Plant regeneration from leaf explants of Aloe barbadensis Mill. and genetic fidelity assessment through DNA markers

An efficient plant regeneration protocol was developed from leaf explants of Aloe barbadensis Mill on Murashige and Skoog’s (MS) medium supplemented with 2.0 mg/l 6-benzyladenine (BA) or Kinetin (Kn), 0.25–0.5 mg/l NAA (1-napthalene acetic acid) and 3 % (w/v) sucrose within 4 weeks of culture. The maximum number of shoot buds were obtained on MS medium supplemented with 2.0 mg/l BA, 0.5 mg/l NAA, 40 mg/l Ads (adenine sulphate) within 4–6 weeks of subculture. Inclusion of 0.25–0.50 mg/l gibberellic acid into the medium, the shoot buds became elongated. Repeated subculture on regeneration medium induces higher rate of shoot regeneration. The root induction from excised microshoots was achieved on half-strength MS medium supplemented with 0.25–1.0 mg/l NAA or indole-3-butyric acid (IBA) and 2 % (w/v) sucrose. Maximum percentage of rooting was achieved on medium having 0.5 mg/l NAA with 3 % (w/v) sucrose. About 80 % of in vitro raised plantlets were hardened in the greenhouse and successfully established in the soil. Both Random Amplified Polymorphic DNA (RAPD) and Inter Simple Sequence Repeat (ISSR) markers were used to detect the variability among the regenerated plants developed in vitro. The results showed that there was no polymorphism among the regenerated plantlets. This study will help for propagation of quality planting material of Aloe barbadensis for commercialization.     

An improved micropropagation and assessment of genetic stability of micropropagated Salvadora oleoides using RAPD and ISSR markers

An improved micropropagation method has been developed for Salvadora oleoides, a valuable tree species of alkaline and arid regions. Nodal explant obtained from a mature tree (30- to 35-year-old) responded optimally (80.0 %) on BAP (2.0 mg l^?1) and produced (4.56 ± 0.52) shoots. Shoots were further multiplied by subculturing the in vitro excised shoots and transferring them to MS medium containing either BAP (0.0–2.0 mg l^?1) alone or in combination with lower concentrations of an auxin (IAA or NAA 0.05–0.4 mg l^?1). Among all the PGRs combination tested, MS medium supplemented with BAP (0.5 mg l^?1) and IAA (0.1 mg l^?1) formed the maximum number of shoots (68.40 ± 2.74 per culture bottle) with an average height (6.59 ± 0.30 cm), after 6 weeks of culture. Rooting in regenerated shoots was achieved by ex vitro methods and about 92.5 % of shoots were rooted with 5.25 ± 0.64 roots per shoot and an average length of 2.76 ± 0.53 cm after 3 weeks of incubation in the green house. More than (80 %) of hardened plantlets survived in the field conditions. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD and ISSR. Of the 10 RAPD primers finally selected, a total of 42 bands (out of 43) were monomorphic and one polymorphic, whereas from 10 ISSR primers selected, all the 43 bands were monomorphic revealing a high level of genetic homogeneity in the regenerated plants and the donor plant. In the present investigation, we achieved significantly more number of shoots during multiplication, which are higher than all previous reports and further evaluated the genetic fidelity of protocol for the first time in S. oleoides, which concludes the clonal (true-to-type) nature of micropropagated plantlets.     

Plantlet regeneration through indirect shoot organogenesis and somatic embryogenesis in Justicia gendarussa Burm. f., a medicinal plant

A protocol for the regeneration of a large number of plantlets via indirect shoot organogenesis and somatic embryogenesis has been developed from the stem and leaf explants of Justicia gendarussa Burm. f. The callus was efficiently induced from the explants using Murashige and Skoog (MS) medium supplemented with α-Naphthalene acetic acid (NAA) + Benzyl amino purine (BAP) (1.0?+?0.1 mg/l). The highest number of plantlets through indirect shoot organogenesis was obtained when the callus was subcultured to MS medium with BAP + NAA (0.1?+?1.0 mg/l). The maximum number of plantlets via somatic embryos was obtained in the medium with BAP + NAA (1.0?+?0.1 mg/l) for stem derived calli and Kinetin (Kn) + NAA (2.0?+?0.1 mg/l) for leaf derived calli. The in vitro developed shoots were rooted well in half strength MS medium supplemented with 0.5 mg/l of Indole-3-acetic acid (IAA). The in vitro regenerated plantlets were hardened using a mixture of sterile sand:soil:manure (1:1:1). The present study is the first report on the regeneration of plants through somatic embryogenesis from stem and leaf derived calli of J. gendarussa.     

Highly efficient in vitro proliferation and genetic stability analysis of micropropagated Ceropegiaevansii by RAPD and ISSR markers: A critically endangered plant of Western Ghats

Ceropegiaevansii McCann (family: Asclepiadaceae), a critically endangered plant of Western Ghats has acquired significant importance due to its medicinal implications, edible tubers, and ornamental flowers. This study deals with the optimization of axillary bud proliferation using nodal explants followed by genetic stability analysis of regenerants. Maximum number of shoots (11.6 ± 1.1) was observed on the Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (4.0 mg/l) and indole-3-acetic acid (0.3 mg/l) with 85% shoot multiplication frequency. In vitro-grown shoots were rooted best in 1/2 MS medium supplemented with indole-3-butyric acid (1.0 mg/l) with an average of 10.3 ± 0.9 roots per shoot and 92% rooting frequency. Plantlets were acclimatized best (90%) in a mixture of sterile soil, sand, and coco peat (1:2:1). Micropropagated plants were subjected to random amplified polymorphic DNA and inter simple sequence repeat markers analyses. Collectively, 759 bands were generated which were monomorphic and similar to the mother plant. Findings of this study are the first report on micropropagation and assessment of genetic stability of micropropagated plantlets in C. evansii which suggests that axillary shoot proliferation can safely be used as an effective tool for propagation and conservation of C. evansii.     

In vitro propagation and assessment of genetic integrity of Talinum triangulare (Jacq.) Willd: a valuable medicinal herb

Talinum triangulare is a medicinally important herb and various parts of the plant are used pharmaceutically for the treatment of different diseases. In our study, a rapid and efficient protocol for micropropagation has been developed from shoot tip and nodal explants of T. triangulare. High shooting frequency (93.33?%) was achieved with shoot tip explants when cultured on Murashige and Skoog??s (MS) medium supplemented with 1.0?mg/L 6-benzyl amino purine (BAP) producing an average of 12.50?±?0.23 shoots and 5.07?±?0.02?cm shoot length per explant. A combination of 0.5?mg/L BAP and 0.5?mg/L kinetin was found to be more effective by producing 15.67?±?0.25 shoots and 6.22?±?0.02?cm shoot length per explant. The microshoots were excised and cultured on half-strength MS and full-strength MS medium containing different concentrations of indole-3-acetic acid and indole-3-butyric acid (IBA) for root induction. More number of roots (45.10?±?0.96) with an average length of 5.46?±?0.08?cm was obtained on half-strength MS medium supplemented with 0.5?mg/L IBA. The rooted shoots were successfully transplanted from different planting substrates to the field with a 100?% survival rate. Random amplified polymorphic DNA analysis was carried out using four random decamer primers. The amplification products were monomorphic in the micropropagated plants and were similar to the mother plant. Absence of polymorphism revealed that no variation was induced, thus maintaining the genetic integrity of the micropropagated plants of T. triangulare.     

Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb.

Somatic embryogenesis (SE) has been achieved from hypocotyl-derived callus culture in Pterocarpus marsupium. Ninety percent of hypocotyl explants (excised from 12-day-old in vitro germinated axenic seedlings) produced callus on Murashige and Skoog medium supplemented with 5 μM 2,4-dichlorophenoxyacetic acid and 1 μM a 6-benzyladenine (BA). Induction of SE occurred after transfer of callus clumps (200 ± 20 mg fresh mass) to MS medium supplemented with BA at 2.0 μM, where a maximum of 23.0 ± 0.88 globular stage embryos per callus clump were observed after 4 weeks of culture. Subculturing of these embryos on MS medium supplemented with 0.5 μM BA, 0.1 μM α-naphthalene acetic acid and 10 μM abscisic acid significantly enhanced the maturation of somatic embryos to early cotyledonary stage, where 21.4 ± 0.32 embryos per callus clump were recorded after 4 weeks of culture. Of 30-well developed somatic embryos, 16.6 ± 0.33 germinated and subsequently converted into plantlets on half-strength MS medium supplemented with 1.0 μM BA. The morphologically normal plantlets with well-developed roots were first transferred to 1/4-liquid MS medium for 48 h and then to pots containing autoclaved soilrite and acclimatized in a culture room. Thereafter, they were transferred to a greenhouse, where 60% of them survived.     

Primary and secondary somatic embryogenesis in Chrysanthemum cv. Euro

Somatic embryogenesis was induced from in vivo grown leaf explants of Chrysanthemum cv. Euro incubated on Murashige and Skoog (MS) medium supplemented with 2.0 mg/L 2,4-dichlorophenoxyacetic acid and 2.0 mg/L Kinetin, yielding the highest mean number of embryos (42 ± 5.97) per explant after 5 weeks of culture. We evaluated the effects of basal medium, various concentrations of sucrose, and timentin on the proliferation of secondary somatic embryos. MS medium was observed to be the more effective in promoting the proliferation of somatic embryos than half-strength Murashige and Skoog (1/2MS). In addition, timentin was also more efficient in induction of secondary embryogenesis than sucrose. Whole plantlets were obtained by culturing of secondary embryos on hormone-free MS medium and successfully acclimated in the green house.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated genetic transformation of <Emphasis Type="Italic">Acacia mangium</Emphasis>

A protocol was developed for Agrobacterium-mediated genetic transformation of Acacia mangium using rejuvenated shoots as the explant. Axillary buds and shoot apices of adult trees were rejuvenated by culturing them on Murashige and Skoog (MS) medium, and stem segments of rejuvenated shoots were co-cultured with Agrobacterium tumefaciens strain LBA4404 harbouring binary vector pBI121. The selection for transgenic shoots was performed through five consecutive steps on MS medium supplemented with 1.0 mg/l thidiazuron, 0.25 mg/l indole-3-acetic acid and different concentrations of geneticin (G418; 12–30 mg/l) and timentin (T; 50–300 mg/l) in the following order: 12 mg/l G418 and 300 mg/l T for 30 days, 20 mg/l G418 and 200 mg/l T for 60 days, 30 mg/l G418 and 100 mg/l T for 30 days, 12 mg/l G418 and 50 mg/l T for 30 days, and finally 15 mg/l G418 and 5 mg/l gibberellic acid (GA₃) for 60 days. Thirty-four percent of the stem segments produced resistant multiple adventitious shoot buds, of which 30% expressed the β-glucuronidase gene. The shoot buds were subjected to repeated selection on MS medium supplemented with 2.0 mg/l 6-benzylaminopurine, 2.5 mg/l GA₃ and 20 mg/l G418. Transgenic plants were obtained after rooting on half-strength MS medium supplemented with 2.0 mg/l α-naphthaleneacetic acid, 0.1 mg/l kinetin and 20 mg/l G418. Genomic Southern blot hybridization confirmed the incorporation of the NPTII gene into the host genome.     

Direct shoot regeneration from basal leaf segments of Lilium and assessment of genetic stability in regenerants by ISSR and AFLP markers

Here, we report a widely applicable procedure for direct shoot regeneration via basal leaf segments of Lilium. Leaf segments (0.8–1.0 cm long and 0.4 cm wide) were excised from leaves on shoot nodes 3 to 6 of 4-wk-old in vitro stock shoot cultures. The segments were wounded by three transverse cuts across the midvein on the abaxial side, with 1 mm between cuts, and cultured with the abaxial side in contact with a shoot regeneration medium composed of half-strength Murashige and Skoog medium supplemented with 1 mg/l naphthaleneacetic acid, 0.5 mg/l thidiazuron, 30 g/l sucrose, and 7 g/l agar (pH?5.8). The cultures were incubated for 4 wk under a 16-h photoperiod at 23?±?2°C for adventitious shoot regeneration. With this procedure, a mean shoot regeneration frequency of 92–100% and mean number of shoots of 4.7–7.0 per segment were obtained in five Lilium species and hybrids, which represent diverse genotypes of Lilium and are commercially popular lilies. Histological studies with Lilium Oriental hybrid “Siberia” revealed that meristemoids initiated from subepidermal cells on the adaxial side of the explant and eventually developed into adventitious buds, without callus formation. In an assessment of genetic stability in the regenerants of “Siberia”, no polymorphic bands were detected by intersimple sequence repeat and only 0.73% polymorphic bands were detected by amplified fragment length polymorphism. The morphologies of the regenerants were identical to those of the control. These results demonstrated that the regenerants were genetically and morphological stable. Thus, this procedure has great potential application for micropropagation, genetic transformation, and preparation of shoot tips for cryopreservation and cryotherapy for virus eradication of Lilium.     

Efficiency of direct and indirect shoot organogenesis, molecular profiling, secondary metabolite production and antioxidant activity of micropropagated Ceropegia santapaui

Ceropegias has acquired significant importance due to their medicinal properties, edible tubers, and its ornamental flowers. The aim of this study was to optimize direct shoot organogenesis (DSO), indirect shoot organogenesis (ISO) and plant regeneration of threatened medicinal plant Ceropegia santapaui, followed by analysis of genetic status and biochemical characterization of micropropagated plantlets. For optimization, cotyledonary nodes and cotyledons were used as source of explants in DSO and ISO respectively. The highest frequency of regeneration (88.0 %) for DSO with 8.1 ± 0.6 shoots per explant was obtained from cotyledonary nodes cultured on Murashige and Skoog’s (MS) medium containing 2.0 mg L^?1 2iP. The best response for callus induction and proliferation was achieved with 1.5 mg L^?1 PR (picloram) in which 97.5 % of cultures produced an average of 913 ± 10.9 mg (fresh weight) of callus. The highest frequency of shoot formation (92.5 %) with an average of 19.7 ± 0.3 shoots in ISO was obtained when calli were transferred to MS medium supplemented with 2.5 mg L^?1 BAP and 0.4 mg L^?1 IBA. Regenerated shoots were best rooted in half-strength MS medium with 2.0 mg L^?1 NAA. Plantlets successfully acclimatized were morphologically indistinguishable from the source plant. Micropropagated plantlets subjected to random amplified polymorphic DNA and inter simple sequence repeats (ISSR) marker based profiling reveled uniform banding pattern in DSO-derived plantlets which was similar to mother plant. ISSR fingerprints of ISO-derived plants showed low variation. Method of regeneration, plant part and solvent system significantly affected the levels of total phenolics, flavonoids and antioxidant capacity. Assay of antioxidant activity of different tissues revealed that significantly higher antioxidant activity was observed in ISO-derived tissues than DSO-derived and mother tissues. RP-HPLC analysis of micropropagated plantlets showed the presence of three major phenolic compounds which were similar to those detected in mother plant. Rapid multiplication rate, genetic stability and biochemical parameter ensures the efficacy of the protocol developed for the propagation of this threatened medicinal plant.     

Micropropagation of Ajuga bracteosa, a medicinal herb

For conservation and genetic transformation, a successful in vitro micropropagation protocol for Ajuga bracteosa, a medicinal herb has been established for the first time. MS medium supplemented with IAA (2 mg/L) and BA (5 mg/L) induced 100 % shoot regeneration with an average of 41.4 shoots of 8.4 cm per culture. Excised in vitro shoots when transferred to MS + IBA (0.5 mg/L) produced 20 roots/shoot of 20.2 cm average length in 100 % cultures. Of the three explants, leaf, petiole and root, leaf displayed quickest response followed by petiole while root was the slowest. Hardening of plantlets was achieved with 82 % survival. The hardened plants were maintained in pots with garden soil under controlled (Temp. 25?±?2 °C) conditions. RAPD exhibited genetic fidelity with 100 % monomorphism in regenerants.     

In vitro propagation of Homalomena aromatica Schott., an endangered aromatic medicinal herb of Northeast India

A successful report on the in vitro propagation of Homalomena aromatica via rhizome axillary bud multiplication is presented. Rhizome bud explants were cultured on Murashige and Skoog medium supplemented with various concentrations of cytokinins to induce multiple shoot formation for micropropagation. The highest number of shoots was achieved in MS medium supplemented with 2.0 mg?l^?1 6-benzylaminopurine. The regenerated shoots rooted most efficiently on half-strength MS medium supplemented with 0.5 mg?l^?1 α-naphthalene acetic acid. The regenerated plantlets showed no morphological differences from the parent plant. This protocol takes approximately 6 months to reach the acclimatization stage from the initiation stage and facilitates commercial and rapid propagation of H. aromatica.     

Somatic embryogenesis and organogenesis in Dendrocalamus hamiltonii

In this study, mature zygotic embryos, plant growth regulators, and various media were tested with the aim of developing an efficient regeneration system for plantlets of the bamboo species Dendrocalamus hamiltonii. Callus formation was induced in explants cultured in Murashige and Skoog (MS) medium supplemented with 1.0–3.0 mg/l 2,4-dichlorophenoxyacetic acid. Optimal shoot differentiation and subsequent shoot growth were also obtained in MS medium supplemented with 2 mg/l benzyladenine, 1 mg/l kinetin, and 1 mg/l naphthaleneacetic acid. Root induction was enhanced by the addition of 5 mg/l indole-3-butyric acid to the culture medium. Histological analysis revealed that both somatic embryogenesis and organogenesis were induced during callus initiation, shoot differentiation, and the development of plantlets from the mature zygotic embryos. Our data provide a useful basis for developing culture protocols for the regeneration of bamboo plants.     

In vitro organogenesis from leaf and transverse thin cell layer derived callus cultures of Talinum triangulare (Jacq.) Willd.

Talinum triangulare is an important medicinal herb used traditionally in the treatment of various diseases. The present study was intended to develop a rapid and efficient protocol for indirect organogenesis from leaf discs and transverse thin cell layer (tTCL) of internodal explants of T. triangulare. Best callusing response (100 %) was observed with tTCL explants on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyl amino purine and 5.37 μM α-naphthalene acetic acid (NAA). High frequency shoot regeneration (96.67 %) was obtained from tTCL derived calli on MS medium supplemented with a combination of 0.45 μM thidiazuron and 0.27 μM NAA, by producing 9.20 ± 0.35 shoots with a shoot length of 2.74 ± 0.03 cm. In vitro rooting of the microshoots was recorded on half-strength MS medium containing 2.46 μM indole-3-butyric acid by eliciting 15.20 ± 0.27 roots with a length of 4.25 ± 0.11 cm. The rooted shoots were acclimatized on garden soil, sand and coco pith (1:1:3 v/v) planting substrate. The plantlets were successfully established under field conditions with 100 % survival rate. The hardened plants exhibited homogeneity and no observable morphological variations were detected among the regenerants and the mother plants of T. triangulare.     

An improved method for micropropagation and regeneration of date palm (Phoenix dactylifera L.)

We developed a novel large-scale micropropagation pathway for date palm (Phoenix dactylifera L.) based on organogenesis. We obtained organogenic stems from shoot tip explants of the Moroccan date palm cultivar Najda, and investigated shoot proliferation from these organogenic stems in vitro on various media; Beauchesne medium (BM) and Murashige and Skoog medium (MS) at full-strength, half-strength, and one-third-strength, containing various concentrations (0, 0.25, 0.5, and 1 mg/L) of 2-naphthoxyacetic acid (NOAA) and kinetin. The optimal medium during the multiplication phase was half-strength Murashige and Skoog medium (MS/2) supplemented with 0.5 mg/L NOAA and 0.5 mg/L kinetin (23.5 morphologically superior shoots per explant, with low vitrification rates). For the shoot elongation phase, shoots were transferred to the same proliferation medium, or to MS or MS/2 media without plant growth regulators (PGRs). Shoots elongated rapidly and showed a high rate of root formation on media supplemented with PGRs. For example, on MS/2 medium containing 1 mg/L NOAA and 1 mg/L kinetin, the average shoot length was 15.1 cm, the average number of roots per shoot was 6.2, and their average length was 3.4 cm. On PGR-free media, shoots were shorter with wider and greener leaves, and had fewer roots. The plantlets were transferred to a greenhouse for acclimation. The survival rate after 2 months was related to the medium used during the elongation phase; >90 % of shoots that were cultured on PGR-free media survived, while there was a poor survival rate of shoots that had been cultured on media containing PGRs.     

Shoot differentiation, regeneration of cauliflower and analysis of somaclonal variation by RAPD

Our major goal in this study was to establish and characterize an efficient callus induction and shoot regeneration protocol for cauliflower (Brassica oleracea var. botrytis). The effects of induction medium, growth regulator combination, organic component, AgNO(3), genotype and explants type on shoot differentiation on the cauliflower regeneration process were taken in account and hereby evaluated. The optimal media for shoot differentiation and rooting were modified MS medium (MS with PG-96 organic components) supplemented with NAA at 0.5 mg l(-1), TDZ at 0.25 mg l(-1), BA at 3.0 mg l(-1), AgNO(3) at 2.0 mg l(-1) and MS supplemented with IBA at 0.4 mg l(-1), respectively. Among the four varieties tested, Saixue gave the best shoot differentiation response (average over 18 shoots per explant) from the upper section of the hypocotyls. We have so far obtained over 500 regenerated plants under this improved protocol. We have further analyzed the somaclonal variation of regenerated plants at the DNA level by using the RAPD molecular markers. By PCR amplification, we were able to amplify 75 scoreable bands from 15 primers out of 40 arbitrary primers screened, where 35 of them were monomorphic and 40 polymorphic bands (53.3%) in four varieties studied. The absence of polymorphism among regenerated plants from the same variety indicated the conformity of the regeneration protocol.     

In vitro organogenesis from internode derived callus cultures of Capsicum annuum L.

An efficient protocol of shoot organogenesis and plant regeneration from internode derived callus has been developed for Capsicum annuum. Optimal callus was developed from internodal segments on Murashige and Skoog (MS) medium supplemented with 10 μM 2,4-dichlorophenoxy acetic acid (2,4-D) and 2.0 μM 6-benzyladenine (BA). Shoot differentiation was achieved from the surface of callus when transferred on shoot induction medium containing BA and thidiazuron (TDZ) alone or in combination. The highest number of de novo adventitious shoots (25.4?±?1.42) and shoot length (4.6?±?0.37 cm) was recorded on MS medium supplemented with 5.0 μM BA and 2.5 μM TDZ. The individual elongated shoots were rooted well on MS medium supplemented with 1.0 μM Indole-3-butyric acid (IBA). The in vitro raised plantlets with properly developed shoot and roots were acclimatized successfully and grew well in the greenhouse. All the regenerated plants appeared normal with respect to morphology and growth characteristics with 85% survival rate.     

Plant regeneration via somatic embryogenesis in Drimia robusta

A simple efficient in vitro plant regeneration system was developed by direct and indirect somatic embryogenesis of Drimia robusta, a medicinal plant extensively used in South African traditional medicine. Different developmental stages of somatic embryos (SEs: globular embryos, partial pear-shaped embryos and club-shaped embryos), club-shaped cotyledon initiation, plumule initiation and plantlets were directly obtained from leaf explants on Murashige and Skoog (MS) medium containing 3.5 % (w/v) sucrose and different plant growth regulators (PGRs). In MS medium containing 3.5 % (w/v) sucrose and supplemented with 10 μM picloram, 1 μM thidiazuron (TDZ) and 20 μM glutamine, a higher number of SEs and plantlets were achieved. These were established onto half-strength MS medium followed by successful acclimatization (100 %) in the greenhouse. Liquid somatic embryo medium (SEM_L) containing 500 mg of friable embryogenic callus on MS medium supplemented with different concentrations and combinations of PGRs and organic elicitors produced different stages of SEs. Somatic embryo production was enhanced by 0.5 μM picloram, 1 μM TDZ and mebendazole treatment. The highest number of plantlets (9.0 ± 0.70) was obtained in SEM_L containing 0.5 μM picloram, 1 μM TDZ and 25 mg l^?1 haemoglobin. All the cotyledon and plumule embryos germinated on half-strength MS medium, however 90 % of SEs germinated on half-strength MS medium containing 0.5 μM naphthaleneacetic acid. All plantlets were successfully acclimatized in the greenhouse. This first report of D. robusta somatic embryogenesis provides an opportunity to control extinction threats, ensure germplasm conservation and provides a system for analysis of bioactive compounds and bioactivity.     

Efficient in vitro plant regeneration from seedling-derived explants and genetic stability analysis of regenerated plants of <Emphasis Type="Italic">Simarouba glauca</Emphasis> DC. by RAPD and ISSR markers

Simarouba glauca DC. is a multipurpose tree species known for oil, timber, and medicinal properties. The application of biotechnological methods for genetic improvement of this species depends on the availability of an efficient plant regeneration system. In this study, the shoot regeneration potential of various seedling-derived explants was assessed after culturing on Murashige and Skoog (MS) and woody plant (WP) medium containing different growth regulators. The explants differed in their capacity for shoot bud formation and subsequent shoot elongation on the media tested. Shoot bud induction was achieved at a high frequency (44.8–76.2%) from different explants on MS medium with 2 mg L^?1 6-benzylaminopurine (BAP) as compared to other media tested. Cotyledons exhibited the highest capacity for shoot bud induction (76.2%) and shoot elongation (9.1 elongated shoots per explant). The in vitro-regenerated shoots rooted at a frequency of 66.7% after pulse treatment in 10 mg mL^?1 indole-3-butyric acid (IBA) solution for 5 min followed by culture on half-strength WP medium with 0.2 mg L^?1 IBA. The regenerated plants were acclimatized and established in the glasshouse with a survival rate of 80%. Molecular characterization of regenerated plants using 14 random amplified polymorphic DNA (RAPD) and 15 intersimple sequence repeat (ISSR) primers revealed a high number of monomorphic bands, with only 1.6–2.6% of the bands being polymorphic. The regeneration system established in the study has the potential to be used for rapid multiplication, conservation, and genetic transformation of this species.     

In vitro morphogenic response of different explants of Gentiana kurroo Royle from Western Himalayas—an endangered medicinal plant

Micropropagation offers a great potential to produce millions of clonal individuals through tissue culture via induction of morphogenesis. The aim of this work was to obtain an efficient protocol for callus regeneration for Gentiana kurroo Royle. The morphogenic response of different explants (leaves, petioles, roots) varied and responded differently for regeneration according to combinations of growth regulators. The petiole explants were best responding for callus induction and subsequently for indirect and direct regeneration. The callus induction was achieved on MS basal + 1.0 mg/l benzyladenine (BA) and 3.00 mg/l naphthalene acetic acid (NAA). MS medium supplemented with 0.10 mg/l NAA and 1.0 mg/l thidiazuron (TDZ) was recorded as the best medium for indirect regeneration. However, for direct regeneration the maximum number of shoot emergence was observed on MS basal fortified with 0.10 mg/l NAA + 0.75 mg/l TDZ. Half strength MS basal supplemented with indole-3-butyric acid (IBA) 1.00 mg/l gave best response for root induction. Subsequently, the plantlets were transferred and 100 % survival rate was recorded only on autoclaved cocopeat. No morphological variations were recorded in the callus regenerated plantlets.