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1.  In vitro regeneration of Eucalyptus camaldulensis 
An efficient in vitro regeneration protocol enables mass multiplication, genetic modification and germplasm conservation of desired plants. In vitro plant regeneration was achieved from nodal segments of 18-months-old superior genotypes of Eucalyptus camaldulensis trees through direct organogenesis (DO) and direct somatic embryogenesis (DSE) pathways. Initial bud break (BB) stage occurred via DO while shoot multiplication phase followed both DO and DSE pathways. Interestingly, both BB and shoot multiplication stages were achieved on shoot induction and multiplication (SIM) media composed of Murashige and Skoog (MS) basal medium supplemented with 2 mg l−1 benzyl aminopurine (BAP) and 0.1 mg l−1 naphthalene acetic acid (NAA). Best shoot elongation response was observed on half strength MS fortified with 0.5 mg l−1 BAP, while root induction and elongation was superior in 1/2 MS + 1 mg l−1 Indole butyric acid (IBA). Full strength MS fortified with cytokinins (BAP) and weak auxin (NAA) in the ratio of 20:1 favored direct regeneration pathways. Further, half strength MS supported shoot and root development. The absence of intervening callus phase in this protocol can help in minimizing the chance occurrence of somaclones. When compared to other compositions tried, hardening in 100 % coco peat resulted in maximum survival (80 %) of the in vitro raised plantlets. For mass multiplication, fortnight subculturing of a single nodal explants for eight passages on SIM medium resulted in 60–148 shoot initials. Repeated subculturing in SIM medium induced the formation of direct somatic embryos which in turn improved the turnover capacity and enabled large scale clonal multiplication of elite and desirable trees of E. camaldulensis. Following this protocol, it takes a minimum time period of four-months between in vitro explant inoculation to hardening stage. In the present study, DO and DSE pathway of plant regeneration was reported occurring simultaneously in the same nodal explants of E. camaldulensis.
PMCID: PMC3550532  PMID: 23573043
Direct organogenesis; Direct somatic embryogenesis; In vitro regeneration; Nodal stem cuttings; Eucalyptus camaldulensis
2.  In vitro regeneration in Sarcostemma acidum (Roxb.) –an important medicinal plant of semi-arid ecosystem of Rajasthan, India 
An efficient regeneration protocol for Sarcostemma acidum – an important medicinal plant has been established. Callus initiated from nodal explant on MS medium with 2.0 mg L−1 of NAA + additives. Callus initiated was subcultured on MS medium containing various concentrations of NAA or 2,4-D. Out of these combinations, MS medium +1.0 mg L−1 of NAA + additives was found to be effective for the multiplication of callus. Subculture was done after an interval of 20–22 days. For differentiation of callus BAP or Kinetin alone was found to be less effective. Maximum frequency of shoot regeneration recorded on MS medium +1.0 mg L−1 of BAP + 0.5 mg L−1 of Kinetin and 0.1 mg L−1 of NAA + additives. The in vitro differentiated shoots were excised and inoculated on 1/4 strength MS medium +2.0 mg L−1 of IBA + 0.02 % activated charcoal for in vitro rooting. Maximum response (90 %) was recorded on this medium. In vitro differentiated shoots were inoculated on autoclaved soilrite® after treatment with root inducing auxins. Ex vitro rooting in this plant species has been reported for the first time. Eighty five percent of the shoots rooted under ex vitro conditions. Both in vitro and ex vitro rooted plantlets were hardened in a green house.
PMCID: PMC3656190  PMID: 24431495
Adenine sulphate; Ex vitro; Hardening; In vitro; Micropropagation; Sarcostemma acidum
3.  Conservation of an endemic medicinal plant, Berberis tinctoria Lesch. In Nilgiris through micro propagation 
Ancient Science of Life  2004;24(1):22-26.
Berberis tinctoria Lesch. Is an endemic plant to high hills of Nilgiris having lot of medicinal properties. For its better conservation through mass multiplication, attempts have been made to standardize tissue culture technology. The results of the study exhibited that the basal medium containing BAP and NAA each at 0.5 mg/1 was found to be the optimum for callus formation. Shoot proliferation was highly effective in the basal medium supplemented with BAP at 0.5 mg/1. The root initiation was maximum in the basal medium containing the NAA at 1.0 mg/1 and the plantlet establishment was successful in the hardening medium composed by vermiculite and soil in the ration of 1:1.
PMCID: PMC3330911  PMID: 22557146
4.  In vitro clonal propagation of Achyranthes aspera L. and Achyranthes bidentata Blume using nodal explants 
To develop the reproducible in vitro propagation protocols for the medicinally important plants viz., Achyranthes aspera (A. aspera) L. and Achyranthes bidentata (A. bidentata) Blume using nodal segments as explants.
Young shoots of A. aspera and A. bidentata were harvested and washed with running tap water and treated with 0.1% bavistin and rinsed twice with distilled water. Then the explants were surface sterilized with 0.1% (w/v) HgCl2 solutions for 1 min. After rinsing with sterile distilled water for 3–4 times, nodal segments were cut into smaller segments (1 cm) and used as the explants. The explants were placed horizontally as well as vertically on solid basal Murashige and Skoog (MS) medium supplemented with 3% sucrose, 0.6% (w/v) agar (Hi-Media, Mumbai) and different concentration and combination of 6-benzyl amino purine (BAP), kinetin (Kin), naphthalene acetic acid (NAA) and indole acetic acid (IAA) for direct regeneration.
Adventitious proliferation was obtained from A. aspera and A. bidentata nodal segments inoculated on MS basal medium with 3% sucrose and augmented with BAP and Kin with varied frequency. MS medium augmented with 3.0 mg/L of BAP showed the highest percentage (93.60±0.71) of shootlets formation for A. aspera and (94.70±0.53) percentages for A. bidentata. Maximum number of shoots/explants (10.60±0.36) for A. aspera and (9.50±0.56) for A. bidentata was observed in MS medium fortified with 5.0 mg/L of BAP. For A. aspera, maximum mean length (5.50±0.34) of shootlets was obtained in MS medium augmented with 3.0 mg/L of Kin and for A. bidentata (5.40±0.61) was observed in the very same concentration. The highest percentage, maximum number of rootlets/shootlet and mean length of rootlets were observed in 1/2 MS medium supplemented with 1.0 mg/L of IBA. Seventy percentages of plants were successfully established in polycups. Sixty eight percentages of plants were well established in the green house condition. Sixty five percentages of plants were established in the field.
The results have shown that use of nodal buds is an alternative reproducible and dependable method for clonal propagation of A. aspera and A. bidentata. The high rate of direct shoot-root multiplication and their high rate of post-hardening survival indicate that this protocol can be easily adopted for commercial large scale cultivation.
PMCID: PMC3609210  PMID: 23569824
In vitro; Plant regeneration; Tissue culture; Nodal culture; Achyranthes aspera; Achyranthes bidentata; Clonal propagation; Nodal explant; Reproducible; MS medium; Shoots; BAP; NAA; IAA; Kinetin; Nodal segment; Shootlet; Rootlet
5.  Indirect shoot organogenesis from leaf explants of Adhatoda vasica Nees 
SpringerPlus  2014;3:648.
A novel protocol for indirect shoot organogenesis of Adhatoda vasica was developed using petiole explants derived from mature shrubby plants. Media with concentrations of cytokinins in combination with auxins were used to induce callus formation in two explants types: petiole and leaf segment. The frequency of callus formation from petiole and leaf segment explants on Murashige and Skoog (MS) basal medium supplemented with 0.25 mg l−1 thidiazuron (TDZ) and 0.25 mg l−1 α-naphthaleneacetic acid (NAA) was 100 ± 0.0 and 83.70 ± 0.52% respectively, while on this medium supplemented with 0.25 mg l−1 6-(γ-γ, dimethylallyamino purine) (2iP) and 0.25 mg l−1 NAA, the callus frequency was 100 ± 0.0 and 96.70 ± 0.67% respectively. The highest shoot regeneration (90.60 ± 0.52%) response and the maximum shoots (8.10 ± 0.28) per callus were achieved from petiole explants on MS medium containing 0.25 mg l−1 TDZ and 0.25 mg l−1 NAA. On the contrary, on Schenk & Hildebrandt (SH) basal medium supplemented with 0.25 mg l−1 TDZ and 0.25 mg l−1 NAA, the frequency of callus formation from petiole and leaf segment explants was 100 ± 0.0 and 90.50 ± 0.89% respectively while the callus frequency on this medium containing 0.25 mg l−1 2iP and 0.25 mg l−1 NAA was 100 ± 0.0 and 89.90 ± 0.72% respectively. The shoot regeneration frequency for petiole explants was 89.90 ± 0.46% producing 6.00 ± 0.21 shoots per callus on SH basal medium supplemented with 0.25 mg l−1 TDZ and 0.25 mg l−1 NAA. Whereas petiole explants could induce 83.70 ± 0.50% shoot regeneration and 7.3 ± 1.05 shoots per callus on SH medium containing 0.25 mg l−1 indole-3-butyric acid (IBA), 0.5 mg l−1 6-benzyladenine (BA) and 0.5 mg l−1 2iP. Elongation of regenerated shoot was obtained on MS basal medium supplemented with 0.25 mg l−1 TDZ. All regenerated shoots developed adventitious roots within 4 weeks when transferred to rooting medium containing SH medium supplemented with 0.5 mg l−1 IBA. Total nine rooted plantlets were transferred from in vitro to in vivo conditions and eight plants survived and successfully acclimatized in the shaded greenhouse 12 weeks after transplanting.
PMCID: PMC4230685  PMID: 25485191
Adhatoda vasica; Adventitious shoots; Callus induction; Organogenesis; Shoot regeneration
6.  In vitro callus induction and plantlet regeneration of Achyranthes aspera L., a high value medicinal plant 
To study callus induction from different explants (internode, leaf, root) and in vitro plantlets propagation from medicinally important plant Achyranthes aspera L.
Sterilized explants were prepared by using 0.1% HgCl2 and 0.5% Bavistin and callus was obtained when cultured onto Murashige Skoog's (MS) medium by using different concentrations and combination of 2,4-D, NAA, BAP, IAA, IBA with 3% sucrose and 0.8% agar. Induced callus was immediately transferred to MS medium containing at different concentrations of phytohormones for shootlets and rootlets induction respectively.
Sterilization treatment of 0.1% HgCl2 for 2-3 min and Bavistin 0.5% for 10-12 min showed the highest percentage of asepsis and survival rate. Maximum induction of callus was obtained from a combination of 2.0 mg/L 2,4-D and 0.5 mg/L NAA from leaf. Highest shootlets number (4.83±0.17) and length (3.8±0.16) cm were observed on full strength MS medium when fortified with BAP 4.0 mg/L and KIN 0.5 mg/L. Concerted efforts of BAP 2.0 mg/L and NAA 0.5 mg/L on full strength MS medium showed highest leaf number (6.77±0.94). In vitro raised shoots were allowed to root on different strengths of MS medium fortified with IAA and IBA at different concentrations. Experimentally, 3.0 mg/L IBA was enabled to induce maximum rootlets number (10.0±9.82) on full strength MS medium. Afterwards, regenerated shoots with well developed roots were successfully subjected to hardening process and were acclimatized. The survived plantlets showed 66.67% survival frequency without any morphological abnormality.
The results demonstrated that different explants were good source of callus induction, morphology analysis as well as indirect plantlets regeneration.
PMCID: PMC3819494  PMID: 24144129
Achyranthes aspera; Callus induction; Plantlets propagation; Murashige Skoog's; Micropropagation
7.  Assessment of clonal fidelity of Tylophora indica (Burm. f.) Merrill “in vitro” plantlets by ISSR molecular markers 
SpringerPlus  2014;3:400.
Tylophora indica Burm F. Merrill. is widely used against various diseases owing to the presence an array of medicinally important secondary metabolites. Its stem is bitter, stomachic, stimulates bile secretion, enriches the blood and cures diseases like diabetes, fever, flatulence, hypertension, jaundice, leucorrhoea, urinary disease and upper respiratory tract infection. It is neglected for tissue culture work because of deciduous nature of climbing shrub, facing problems for micropropagation. Hence, in vitro regeneration of complete plantlets was done through indirect organogenesis in Tylophora indica. Calli were produced from in vivo leaves of T. indica on MS medium supplemented with 6-Benzylaminopurine (BAP: 2.0 mg l-1) and Indole-3-butyric acid (IBA: 0.5 mg l-1). The multiple shoots (12.00 ± 1.50) emerged and elongated on MS medium fortified with Thidiazuron (TDZ: 0.1 mg l-1). They were rooted on half strength MS medium having IBA (0.5 mg l-1) (7.75 ± 0.25) after 20 days of sub-culturing followed by hardening and acclimatization. During indirect regeneration of plants, chances of somaclonal variations may arise. These variations should be identified to produce true to type plants. Plantlets raised through tissue culture were used to validate the clonal fidelity through Inter simple sequence repeat markers (ISSR). Clonal fidelity is a major consideration in commercial micropropagation using in vitro tissue culture methods. During the study, total 71 clear and distinct bands were produced using 6 primers. The banding pattern of each primer was uniform and comparable to mother plant and showed about 93% homology using un-weighted pair group method with arithmetic averaging (UPGMA). ISSR analysis confirmed the genetic stability of in vitro raised plants.
PMCID: PMC4147081  PMID: 25170431
Clonal fidelity; ISSR markers; Organogenic calli; Tylophora indica; UPGMA
8.  Regeneration of Stevia Plant Through Callus Culture 
Stevia rebaudiana Bertoni that conventionally propagated by seed or by cuttings or clump division which has a limitation of quality and quantity seed material. In present study, callus culture technique was tried to achieve rapid plant multiplication for quality seed material. Callus induction and multiplication medium was standardized from nodal as well as leaf sagments. It is possible to maintain callus on Murashige and Skoog medium supplemented with 6-benzyl amino purine and naphthalene acetic acid. Maximum callus induction was obtained on Murashige and Skoog medium incorporated with 6-benzyl amino purine (2.0-3.0 mg/l) and naphthalene acetic acid (2.0 mg/l) treatments. However, Murashige and Skoog medium containing 2.0 mg/l 6-benzyl amino purine+2.0 mg/l naphthalene acetic acid was found to be the best for callus induction. Higher regeneration frequency was noticed with Murashige and Skoog medium supplemented with 2.0 mg/l 6-benzyl amino purine+0.2 mg/l naphthalene acetic acid. Regenerated plants were rooted better on ¼ Murashige and Skoog strength supplemented with 0.1 mg/l indole-3-butyric acid. The rooted plantlets were hardened successfully in tera care medium with 63 per cent survival rate. The developed protocol can be utilized for mass production of true to type planting material on large scale independent of season, i.e. external environmental conditions.
PMCID: PMC2810047  PMID: 20177455
Stevia; regeneration; glucose; glycoside; explant; callus; hardening
9.  An efficient and rapid regeneration via multiple shoot induction from mature seed derived embryogenic and organogenic callus of Indian maize (Zea mays L.) 
Plant Signaling & Behavior  2013;8(10):e25891.
An efficient method for in vitro micro propagation and genetic transformation of plants are crucial for both basic and applied research. Maize is one of the most important cereal crops around the world. Regeneration from immature embryo is hampered due to its unavailability round the year. On the contrary mature embryo especially tropical maize is recalcitrant toward tissue culture. Here we report a highly efficient regeneration (90%) system for maize by using 2 different approaches i.e., embryogenic and organogenic callus cultures. Seeds were germinated on MS medium supplemented with 5 mg/l 2,4-D and 3 mg/l BAP. Nodal regions of 2 wks old seedlings were longitudinally split upon isolation and subsequently placed on callus initiation medium. The maximum frequency of embryogenic callus formation (90%) was obtained on MS medium supplemented with 2 mg/l 2,4-D and 1 mg/l BAP in the dark conditions. The compact granular organogenic callus formation (85% frequency) was obtained on MS medium supplemented with 2.5 mg/l 2,4-D and 1.5 mg/l BAP at light conditions. MS medium supplemented with 2 mg/l BAP, 1 mg/l Kinetin and 0.5 mg/l NAA promoted the highest frequency of shoot induction. The highest frequency of root formation was observed when shoots were grown on MS medium. The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. The important advantage of this improved method is shortening of regeneration time by providing an efficient and rapid regeneration tool for obtaining more stable transformants from mature seeds of Indian tropical maize cultivar (HQPM-1).
PMCID: PMC4091112  PMID: 23921544
embryogenic callus; immature embryo; maize; mature seeds; multiple shoots induction; organogenic callus; regeneration
10.  Establishment of an Efficient In Vitro Regeneration Protocol for Rapid and Mass Propagation of Dendrobium chrysotoxum Lindl. Using Seed Culture 
The Scientific World Journal  2014;2014:740150.
An efficient in vitro regeneration protocol from seed culture has been established successfully for Dendrobium chrysotoxum, an epiphytic orchid having tremendous ornamental and medicinal values. Seed germination response was encouraging in Mitra (M) medium enriched with different combinations of auxins and cytokinins. Medium supplemented with 0.4% activated charcoal (AC), 2 mg/L 6-benzyl amino purine (BAP), and 2 mg/L indole-3-acetic acid (IAA) produced best seed germination percentage in 2 weeks of culture. Incorporation of higher concentration of kinetin (KN) or BAP in combination with low auxin in medium induced pronounced shooting and leaf formation. Reduction in leaf development was evident when cytokinins exist singly in medium indicating synergistic effect of auxin and cytokinin in leaf induction. Presence of elevated level of indole-3-butyric acid (IBA) or 1-naphthalene acetic acid (NAA) with low cytokinin content in medium generated more in vitro rooting, though IBA was found to be more effective in rooting induction as compared to NAA. The in vitro protocol for asymbiotic seed germination developed from the present investigation can be used for rapid mass propagation of this highly important Dendrobium orchid species.
PMCID: PMC4221871  PMID: 25401154
11.  Shoot differentiation from protocorm callus cultures of Vanilla planifolia (Orchidaceae): proteomic and metabolic responses at early stage 
BMC Plant Biology  2010;10:82.
Vanilla planifolia is an important Orchid commercially cultivated for the production of natural vanilla flavour. Vanilla plants are conventionally propagated by stem cuttings and thus causing injury to the mother plants. Regeneration and in vitro mass multiplication are proposed as an alternative to minimize damage to mother plants. Because mass production of V. planifolia through indirect shoot differentiation from callus culture is rare and may be a successful use of in vitro techniques for producing somaclonal variants, we have established a novel protocol for the regeneration of vanilla plants and investigated the initial biochemical and molecular mechanisms that trigger shoot organogenesis from embryogenic/organogenic callus.
For embryogenic callus induction, seeds obtained from 7-month-old green pods of V. planifolia were inoculated on MS basal medium (BM) containing TDZ (0.5 mg l-1). Germination of unorganized mass callus such as protocorm -like structure (PLS) arising from each seed has been observed. The primary embryogenic calli have been formed after transferring on BM containing IAA (0.5 mg l-1) and TDZ (0.5 mg l-1). These calli were maintained by subculturing on BM containing IAA (0.5 mg l-1) and TDZ (0.3 mg l-1) during 6 months and formed embryogenic/organogenic calli. Histological analysis showed that shoot organogenesis was induced between 15 and 20 days after embryogenic/organogenic calli were transferred onto MS basal medium with NAA (0.5 mg l-1). By associating proteomics and metabolomics analyses, the biochemical and molecular markers responsible for shoot induction have been studied in 15-day-old calli at the stage where no differentiating part was visible on organogenic calli. Two-dimensional electrophoresis followed by matrix-assisted laser desorption ionization time-of-flight-tandem mass spectrometry (MALDI-TOF-TOF-MS) analysis revealed that 15 protein spots are significantly expressed (P < 0.05) at earlier stages of shoot differentiation. The majority of these proteins are involved in amino acid-protein metabolism and photosynthetic activity. In accordance with proteomic analysis, metabolic profiling using 1D and 2D NMR techniques showed the importance of numerous compounds related with sugar mobilization and nitrogen metabolism. NMR analysis techniques also allowed the identification of some secondary metabolites such as phenolic compounds whose accumulation was enhanced during shoot differentiation.
The subculture of embryogenic/organogenic calli onto shoot differentiation medium triggers the stimulation of cell metabolism principally at three levels namely (i) initiation of photosynthesis, glycolysis and phenolic compounds synthesis; (ii) amino acid - protein synthesis, and protein stabilization; (iii) sugar degradation. These biochemical mechanisms associated with the initiation of shoot formation during protocorm - like body (PLB) organogenesis could be coordinated by the removal of TDZ in callus maintenance medium. These results might contribute to elucidate the complex mechanism that leads to vanilla callus differentiation and subsequent shoot formation into PLB organogenesis. Moreover, our results highlight an early intermediate metabolic event in vanillin biosynthetic pathway with respect to secondary metabolism. Indeed, for the first time in vanilla tissue culture, phenolic compounds such as glucoside A and glucoside B were identified. The degradation of these compounds in specialized tissue (i.e. young green beans) probably contributes to the biosynthesis of glucovanillin, the parent compound of vanillin.
PMCID: PMC3095354  PMID: 20444255
12.  Effect of different plant growth regulators on micro-tuber induction and plant regeneration of Pinellia ternate (Thunb) Briet 
An efficient micropropagation system for Pinellia ternate (Thunb) Briet, a traditional Chinese medicinal plant, has been developed. Petiole and lamina of P. ternate were used as explants and cultured on Murashige and Skoog (MS) medium containing different concentrations of different plant growth regulators. The results indicated that low concentration of 2,4-dicholorophenoxy acetic acid (2,4-D), indole-3-acetic acid (IAA) and α-naphthalene acetic acid (NAA) were suitable for micro-tuber induction, but callus induction rate increased with increasing concentrations of growth regulators. Tubers induction rates of petiole and leaf were (81.8 %–100 %) and (89.4 %–96.0 %) respectively, when 0.2 mg l−1 2, 4-dicholorophenoxy acetic acid, indole-3-acetic acid or α-naphthalene acetic acid were present in the medium. Tubers induction rates of petiole and leaf cultured on MS medium supplemented with 0.2–0.5 mg l−1 6-benzyl amino purine (6-BAP) were (94.1 %–100 %) and (96.0 %–100 %) respectively. When the concentration of 2,4-dicholorophenoxy acetic acid, α-naphthalene acetic acid and 6-benzyl amino purine was increased to 2.0 mg l−1, callus induction rates of petiole and leaf were 100 % and 98.2 %, 91.0 % and 36.0 %, 62.3 % and 70.0 %, respectively. Different concentration of kinetin (KT) and zeatin (ZT) had no significant effect on micro-tuber induction of petiole. Most petioles showed polarity during the cultivation of explants, when supplemented with different concentrations of auxin or cytokinin in the MS medium.
PMCID: PMC3550348  PMID: 23572946
Pinellia ternate (Thunb) Briet; Plant growth regulator; Micro-tuber; Regeneration
13.  Regeneration of Centella asiatica plants from non-embryogenic cell lines and evaluation of antibacterial and antifungal properties of regenerated calli and plants 
The threatened plant Centella asiatica L. is traditionallyused for a number of remedies. In vitro plant propagation and enhanced metabolite production of active metabolites through biotechnological approaches has gained attention in recent years.
Present study reveals that 6-benzyladenine (BA) either alone or in combination with 1-naphthalene acetic acid (NAA) supplemented in Murashige and Skoog (MS) medium at different concentrations produced good quality callus from leaf explants of C. asiatica. The calli produced on different plant growth regulators at different concentrations were mostly embryogenic and green. Highest shoot regeneration efficiency; 10 shoots per callus explant, from non-embryogenic callus was observed on 4.42 μM BA with 5.37 μM NAA. Best rooting response was observed at 5.37 and 10.74 μM NAA with 20 average number of roots per explant. Calli and regenerated plants extracts inhibited bacterial growth with mean zone of inhibition 9-13 mm diameter when tested against six bacterial strains using agar well diffusion method. Agar tube dilution method for antifungal assay showed 3.2-76% growth inhibition of Mucor species, Aspergillus fumigatus and Fusarium moliniformes.
The present investigation reveals that non-embryogenic callus can be turned into embryos and plantlets if cultured on appropriate medium. Furthermore, callus from leaf explant of C. asiatica can be a good source for production of antimicrobial compounds through bioreactor.
PMCID: PMC3238213  PMID: 21989222
Antimicrobial; Centella asiatica; organogenesis; plant growth regulators
14.  Flying between Sky Islands: The Effect of Naturally Fragmented Habitat on Butterfly Population Structure 
PLoS ONE  2013;8(8):e71573.
High elevation montane areas are called “sky islands” when they occur as a series of high mountains separated by lowland valleys. Different climatic conditions at high elevations makes sky islands a specialized type of habitat, rendering them naturally fragmented compared to more continuous habitat at lower elevations. Species in sky islands face unsuitable climate in the intervening valleys when moving from one montane area to another. The high elevation shola-grassland mosaic in the Western Ghats of southern India form one such sky island complex. The fragmented patches make this area ideal to study the effect of the spatial orientation of suitable habitat patches on population genetic structure of species found in these areas. Past studies have suggested that sky islands tend to have genetically structured populations, possibly due to reduced gene flow between montane areas. To test this hypothesis, we adopted the comparative approach. Using Amplified Fragment Length Polymorphisms, we compared population genetic structures of two closely related, similar sized butterfly species: Heteropsis oculus, a high elevation shola-grassland specialist restricted to the southern Western Ghats, and Mycalesis patnia, found more continuously distributed in lower elevations. In all analyses, as per expectation the sky island specialist H. oculus exhibited a greater degree of population genetic structure than M. patnia, implying a difference in geneflow. This difference in geneflow in turn appears to be due to the natural fragmentation of the sky island complexes. Detailed analysis of a subset of H. oculus samples from one sky island complex (the Anamalais) showed a surprising genetic break. A possible reason for this break could be unsuitable conditions of higher temperature and lower rainfall in the intervening valley region. Thus, sky island species are not only restricted by lack of habitat continuity between montane areas, but also by the nature of the intervening habitat.
PMCID: PMC3731288  PMID: 23936518
15.  Optimization of Culture Conditions (Sucrose, pH, and Photoperiod) for In Vitro Regeneration and Early Detection of Somaclonal Variation in Ginger Lime (Citrus assamensis) 
The Scientific World Journal  2014;2014:262710.
Various explants (stem, leaf, and root) of Citrus assamensis were cultured on MS media supplemented with various combinations and concentrations (0.5–2.0 mgL−1) of NAA and BAP. Optimum shoot and root regeneration were obtained from stem cultures supplemented with 1.5 mgL−1 NAA and 2.0 mgL−1 BAP, respectively. Explant type affects the success of tissue culture of this species, whereby stem explants were observed to be the most responsive. Addition of 30 gL−1 sucrose and pH of 5.8 was most optimum for in vitro regeneration of this species. Photoperiod of 16 hours of light and 8 hours of darkness was most optimum for shoot regeneration, but photoperiod of 24 hours of darkness was beneficial for production of callus. The morphology (macro and micro) and anatomy of in vivo and in vitro/ex vitro Citrus assamensis were also observed to elucidate any irregularities (or somaclonal variation) that may arise due to tissue culture protocols. Several minor micromorphological and anatomical differences were observed, possibly due to stress of tissue culture, but in vitro plantlets are expected to revert back to normal phenotype following full adaptation to the natural environment.
PMCID: PMC3995104  PMID: 24977187
16.  Effect of Growth Regulators on In Vitro Morphogenic Response of Boscia senegalensis (Pers.) Lam. Poir. Using Mature Zygotic Embryos Explants 
The percent study describes the in vitro responses of mature zygotic embryos of Boscia senegalensis to different concentrations (0.0–5.0 mg/L) of 6-benzyladnine (BA), Thidiazuron (TDZ), α-Naphthalene acetic acid (NAA), and 2, 4-Dichlorophenoxyacetic acid (2, 4-D) supplemented on Murashige and Skoog medium (MS). The plant growth regulators (PGRs) were considerably affected the morphogenetic responses. BA produced adventitious shoots through two ways: direct organogenesis and auxiliary shoot formation. Both 2, 4-D and TDZ tend to produce callus, whereas NAA improve the development of embryos to seedlings. Maximum number of shoots/explant (14.8 ± 0.6) was obtained on MS medium supplemented with 3.0 mg/L BA. 67.0% of excised shoots were rooted either on 1/2 MS medium augmented with or without 0.25 mg/L IBA. The highest number of roots (1.2 ± 0.4) and root length (0.5 ± 0.2 cm) was produced on 0.25 mg/L IBA-containing medium. Regenerated plants were successfully acclimatized and transferred to the green house with 70% survival rate. All the plants appeared morphologically uniform with normal growth pattern. A rapid (30 days), efficient and without subculturing protocol for in vitro regeneration of B. senegalensis was developed.
PMCID: PMC3113285  PMID: 21687567
17.  Multiple shoot regeneration and alkaloid cerpegin accumulation in callus culture of Ceropegia juncea Roxb. 
This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of “Soma” drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog’s) medium fortified with either IAA or NAA (0.5–2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.
PMCID: PMC3550381  PMID: 23572914
In vitro propagation; Pyridone alkaloid; Cerpegin; Callus; Ceropegia juncea
18.  High frequency plant regeneration from embryogenic callus of a popular indica rice (Oryza sativa L.) 
The aim of the study is to establish a routine procedure for high frequency plant regeneration from in vitro raised embryogenic callus of abiotic salt sensitive indica rice (Oryza sativa L.) cultivar ADT 43. The effect of synthetic auxin 2,4-D on callus induction was optimized to achieve high frequency plant regeneration from fresh embryogenic callus without further subculture. Friable, nodular and creamish-white embryogenic callus cultures were raised from mature rice seeds on LS medium supplemented with 2.5 mg L−1 2,4-D and 1.0 mg L−1 thiamine-HCL. Plant regeneration was achieved by the 24 days old embryogenic callus on MS medium supplemented with 1.0 mg L−1 BAP and 1.5 mg L−1NAA. In vitro regenerated plants with multiple tillers and roots were transferred to sterile soil and maintained in the growth chamber. The regenerated plants exhibited normal growth and were phenotypically similar to plants maintained in the garden. Using the present protocol, 25–30 plantlets were regenerated from 50 individual mature seed derived callus within two to three months. This protocol has the potential for large-scale production of elite plants after genetic transformation.
PMCID: PMC3550345  PMID: 23572948
Oryza sativa; 2,4-D; Regeneration; Mature seed; Embryogenic callus
19.  High-frequency regeneration via multiple shoot induction of an elite recalcitrant cotton (Gossypium hirsutum L. cv Narashima) by using embryo apex 
Plant Signaling & Behavior  2012;8(1):e22763.
Cotton (Gossypium hirsutum L.) is one of the most commercially important fiber crops in the world. Compared with other crops, cotton represents a recalcitrant species for regeneration protocols. The development of efficient and rapid regeneration protocol for elite Indian cotton variety could help improve the quality characteristics and biotic or abiotic stress tolerance. Here we report a novel regeneration protocol in Indian cotton cultivar Narashima. The maximum number of multiple shoots obtained was 16 per explants, performance which has never been achieved in any prior reports. The embryo apex explants were isolated from 2 d old in vitro growing seedlings. Explants were cultured on MS medium containing different plant growth regulator combinations in order to induce multiple shoots. Among the tested combinations, the 2 mg/l of 6-benzylaminopurine (BAP) and 2 mg/l kinetin (KIN) proved to be most suited for achieving the maximum number of multiple shoots. The elongation of multiple shoots was obtained in media supplemented with gibberellic acid (GA3). The regenerated plants were successfully hardened in earthen pots after adequate acclimatization. This method avoids callus tissue, the stage of regeneration which may lead to somaclonal variation. The important feature of the presented method is shortening of regeneration time, as well as the induction of a high number of multiple shoots per explants. The present protocol may provide an efficient and rapid regeneration tool for obtaining more stable transformants from embryo apex explants of Indian cotton cultivar Narashima.
PMCID: PMC3745583  PMID: 23221745
Gossypium hirsutum (cotton); embryo apex; multiple shoots induction; organogenesis; regeneration
20.  In vitro propagation of the medicinal plant Ziziphora tenuior L. and evaluation of its antioxidant activity 
Ziziphora tenuior L. (Lamiaceae) is an aromatic herb used for its medicinal values against fungi, bacteria. Micropropagation can be used for large-scale multiplication of essential oil producing plants thus avoiding an overexploitation of natural resources. This work aims to develop a reliable protocol for the in vitro propagation of Z. tenuior, and to compare the antioxidant activity between in vitro propagated and wild plants.
The explants were sterilized and cultured on MS medium containing different concentrations of growth regulators naphthalene acetic acid (NAA) or indole-3-butyric acid (IBA) with 0.5 mg/L of kinetin (Kin) callus formation was 70.2% after 45 days of incubation in dark on medium supplemented with 1.5 mg/L of NAA. After one month of callus culture on medium supplemented with 2 mg/L BA the shoot number was 5.12 and for the multiplication stage. The shoot number was 4.21 and length was 6.17 cm on medium supplemented with 1 mg/L Kin + 0.1 mg/L NAA.
DPPH• reagent was used to test the antioxidant activity. The aqueous and methanol extracts of in vitro plants which were treated with 1.5 and 1 mg/L of kin plus 0.1 mg/L of NAA showed a strong DPPH• scavenging activity where IC50 was 0.307 and 0.369 mg/ml, respectively, while the IC50 of aqueous and methanol extracts of wild plants was 0.516 and 9.229 mg/ml, respectively. Our results suggested that plant growth regulators and in vitro culture conditions increased the antioxidant activity.
PMCID: PMC4150220  PMID: 25183942
In vitro; Micropropagation; Auxin; Cytokinin; Radical scavenging activity (RSA); Reactive oxygen species (ROS); DPPH• (2,2-diphenyl-1-picrylhydrazyl)
21.  In Vitro Cultures of Schisandra chinensis (Turcz.) Baill. (Chinese Magnolia Vine)—a Potential Biotechnological Rich Source of Therapeutically Important Phenolic Acids 
Applied Biochemistry and Biotechnology  2012;166(8):1941-1948.
The contents of free phenolic acids and cinnamic acid were determined using an HPLC method in methanolic extracts from biomass of Schisandra chinensis (Turcz.) Baill. (Chinese magnolia vine) at different stages of organogenesis, cultured in vitro on a few variants of Murashige and Skoog (MS) medium, containing different concentrations of plant growth regulators 6-benzylaminopurine (BAP) and 1-naphthaleneacetic acid (NAA) (from 0.1 to 3.0 mg/l) and in extracts from overground parts of plants growing in vivo. Six of 12 analysed compounds were detected in all extracts: chlorogenic, p-coumaric, p-hydroxybenzoic, protocatechuic, salicylic and syringic acids. Total contents of the examined metabolites in biomass of shoot-differentiating callus culture cultivated on six MS medium variants were dependent on concentrations of growth regulators in the media and ranged from 14.90 to 60.05 mg/100 g d.w. Total contents of the compounds in biomass extracts from undifferentiating callus culture maintained only on two of six MS medium variants were higher and amounted to 74.54 and 78.24 mg/100 g d.w. Maximum total contents of phenolic acids in both types of in vitro cultures were greater than in fruits (55.73 mg/100 g d.w.) and leaves (4.55 mg/100 g d.w.) of plants gowning in vivo. Chlorogenic acid and salicylic acid were the main compounds identified in biomass extracts of shoot-differentiating callus cultures (max 22.60 and 21.17 mg/100 g d.w., respectively), while chlorogenic acid (max 38.43 mg/100 g d.w.) and protocatechuic acid (max 20.95 mg/100 g d.w.) prevailed in the extracts from undifferentiating callus cultures. Other compounds dominated in fruits, namely p-coumaric acid (23.36 mg/100 g d.w.) and syringic acid (14.96 mg/100 g d.w.). This is the first report on biochemical potential of cells from S. chinensis in vitro cultures to produce the biologically active phenolic acids. These are the first results on the analysis of this group of metabolites in overground parts of plants growing in vivo, too.
PMCID: PMC3324685  PMID: 22399445
Schisandraceae; Shoot-differentiating callus culture; Undifferentiating callus culture; Chlorogenic acid; Protocatechuic acid; Salicylic acid
22.  Plant Regeneration and Somatic Embryogenesis from Immature Embryos Derived through Interspecific Hybridization among Different Carica Species 
Plant regeneration and somatic embryogenesis through interspecific hybridization among different Carica species were studied for the development of a papaya ringspot virus-resistant variety. The maximum fruit sets were recorded from the cross of the native variety C. papaya cv. Shahi with the wild species C. cauliflora. The highest hybrid embryos were recorded at 90 days after pollination and the embryos were aborted at 150 days after pollination. The immature hybrid embryos were used for plant regeneration and somatic embryogenesis. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora showed the highest percentage of germination, as well as plant regeneration on growth regulators free culture medium after 7 days pre-incubation on half-strength MS medium supplemented with 0.2 mg/L BAP, 0.5 mg/L NAA and 60 g/L sucrose. The 90-day-old hybrid embryos from the cross of C. papaya cv. Shahi × C. cauliflora produced maximum callus, as well as somatic embryos when cultured on half-strength MS medium containing 5 mg/L 2,4-D, 100 mg/L glutamine, 100 mg/L casein hydrolysate and 60 g/L sucrose. The somatic embryos were transferred into half-strength MS medium containing 0.5 mg/L BAP and 0.2 mg/L NAA and 60 g/L sucrose for maturation. The highest number of regenerated plants per hybrid embryo (10.33) was recorded from the cross of C. papaya cv. Shahi × C. cauliflora. Isoenzyme and dendrogram cluster analysis using UPGMA of the regenerated F1 plantlets confirmed the presence of the hybrid plantlets.
PMCID: PMC3546739  PMID: 23235330
Carica species; hybridization; plant regeneration; somatic embryogenesis
23.  In vitro micropropagation in Polygonatum verticillatum (L.) All. an important threatened medicinal herb of Northern India 
An ideal micropropagation method of Polygonatum verticillatum has been developed using stem disc explants. Multiple shoots were initiated from stem disc explants on Murashige and Skoog (MS) medium fortified with different concentrations (0.25–10.0 mgl−1) and combinations of cytokinins (BAP, Kn and TDZ) along with (0.5–1.0 mgl−1) auxins (NAA/IBA/IAA). 1.0 mgl−1 BAP with 0.5 mgl−1 NAA was found to be the most effective in producing maximum number of shoots. Regular subculturing of these in vitro multiple shoots induced profuse growth of lateral roots in the same medium. Individual shoots were excised and rooted in vitro on half strength MS medium with 1.0 mgl−1 NAA. Regenerants were hardened in growth chamber with high humidity and showed a high rate of survival.
PMCID: PMC3550528  PMID: 23573044
Liliaceae; Meda/Mahameda; Micropropagation
24.  Influence of plant growth regulators on callus mediated regeneration and secondary metabolites synthesis in Withania somnifera (L.) Dunal 
Withania somnifera (L.) Dunal, is an important medicinal plant being the source of extremely important compounds like withanolides and withaferin. Influence of different plant growth regulators (PGRs) were evaluated for induction of callus, callus mediated regeneration and production of secondary metabolites in them. Explants for callusing were collected from plants grown in vitro and maximum callusing (98 %) was obtained on MS medium supplemented with a combination of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5 mg l-1) and kinetin (KN) (0.2 mg l−1). Among different types of calli, best shoot regeneration was observed on green, compact calli produced on MS medium with a combination of 6-benzylamino purine (BAP) and indole butyric acid (IBA). MS medium supplemented with BAP (2 mg l−1) showed highest frequency (98 %) of shoot bud regeneration. The micro-shoots were efficiently rooted on MS media supplemented with 0.5 mg l−1 IBA. Rooted plants were transferred to soil-vermi-compost (1:3; w/w) medium in greenhouse for acclimatization. Presence of withanolide A and withaferin A in calli was validated through high performance thin layer chromatography (HPTLC). It was interesting to observe that the PGRs showed significant influence on the secondary metabolites production in callus and 2,4-D having the least effect. Histological studies revealed the origin of shoot tip in the callus during regeneration.
PMCID: PMC3550684  PMID: 24381443
Callusing; High performance thin layer chromatography; Histology; Plant growth regulator; Shoot bud regeneration; Withaferin A; Withanolide A
25.  In vitro propagation of an endangered medicinal herb Chlorophytum borivilianum Sant. et Fernand. through somatic embryogenesis 
Tuberous roots of Chlorophytum borivilianum Sant. et Fernand. which are a source of steroidal saponins, possess immunomodulatory, adaptogenic, aphrodisiac, antipyretic, diuretic, hemostatic and anti-tumour properties. Poor seed setting and germination and slow growth in conventional vegetative propagation are major constraints in the large-scale cultivation of this commercially important medicinal plant. In the present study, a procedure for in vitro propagation of this endangered herb through somatic embryogenesis has been established. Seeds of Chlorophytum borivilianum were germinated on MS medium supplemented with 57.74 μM gibberellic acid and hypocotyl portion from germinated seedling was used as explant for callus induction. Moderate to good callus induction was observed on MS medium containing 1.16 μM kinetin and 1.13–2.26 μM 2,4-dichlorophenoxyacetic acid. Regular subculturing of callus on kinetin (1.16 μM) and 2,4-dichlorophenoxyacetic acid (1.13 μM) supplemented medium induced somatic embryogenesis. In modified MS medium, 1.79 mM NH4NO3 and 10.72 mM KNO3 was optimal for somatic embryogenesis. 7.38 μM 2-isopentenyladenine supplemented to modified MS medium, showed best response for somatic embryogenesis while proline (0.76 mM) as an amino acid supplement gave better response than glutamine. 30% germination of mature somatic embryos was achieved on MS medium supplemented with 15.54 μM 6-benzylaminopurine. Multiplication of C. borivilianum through somatic embryogenesis may offer a better approach compared to organogenesis for developing scale-up technology employing bioreactors for its mass propagation.
PMCID: PMC3550677  PMID: 23572975
Liliaceae; Safed musli; Saponins; Somatic embryogenesis; Proline

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