𝜇M NAA, whereas the highest number of roots per shoot (6.66 ± 0.33) was induced at MS + 5.0 𝜇M NAA. The highest length of roots (3.66 cm) was recorded at basal MS medium fortified with 5.0 𝜇M NAA and 7.5 𝜇M NAA. Novelty: The standardized protocol method is of first of its kind, a quick and suitable approach for the high quantity production of Anodendron paniculatum with appropriate concentrations of plant growth regulators that meet thecommercial demand of endangered medicinal plants. Keywords: Anodendron paniculatum; Endangered plant; Plant growth regulators; Regeneration; Protocol standardization

Indirect in vitro Regeneration in Four Varieties of Potato (Solanum tuberosum L.) from Internodal Segments and Leaf Explants

Internodal segments and leaf explants from four varieties of potato (Solanum tuberosum L.) cultivated in Bangladesh, namely, Asterix, Diamant, Granola and Lady Rosseta were utilized for indirect in vitro regeneration of shoots through callus culture. The best responses regarding the reaeration of shoots were obtained when the explants were cultured on MS medium supplemented with 4.0 mg/l BAP and 1.0 mg/l IAA and 0.5 mg/l GA3. The highest frequency of multiple shoot regeneration was recorded from internodal segments rather than from the leaf explants. Hormone free MS medium was found to be the most effective for healthy root induction from the in vitro raised excised shoots. Following adequate root induction, the in vitro regenerated plantlets were successfully transplanted and established to soil for further growth and development of tubers. Plant Tissue Cult. & Biotech. 34(1): 83-92, 2024 (June)

Efficient in vitro Regeneration of Brassica napus L. Var. BARI Sorisha-18

Brassica is one of the most nutritious and significant oil yielding crop plants around the world, including in Bangladesh. However, the development of a smart variety with a short maturity duration and high yield is a need of the present day. To achieve that, both genetic transformation and genome editing can be promising technologies for improvement, and establishment of suitable regeneration systems is a pre-requisite to deploy such technique. For this reason, an efficient and reproducible in vitro regeneration protocol for Brassica napus L. var. BARI Sorisha-18 was developed using MS medium supplemented with various concentrations and combinations of 2, 4-D, BAP and NAA. The highest frequency of in vitro regeneration of multiple shoots was obtained on MS medium supplemented with 1.0 mg/l BAP and 0.1 mg/l NAA from both cotyledonary leaf attached with petiole and hypocotyl explants. The elongation of shoots was also achieved with the same media composition. Half strength of MS medium supplemented with 0.2 mg/l IBA was found to be more effective in producing effective roots than other hormonal supplements applied. Following the development of effective roots, in vitro raised plantlets were successfully transplanted into the soil. Plant Tissue Cult. & Biotech. 33(2): 107-114, 2023 (December)

In vitro Regeneration of Multiple Shoots in Abelmoschus esculentus (L.) Moench (Okra) via Apical Shoot Meristem Culture

Introduction: To develop the efficient protocol for multiple shoot regeneration of A.esculentus by apical root culture method. Abelmoschus esculentus L., commonly known as okra, is a member of the Malvaceae family, which is widely consumed due to its high nutrient content and potential therapeutic properties. Okra contains various bioactive compounds that ex-hibit antibacterial properties and may be useful in treating type-2 diabetes, digestive diseases, and liver detoxification. To select the plant for the present and prepare the efficient protocol for the development of multiple shoot regeneration culture method. Methods: In this study, we developed an efficient protocol for multiple shoot regeneration of A. esculentus using the apical shoot culture method. Mature shoot apex explants of the germinated A. esculentus genotype CoBhH1 were cultured on Murashige and Skoog (MS) medium supple-mented with different concentrations of cytokinins (BAP and TDZ) and auxins (IAA and NAA) to determine the optimal conditions for shoot induction. Results: The highest number of multiple shoots (27.04 shoots) was obtained with 0.8 mg/L TDZ. Excised shoots were cultured on MS medium supplemented with GA3, which induced elongation of the shoots to a maximum of 8-10 cm. Regenerated plantlets were successfully transferred to soil, with a 100% survival rate and no differences in morphology or growth characteristics com-pared to control plants. Rooting was achieved with 1 mg/L IBA. Conclusion: This study provides an efficient protocol for multiple shoot regeneration of A. esculentus through apical shoot culture, which has potential applications in plant breeding and genetic engineering.

Thidiazuron Induced In Vitro Plant Regeneration, Phenolic Contents, Antioxidant Potential, GC-MS Profiles and Nuclear Genome Stability of Plectranthus amboinicus (Lour.) Spreng

Plectranthus amboinicus (Lour.) Spreng is a perennial plant from the mint family with aromatic, succulent leaves and several health benefits. Multiple shoot regeneration was accomplished in vitro using nodal segments (NS) explants of P. amboinicus pretreated with 0, 0.5, 5, 25, 50, and 100 μM thidiazuron (TDZ) for 4 h, then transferred to a growth regulator-free media. After 8 weeks of growth, NS explants pre-treated with 25 μM TDZ for 4 h and then transferred to TDZ-free Murashige and Skoog (MS) media produced the greatest number of shoots (27.3 per NS) with the longest average shoot length (4.9 mm) in 97.2% of cultures. On the same medium, regeneration of roots in most of the P. amboinicus shoots occurred spontaneously. The in vitro-regenerated P. amboinicus plantlets were adequately hardened off and adapted to the ex-vitro environment with a 90% survival rate. Total phenolic, tannin, and flavonoid contents, as well as 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging/antioxidant activity, were significantly higher in in vitro-regenerated plants than in ex vitro-plants. Flow cytometry (FCM) analysis validated the nuclear genome stability of the in vitro generated plants, which assessed their nuclear DNA content and found it to be comparable in genome size to that of the field-grown plants. The study found a quick and efficient method for in vitro multiplication of P. amboinicus which can aid to increased availability and accessibility of this plant species for various purposes. The genetic and phytochemical analysis of the in vitro propagated plants can also provide valuable insight into the plant’s properties and potential applications, which can further assist in its preservation and sustainable usage.

EVALUATION OF SOMACLONES OF POTATO (SOLANUM TUBEROSUM L.)

Evaluation of somaclones in the field of three potato varieties (Diamant, Multa and Cardinal) were examined through callus culture using node, internode and leaf segments. Multiple shoot regeneration from callus at various frequencies was observed using different concentrations and combinations of growth regulators. The highest percentage of callus induction from internode was observed in MS medium containing 3.0 mg l-1 of 2,4-D + 150 mg l-1 of L-asparagine. In this combination, the percentage of callusing was 90% for Diamant, 80% for Multa and 70% for Cardinal. The best response for multiple shoot formation was observed in MS medium supplemented with 4.0 mg l-1 of KIN + 0.5 mg l-1 of NAA. Diamant responded better than Multa and Cardinal for shoot regeneration from callus. Best rooting was observed from node or internode derived plantlets of primary calliclone in MS medium supplemented with IBA. Rooted calliclones were transplanted in the field and evaluated somaclones variation using parameters viz. plant height, number of leaves/plant, number of tuber/plant and tuber weight/plant. The result of statistical analysis on individual character supports existence of significant variation among the different somaclones.

Callus induction and regeneration in high-altitude Himalayan rice genotype SR4 via seed explant

SR4 genotype of rice is high altitude Himalayan rice prone to various abiotic stresses such as cold stress and therefore gives a poor yield. An efficient protocol for callusing and regeneration via direct and indirect means was established using mature seeds as an explant which can be utilized for molecular studies for genetic advancement of Himalayan rice genotype SR4 through transformation. Highest frequency (96.6%) of callus induction was obtained on MS media 3.0 mg/L 2, 4-D. While maximum regeneration frequency (100%), number of shoots with maximum length 9.14 ± 0.204 (cm) from callus was recovered from MS media amended with 5.0 mg/L BAP in combination with 0.5 mg/L NAA with highest number of shoots having an average shoot length 9.14 ± 0.204 (cm) after four weeks of culture. Direct multiple shoot regeneration from seed explants was obtained using various concentrations of TDZ and BAP with highest regeneration frequency was observed on MS media fortified with 6 mg/L of TDZ with maximum number of shoots. The shoots developed roots on MS media supplemented with 0.6 mg/L IBA.

Simple and robust multiple shoot regeneration and root induction cycle from different explants of Hypericum perforatum L. genotypes

Hypericum perforatum L. commonly known as Saint John’s Wort (SJW) is an economically important medicinal plant known for accumulating its valuable bioactive compounds in a compartmentalized fashion. The dark glands are very rich in hypericin, and translucent glands are filled with hyperforin. The antibiotic properties of the afore mentioned bioactive compounds make it hard to establish tissue regeneration protocols essential to put in place a transformation platform that is required for testing gene function in this challenging species. In this study, we report the establishment of a regeneration and root induction cycle from different types of explants. The regeneration cycle was set up for the continuous supply of roots and leaf explants for downstream transformation experiments. The most effective medium to obtain multiple shoot-buds from node cultures was MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium supplemented with 0.5 mg L−1 6-Benzylaminopurine (BAP) and 0.5 mg L−1 indole-3-butyric acid (IBA). The same combination yielded copious amounts of shoots from root and leaf explants as well. For rooting the elongated shoots, MS medium devoid of plant growth regulators (PGRs) was sufficient. Nevertheless, addition of a low amount of IBA improved the quantity and quality of roots induced. Additionally, the roots obtained on a medium containing IBA readily developed shoot buds.

In vitro Regeneration of two BINA Tomato (Lycopersicon esculentum Mill.) Varieties of Bangladesh

Cotyledonary leaf from 12 days old aseptically grown seedlings were used as explant. Cotyledonary leaf explants of two BINA varieties of tomato were cultured on MS medium containing different concentrations and combinations of BAP, Kn and IAA for multiple shoot formation via organogenesis. 100% shoot induction was observed when the explants were cultured on MS medium supplemented with BAP, Kn and IAA. Earliest shoot induction was observed within four weeks. The MS media supplemented with 1.5 mg/l BAP + 0.5 mg/l Kn + 0.2 mg/l IAA found to be the best medium with regards to number of days required for the initiation of regeneration, no. of multiple shoot formation and regeneration frequency in case of both the varieties. Shoot elongation was obtained in the same regeneration medium. The elongated shoots were rooted on half strength MS medium supplanted with 0.2 mg/l IBA. The in vitro rooted plantlets were successfully established in soil with 97% survival rate. Subsequently, the plantlets were transferred to large earthen pot for acclimatization and finally, transferred to the open field for further growth and development. Plant Tissue Cult. & Biotech. 32(1): 43-51, 2022 (June)

PHYTOCHEMICAL ASSESSMENT FROM CALLUS AND SHOOT CULTURES OF A POTENTIAL MEDICINAL HERB: PHYLLANTHUS AMARUS (Schum. & Thonn)

This study was undertaken to evaluate the performance of cut Dendrobium pink sunshine under various holding solutions at post graduate lab Department of Horticulture, Faculty of Agriculture, Annamalai University, Tamil Nadu, India, during February 2016. The treatments with three replications were carried out in completely randomized design with 3 spikes in each replication. Nine chemical preservative solutions were used for extending the vase life and the treatments are T1 8-HQC (200-ppm), T2 Citric Acid (200-ppm), T3 STS (200-ppm), T4 GA (200-ppm), T5 Al2 (SO4)3 (10-ppm), T6 8-HQC (200-ppm) + 5% sucrose, T7 Citric Acid (200- ppm + 5% sucrose, T8 STS (200-ppm) + 5% sucrose, T9 GA (200-ppm) + 5% sucrose,T10 Al2(SO4)3 (200-ppm) + 5% sucrose and T11 Distilled water. Postharvest observations including bud open (%), flower drop (%), flower colour retention (days), flower diameter (cm), vase life and water uptake (ml). Among the treatments (T10) Al2(SO4)3 (200-ppm) + 5% sucrose recorded maximum performances in the postharvest studies followed by (T8) STS (200-ppm) + 5% sucrose. This study reports the phytochemical production from the callus and shoot cultures of Phyllanthus amars. Callus and shoot cultures were established from the nodal explants. Murashige and Skoog medium supplemented with N 6 - benzylaminopurine was found best medium for multiple shoot regeneration from the node explant and further maintenance of the shoot cultures. Callus induction was favoured on medium supplemented with 4.50 µM 2,4- dichlorophenoxy acetic acid. In vitro regenerated shoots and callus cultures were dried at room temperature and after measuring the dry biomass subjected for phytochemical extraction by using methanol and water. The extracts were quantified tested for total phenolics and flavonoids. The methanolic extract of in vitro and in vivo shoot showed the antioxidant activity.

Multiple Shoot Regeneration from Detached Embryonic Axis in Greengram (Vigna radiata) cv.SML 668

Background: Under prevailing regime of climate change and dynamic pest-crop interactions, greengram genetic improvement requires a speedier and precise tool such as genetic transformation to develop improved lines. In this context, an in vitro multiple shoot regeneration system via organogenesis was demonstrated in mungbean cv. SML668, a parent cultivar used in crop improvement programs, using double cotyledonary node (DCN) and detached embryonic axis (EA) explants with 1.0 mg l-1 BAP. Methods: While both the explants responded to in vitro regeneration, number of shoots regenerated was higher with EA (4.02±0.19) than with DCN (3.1±0.08). 6-benzyl aminopurine (BAP) was found to be most effective for inducing and regenerating multiple shoots, in comparison to all other phytohormones (NAA, IAA and TDZ) and supplements (amino acids) tested. Sub culturing twice on BAP supplemented media followed by two subcultures on basal media was optimal for multiple shoot regeneration. Rhizogenesis was obtained on basal media devoid of any phytohormones in EA explants and in 1.0 mg l-1 IAA for DCN explants. The in vitro regenerated plantlets were successfully hardened in a mixture of soil, sand and vermiculite that flowered, produced pods and viable seeds on maturity. Result: The study revealed that detached embryonic axis was a potential explant for in vitro regeneration in SML668, a cultivar not tested for its in vitro regeneration ability before.

In Vitro Propagation of Aconitum chasmanthum Stapf Ex Holmes: An Endemic and Critically Endangered Plant Species of the Western Himalaya

Aconitum chasmanthum Stapf ex Holmes, a highly valued medicinal plant, is a critically endangered plant species with restricted global distribution. Because there is no published report on the in vitro micropropagation of A. chasmanthum, the present study was undertaken to contribute to the development of an efficient micropropagation protocol for its conservation. Seeds collected from the wild showed enhanced germination after being given a chilling treatment (−4 °C and −20 °C) for different durations (10, 20, 30 and 40 days). Seeds given a chilling treatment of −4 °C for 10 days showed enhanced germination rates of 47.59 ± 0.53% with a mean germination time of 10.78 ± 0.21 days compared to seeds kept at room temperature when grown in an MS basal medium. Nodes, leaves and stems, taken from 20–40-day-old seedlings, were used as an explant for micropropagation. An MS medium supplemented with different concentrations of cytokinins (BAP, Kn), auxins (2,4-D, NAA), and an additive adenine sulphate were tested for callusing, direct shoot regeneration and rooting. Only nodal explants responded and showed direct multiple shoot regeneration with 7 ± 0.36 shoots with an elongation of 5.51 ± 0.26 cm in the MS medium supplemented with BAP 0.5 mg/L, and with a response time (RT) of 10.41 ± 0.51 days and a percentage culture response of 77.77 ± 2.77%. Rhizome formation was observed after 8 weeks, with the highest culture response of 36.66 ± 3.33% in the MS basal media with an RT of 43.75 ± 0.50 days. These rhizomes showed a 60% germination rate within 2 weeks and developed into plantlets. The present in vitro regeneration protocol could be used for the large-scale propagation and conservation of A. chasmanthum.

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.

In Vitro plant regeneration from leaf explants of Tagetes erecta L.

Regeneration of multiple shoots via callus induction and organogenesis was obtained from young leaf explants of the field grown marigold (Tagetes erecta L.). Callus induction and shoot regeneration at various frequencies were observed using different concentrations and combinations of growth regulators. Highest percentage (90%) of callus formation was observed within two weeks on MS medium supplemented with 5.0 mg/l BAP with 2.5 mg/l NAA. The maximum percentage (80%) of shoot bud formation (10±0.5/callus) was obtained from MS medium containing 1.0 mg/l BAP with 0.5 mg/l kinetin. The regenerated shoots developed highest percentages (90%) of roots on half strength MS medium supplemented with 1.0 mg/l IBA. The plantlets when transferred into potsoil 80% survived. Regenerated plants were morphologically uniform with normal leaf shape and growth pattern.
 Bangladesh J. Sci. Ind. Res.56(2), 69-74, 2021

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.

Effect of media concentration and growth hormones on shoot regeneration and in vitro rooting of sugarcane varieties (Saccharum spp. )

The protocol for plantlet induction and regeneration through in vitro culture was standardized for the sugarcane variety Co 06022 with Co 86032. Genotype specific media combinations for establishment and rooting efficiency was identified and standardized. Multiple shoot regeneration was observed at different frequencies, using different concentration and growth regulator combination. In vitro propagation of Saccharum offers opportunities for increasing plant material for cultivation. Apical meristems were cultured on modified MS medium containing different concentrations of auxins. In Co 06022, the most effective concentration was combination of ½ MS medium supplemented with IBA (2.0 mg/l) which induced an average of 3.00±0.14 number of shoots per microshoot with 10.77±0.42 cm shoot length. Among the auxins used, NAA showed better response than IBA for profuse rooting in Co 06022. The number of roots formed was maximum (14.80±0.09) in MS with NAA (2mg/l) which had taken 10 days for root initiation with the root length of 6.45±0.07 cm. However, in Co 86032, the best performance was observed on ½ MS medium supplemented with IBA (2.0 mg/l) with more number of shoots (2.00 ± 0.19) and average shoot length of 12.30± 0.44 cm. Best rooting was observed in ½ MS medium supplemented with NAA (2.0 mg/l) and maximum number of roots were 11.60±0.12 which has taken 8 days for initiation of root primordia with average root length of 1.16±0.12 cm. Thus, it can be deduced that this protocol can be used successfully for in vitro rooting and acclimatization of these genotypes.

Enhanced in vitro shoot proliferation through nodal explants of Staurogyne repens (Nees) Kuntze

In this study, multiple and rapid propagations of Staurogyne repens (Nees) Kuntze with tissue culture techniques were aimed. The nodal explants of S. repens were cultured for multiple shoot regeneration in culture medium supplemented with different rates of 6-Benzilaminopurin (BAP) and Kinetin (KIN) alone (0.25–1.50 mg/L) or combinations with 0.25 mg/L Indole-3-acetic acid (IAA). Generally, shoot regeneration capabilities of the explants were high due to the growth regulators used. Shoot regeneration frequencies were ranked between 77.77% and 100.00% in BAP-IAA and KIN-IAA applications. In BAP-IAA application, the average number of shoots was recorded between 2.53–16.11 shoots/explant and the highest number of shoots per explant (16.11 shoots/explant) was obtained in culture medium containing 1.25 mg/L BAP. As the BAP level used increased, the number of regenerated shoots increased. In KIN-IAA application, mean shoot counts were recorded as 2.66–10.27 shoots/explant, and maximum shoots per explant (10.27 shoots/explant) was determined in culture medium containing 0.50 mg/L KIN. When shoot lengths were examined, longer shoots were seen in KIN-IAA application compared to BAP-IAA application. The longest shoot was obtained as 2.82 cm in the culture medium fortified with 1.50 mg/L KIN. Regenerated shoots for in vitro rooting were transferred to the growth culture containing different concentrations of IAA and 1-Naphthaleneacetic acid (NAA). The best values in terms of number and length of roots per shoot were determined in a growth culture containing 0.25 mg/L IAA. The rooted shoots were accustomed to external conditions successfully.

Micropropagation of Two Varieties of Bell pepper (Capsicum annuum L.)

Cotyledonary leaf and node of two varieties, namely red and yellow of Bell pepper (Capsicum annuum L.) were cultured in MS with different concentrations and combinations of plant growth regulators. The cotyledonary leaf was found to be most responsive towards multiple shoot regeneration. The leaf explants of red variety showed best result towards shoot regeneration in MS with BAP (8.0 mg/l), NAA (0.02 mg/l) and IAA (1.0 mg/l). Eighty per cent explants responded within 13 - 14 days, and the mean number of shoot per explant was was found to be 5.67. The cotyledonary leaf explants of yellow variety showed promising result when the cotyledonary leaf explant was cultured in MS with BAP (8.0 mg/l) and NAA (0.5 mg/l) being the mean number of shoot per explants 5.13 and 70% of them responded within 14 - 15 days. Maximum root induction was found in MS with IBA (0.5 mg/l) in both capsicum varieties. After successful root formation plantlets were transplanted and acclimatized to the soil.
 Plant Tissue Cult. & Biotech. 30(2): 267-275, 2020 (December)

Micropropagation and Screening of Phytocompounds Present among in vitro Raised and Wild Plants of Rauvolfia serpentine

Rauvolfia serpentina, commonly known as Sarpgandha, is a plant well-known for being utilized for several medicinal purposes. Unrestricted collection from natural stands and overexploitation for medicinal and traditional purposes have rendered it endangered, hence there is necessary requirement for the development of cultivation protocols for mass propagation and sustained utilization of the plant. In the present study, in vitro culture of the apical parts of R. serpentina on MS (Murashige and Skoog) medium enriched with NAA resulted in development of callus, whereas multiple shoot regeneration along with callus development was achieved on medium combination MS + NAA + Kn and MS + NAA + BAP. MS + 4µM NAA + 4µM BAP was found to be most optimum media composition for regeneration of shoots and callus. Among different media combinations utilized for in vitro rooting, a maximum of 82.6 % explants developed in vitro roots on ½ MS + 12µM NAA. Gas chromatography-mass spectrometry (GC-MS) analysis of methanolic extract prepared from leaves of wild and micropropagated plants of R. serpentina revealed the presence of 38 and 48 phytocompounds, respectively. 9, 12-Octadecadienoic acid, Methyl linoleate, Methyl stearate, Hexadecanoic acid, methyl ester, Linoleic acid, Ergost-4, 7, and 22-trien-3.alpha.-ol were some of the major compounds found to be present in the leaves of wild plants, and Linoleic acid, methyl ester, Cis-Linoleic Acid Methyl Ester, Methyl elaidate, hexadecanoic acid, and methyl ester were major compounds found to be present in in vitro raised plants. Many of the compounds detected have been known to possess 1 or more biological or pharmacological activities.

Micropropagation of Salacia macrosperma Wight - An Endemic Medicinal Plant of Western Ghats

Salacia macrosperma Wight. - a potent medicinal plant facing the verge of rare and endemic status in the Western Ghats region of southern India. The effective protocol has been standardized for callus induction and multiple shoot regeneration using leaf and nodal explants. The Murashige and Skoog (MS) medium fortified with various plant growth regulators like 2,4-dichlorophenoxyacetic acid (2,4-D), Benzyl amino purine (BAP), Thidiazuron (TDZ), Indole acetic acid (IAA), Kinetin (Kn), Naphthalene acetic acid (NAA) and Indole butyric acid (IBA). The leaf explants produced more calli than nodal explants in MS medium supplemented with 2, 4-D and BAP in combination than individual hormones. Likewise, MS medium with 1.5 mg L-1of 2, 4-D, 2.5 mg L-1 of BAP and 1.5 mg L-1of TDZ along with 1% activated charcoal was apt for multiple shoot regeneration (93.33%) from nodal explants with slight embryogenic callus. Further, each developed plantlets were produced maximum rhizogenesis in liquid MS medium supplemented with 1.0 mg L-1 of IAA. Furthermore, the cytological study of embryogenic callus revealed variations in callus cells such as multinucleate, multi-nucleolate, cytodifferentiation, chromosomal bridges were noticed, besides normal dividing stages. Further, by scanning electron micrograph (SEM) analysis of embryogenic callus different stages of morphogenic developmental features were recorded.