In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with ß-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.     

Indirect organogenesis from leaf explants of Hemidesmus indicus (L.) R. Br.: an important medicinal plant

Hemidesmus indicus (Asclepiadaceae) leaf explants were utilized for establishing culture in MS medium fortified with individual cytokinins, auxins, and their combinations. Optimum response (80%) was observed in N⁶-benzyladenine (BA, 20 μM) + indole-3-acetic acid (IAA, 1 μM) with 19.67 ± 0.81 shoots per explant. Roots were induced in ¼MS + indole-3-butyric acid (IBA, 20 μM).     

In vitro morphogenesis in Selaginella microphylla (Kunth.) Spring

Selaginella, an extant genus of primitive vascular plants, has survived over 400 million years of evolution. In vitro morphogenesis in Selaginella microphylla is considered for the first time to establish a well-documented aseptic culture on half- strength Murashige and Skoog’s basal medium with 2ip (4.92–49.21 μM), or Kn (4.65–46.47 μM) or GA₃ (2.89–28.90 μM) for shoot multiplication, and with different concentrations of IBA (4.9–49 μm) to initiate root cultures. GA₃ was instrumental for shoot multiplication as well as induction of reproductive structures in each and every leaf axil. On the other hand, it is observed that IBA alone in S. microphylla can act as signal molecules for induction of enormous numbers of root masses from a few existing roots. An interesting pattern of re-differentiation has also been observed where apical portions of large numbers of roots were converted to green shoot apical meristems. Further differentiation produced tiny green shoots. Distinct bipolarity was noted in shoots when they were isolated from root masses and appeared as embryo-like structures. Chromosome analysis from in vitro sporophytic plants revealed 2n = 16 chromosomes, indicating chromosomal stability. The interesting in vitro pattern of morphogenesis obtained in S. microphylla may provide new insights into totipotency of plants.     

<Emphasis Type="Italic">In vitro</Emphasis> shoot regeneration and enhanced synthesis of plumbagin in root callus of <Emphasis Type="Italic">Plumbago zeylanica</Emphasis> L.—an important medicinal herb

Plumbago zeylanica L., an important medicinal herb, possesses plumbagin, a valuable secondary metabolite. Roots of this plant, collected from four locations in Himachal Pradesh, India, were screened for plumbagin content with high-performance liquid chromatography. The chemotype collected from Hamirpur yielded the highest content (26.47?±?0.63 mg g^?1 dry weight). Callus cultures were established from nodal explants of this chemotype on Murashige and Skoog (MS) medium augmented with α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), 2,4-dichlorophenoxyacetic acid, (2,4-D), 6-benzyladenine (BA), isopentenyl adenine (2iP), or thidiazuron, (TDZ). After 45 d, 98% of the cultures induced bright-green, compact callus on MS?+?5 μM TDZ. Upon subculturing, this callus differentiated an average of 4.08?±?1.16 shoots in 62.5% of the cultures. After elongation on basal MS medium, excised shoots were transferred to indole-3-acetic acid, NAA, or IBA supplemented MS medium. A maximum of 4.3?±?1.36 roots with an average length of 15.31?±?2.76 cm were recorded on 5 μM IBA. Rooted plantlets were successfully acclimatized in a greenhouse, and their genetic fidelity was evaluated using inter simple sequence repeats and start codon targeted molecular markers, which revealed 97% similarity. A significant increase in plumbagin content (6.5- and 3.4-fold) was achieved in root callus employing 100 mg L^?1 yeast extract (YE) and 25 μM salicylic acid (SA), respectively. This is the first report of large-scale propagation of P. zeylanica and an increase of plumbagin through in vitro root callus.     

Differences in hypericin synthesis between experimentally induced seedling shoot cultures of Hypericum hookerianum Wight & Arn.

During seed germination trials of Hypericum hookerianum, seedlings of Lake View accession from Palni hills of Southern India segregated into green- (97.44 %) and red-pigmented (2.56 %) types. Seedlings cultured in Murashige and Skoog (1962) basal medium developed into fast growing green and slow growing red plant types in 6 weeks, the latter showing increased concentrations of total phenols, anthocyanins and flavonoids and 19-fold higher concentration of hypericin. Hypocotyls/cotyledons of red seedlings cultured using 2.325 μM kinetin (KIN) produced hypericin-rich (4.38 ± 0.18 mg/g DW), stunted (0.5–1.2 cm) shoots which ceased to grow in 8 weeks. Segments (4–6 mm) of these shoots sub-cultured in the dark for 4 weeks followed by 2-week light exposure and repeated subculture enabled mass multiplication of productive (3.93 ± 0.06 mg hypericin/g DW) shoots. Green hypocotyls and cotyledons subjected to 4 + 2 weak dark–light treatment also produced 9.18 ± 2.44 and 4.25 ± 0.96 comparable hypericin-rich (3.73 ± 0.21 mg/g DW) shoots. Red and green seedling explants cultured in basal medium in the dark produced 6.82 ± 0.75 cm etiolated shoots with reduced leaves which synthesized 2.27 ± 0.15 mg hypericin/g DW on illumination. Green cotyledons cultured in the dark using 2.45 μM indole-3 butyric acid (IBA) formed calluses which on illumination formed 12.64 ± 3.8 productive (3.86 ± 0.31 mg hypericin/g DW) 0.5- to 1.5-cm-long shoot clusters. Phenotypic segregation of seedlings, the ability of both red and green seedling explants to multiply in the dark and produce hypericin on illumination, and IBA-induced indirect shoots producing significant amounts of metabolite compared to wild plants (0.35 ± 0.09 mg/g DW) and green shoot cultures (0.91 ± 0.03 mg/g DW) are new to Hypericum.     

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.     

In vitro plantlet regeneration of Capsicum chinense Jacq. cv. ‘Bhut jalakia’: hottest chili of northeastern India

Chili (Capsicum chinense) cv. ‘Bhut jalakia’ is used in India for extraction of oleoresin and capsaicin as it is characterized by a very high capsaicin content. The conventional method of propagation of ‘Bhut jalakia’ is through seeds, but this is beset by short viability and low germination rates. Developing a suitable regeneration protocol for ‘Bhut jalakia’ was the focus of this study; as to date, in vitro regeneration for this cultivar has not been investigated. Cotyledon and shoot tip explants were cultured on Murashige and Skoog (MS) media supplemented with different concentrations of cytokinins and auxins. In the case of cotyledon explants, MS medium supplemented with 6-benzylaminopurine (BAP) at 35 μM and kinetin (KIN) at 15 μM were found to be optimal (4.00?±?0.57) for induction of multiple shoots per explant, whereas BAP at 14.8 μM and KIN at 60 μM were best (5.00?±?0.57) for growth of shoot tip explants. Shoots developed from cotyledon explants produced the maximum (8.67?±?0.32) number of roots on MS medium supplemented with low concentration (2.6 μM) of 2-naphthaleneacetic acid (NAA). Supplementation of indole-3-butyric acid (IBA) at 5 μM was found optimal for root formation (16.67?±?2.60) for shoots derived from of shoot tip explants. One month after transfer of in vitro regenerated plantlets to various potting mixes, the highest survival rate (40%) was observed in a mixture of sand, soil, and cow dung in a ratio of 1:1:1. Thus, both shoot tip and cotyledon explants may be cultured on MS medium modified with BAP, IBA, NAA, and KIN to regenerate ‘Bhut jalakia’ chili plants within 90 d.     

Potential role of cytokinin–auxin synergism, antioxidant enzymes activities and appraisal of genetic stability in Dianthus caryophyllus L.—an important cut flower crop

The regeneration potential, antioxidative enzyme activities, and genetic stability among micropropagated plantlets of Dianthus caryophyllus L. were evaluated. Multiple adventitious shoots were induced from leaf explants on Murashige and Skoog medium incorporated with various combinations and concentrations of plant growth regulators (PGRs). The highest leaf explant response (90%), number of shoots per explant (15.30?±?1.19), and shoot length (6.75?±?0.63 cm) was recorded in response to a combination of 2.5 μM 6-benzyladenine and 0.5 μM α-naphthaleneacetic acid (NAA) after 8 wks culture. Subsequent subculturing for five passages, on a medium with the same composition of PGRs, induced the highest shoot number (42.50?±?1.44), with an average shoot length of 8.06 cm after the fourth subculture. Different concentrations of indole-3-butyric acid (IBA) were tested to determine the optimum conditions for ex vitro rooting of microshoots. The best result was accomplished with a pulse treatment of IBA (100 μM) applied to the basal end of the microshoot for 30 min, followed by transfer to plastic cups containing soilrite, and eventually established in natural soil with an 85% survival rate. The determination of activities of antioxidative enzymes (superoxide dismutase, ascorbate peroxidase, catalase, and glutathione reductase) revealed involvement of these enzymes in shoot differentiation and development. All of these activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. Intersimple sequence repeat DNA analysis was carried out using five primers. The amplification products were monomorphic in micropropagated plants, similar to those of the mother plant. No polymorphisms were detected revealing the genetic integrity of the micropropagated plants.     

Micropropagation and in vitro flowering of endemic and endangered plant Ceropegia attenuata Hook

Factors affecting in vitro propagation were evaluated for Ceropegia attenuata Hook., an endemic and endangered plant having ornamental potential but a limited reproductive capacity. Rapid shoot multiplication from nodal explants was established using varying concentrations of cytokinins and auxins either alone or in combinations. The highest frequency of shoot induction was achieved when nodal explants were inoculated on Murashige and Skoog (MS) medium supplemented with 13.31 μM 6-benzylaminopurine with a mean of 12.9?±?0.5 shoots per explant. High concentrations of TDZ (6.81–11.35 μM) and KN (6.78–11.61 μM) resulted in stunted and vitrified shoots. Factors implicated in the promotion of floral transition of the C. attenuata have been identified which are 4-amino-3, 5, 6-trichloropicolinic acid (picloram), 6-benzylaminopurine, sucrose and photoperiod. The highest frequency of flowering (100%) was obtained when axillary shoot explants were transferred to MS medium supplemented with picloram (4.14 μM) within 4 weeks of culture. Transfer of in vitro regenerated shoots to half strength MS medium with 2.46 μM indole-3-butyric acid (IBA) showed maximum root induction. The in vitro grown plantlets were successfully acclimatized in the glasshouse with 85% of survival and showed normal development. The developed protocol provided a simple, cost-effective approach for the conservation of endangered plant C. attenuata for replenishing its declining populations.     

Rapid plant regeneration and analysis of genetic fidelity in micropropagated plants of Vitex trifolia: an important medicinal plant

An efficient in vitro regeneration protocol of a valuable medicinal plant, Vitex trifolia has been successfully established using nodal segments as explants. Three different cytokinins (BA, Kn, 2iP) and auxins (NAA, IAA, IBA) in different concentrations and combinations, evaluated as supplements to Murashige and Skoog’s medium showed to have a marked influence on the regeneration output. Among all the single cytokinin treatments MS medium supplemented with 5.0 μM BA produced the maximum number of shoots yielding 8.20 ± 0.37 shoots per explant with 4.8 ± 0.43 cm shoot length after 8 weeks of culture. Combined with low auxin concentrations, all the three cytokinins at their optimal concentrations synergistically enhanced the regeneration credentials. However, MS medium enriched with 5.0 μM BA and 0.5 μM NAA yielded the best possible regeneration in the species with a regeneration percentage of 97.33 ± 2.67 % and amounting to 16.80 ± 0.58 shoots per explant with 6.20 ± 0.25 cm mean shoot length at the end of 8 weeks in culture. Ex vitro rooting of in vitro derived microshoots was achieved by 20 min 500 μM IBA treatment followed by transfer to thermocol cups containing sterile soilrite. A 95 % plantlets survived acclimatization procedure to the field. Genetic conformity of the regenerated plants was established through RAPD. All the bands visualized on agarose gels were monomorphic with that of the donor plant indicating the clonal nature of the regenerants.     

TDZ-induced plant regeneration in <Emphasis Type="Italic">Brassica oleracea</Emphasis> L. var. <Emphasis Type="Italic">botrytis</Emphasis>: effect of antioxidative enzyme activity and genetic stability in regenerated plantlets

The effects of various combinations of plant growth regulators on regeneration potential from seedling-derived leaf tissues of Brassica oleracea L. var. botrytis were evaluated. Callus was induced from 2-wk-old leaf explants. The explants were incubated on Gamborg’s (MSB₅) medium. The maximum frequency of callus induction (85.56%) was recorded on MSB₅ medium supplemented with 9.1 μM thidiazuron (TDZ) and 0.5 μM α-naphthaleneacetic acid (NAA). Optimum shoot induction (54.44%) was obtained on MSB₅ medium supplemented with 4.5 μM TDZ and 0.5 μM NAA. The maximum number of shoots per explant (5.33) was recorded on MSB₅ medium with 4.5 μM TDZ and 0.5 μM NAA, whereas the maximum shoot length (4.86 cm) was recorded for shoots cultured on MSB₅ medium supplemented with 4.5 μM TDZ and 5.7 μM gibberellic acid (GA₃). However, optimum root induction (71.11%) occurred on half-strength Murashige and Skoog basal medium supplemented with 4.9 μM indole-3 butyric acid (IBA). Studies on the antioxidant activity of superoxide dismutase, ascorbate peroxidase, and peroxidase in seedlings, callus, regenerated shoots, and regenerated plantlets cultured on 4.5 μM TDZ and 0.5 μM NAA medium revealed the roles of these key antioxidative enzymes in callus induction and regeneration. The genetic stability of the regenerated plantlets was assessed using inter simple sequence repeat primers. The monomorphic amplification products confirmed true-to-type in vitro regenerated plants. This in vitro regeneration method can be useful in the large-scale production of genetically uniform plants, for genetic transformation, and conservation of elite germplasm of plant species.     

Plant regeneration of non-toxic Jatropha curcas—impacts of plant growth regulators, source and type of explants

Jatropha curcas is an oil bearing species with multiple uses and considerable economic potential as a biofuel plant, however, oil and deoiled cake are toxic. A non-toxic variety of J. curcas is reported from Mexico. The present investigation explores the effects of different plant growth regulators (PGRs) viz. 6-benzyl aminopurine (BAP) or thidiazuron (TDZ) individually and in combination with indole-3-butyric acid (IBA), on regeneration from in vitro and field-grown mature leaf explants, in vitro and glasshouse-grown seedlings cotyledonary leaf explants of non-toxic J. curcas. In all the tested parameters maximum regeneration efficiency (81.07%) and the number of shoot buds per explants (20.17) was observed on 9.08 μM TDZ containing Murashige and Skoog’s (MS) medium from in vitro cotyledonary leaf explants. The regenerated shoot buds were transferred to MS medium containing 10 μM kinetin (Kn), 4.5 μM BAP and 5.5 μM α-naphthaleneacetic acid (NAA) for shoot proliferation. The proliferated shoots could be elongated on MS medium supplemented with 2.25 μM BAP and 8.5 μM IAA. Rooting was achieved when the basal cut end of elongated shoots were dipped in half strength MS liquid medium containing different concentrations and combinations of IBA, IAA and NAA for four days followed by transfer to growth regulators free half strength MS medium supplemented 0.25 mg/l activated charcoal. The rooted plants could be established in soil with more than 90% survival rate.     

Encapsulation of internode regenerated adventitious shoot buds of Indian Siris in alginate beads for temporary storage and twofold clonal plant production

This study for the first time demonstrates single bead alginate encapsulation and conversion (multiple shootlets rejuvenation) from adventitious shoot buds (AB) of Albizia lebbeck (L.) Benth. Internodal (IN) segments isolated from field grown 1-year-old plant of A. lebbeck were used for AB induction under in vitro conditions. IN segments incubated on MS medium augmented with 10.0 µM BA exhibited highest adventitious shoot bud induction frequencies (76 %) on all over the surface after 10 weeks of culture. Induced AB were detached from in vitro proliferating cultures and used for encapsulation as an explant to produce large number of synseeds (07–08) from a single IN explant. Four to five AB were encapsulated in a single calcium alginate bead to manage mass propagation, interim storage and germplasm sharing. The finer gel matrix for encapsulation was attained using 3.0 % sodium alginate and 100 mM calcium chloride. Highest percentage of shoot emergence and multiplication (75 %) from synseed was obtained on MS + 10.0 µM BA + 1.0 µM NAA (RM) after 10 weeks of culture. Encapsulated adventitious buds stored at 4 °C for 1–8 weeks (2 months) too showed thriving shoot emergence (68 %) and multiplication in encapsulated AB and development into complete plantlets when returned to RM. Hence, 4–5 encapsulated AB stored at 4 °C, when cultured back to RM also showed shoot induction resulting in up to 10 shoots per synseeds after 10 weeks of culture. Healthy root formation (½ MS + 2.0 µM IBA) and acclimatization were optimized by using previously standardized protocol (Perveen et al. in J For Res 22:47–52, 2011). Genetic stability of synseed-derived plantlets acclimatized under ex vitro was assessed and compared with mother plant using inter-simple sequence repeats (ISSR) analysis. The synthetic seeds have the achievability of being a substitute planting material for the forestry sector in future, especially for the multipurpose plant species.     

Multiple shoot induction and regeneration of whole plants from cotyledonary node and nodal explants of Sterculia urens Roxb., a gum yielding tree

Plant regeneration from the nodal explants of 1-month-old in vitro grown plants and cotyledonary node explants of 15-days-old seedlings of Sterculia urens is reported. Nodal explants were grown on MS medium supplemented with various growth regulators like BA, KIN and TDZ. For shoot induction 13.3 μM BA, 0.9 μM TDZ and 9.3 μM KIN were found optimum. Among the three growth regulators 0.90 μM TDZ was used for the growth of cotyledonary node explants. An average of 8.6 shoots per node and 11.2 shoots per cotyledonary node were observed in 4 to 5 weeks. These shoots were subsequently rooted in vitro on half strength MS medium containing various concentrations of auxins like IBA and NAA. The best concentrations for rooting of shoots were 19.7 μM IBA and 16.1 μM NAA. Plantlets were acclimatized to ex vitro conditions and established in the field.     

Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae)

In vitro propagation for Mesomelaena pseudostygia a difficult-to-propagate dryland sedge species (Cyperaceae) endemic to Western Australia is described. Multiple avenues to in vitro propagation were investigated: shoot culture, organogenesis and somatic embryogenesis, with zygotic embryos as initiation material. The highest multiplication rate for shoots was 3.4?±?1.0 after 6 wk on basal medium (1/2 strength Murashige and Skoog) with 2.5 μM kinetin and 0.5 μM 6-benzylaminopurine. Shoots achieved peak rooting (83%) following a pulse treatment on basal medium containing 10 μM indolebutyric acid and 2 μM α-naphthaleneacetic acid for 7 wk, followed by transfer to medium (without growth regulators) for a further 7 wk. Alternatively, in vitro grown shoots were pulse treated on basal medium with both 100 μM indolebutyric acid and 20 μM α-naphthaleneacetic acid for 1 wk then placed in Rockwool plugs (under propagation house conditions) for another 7 wk resulting in 63% root induction. Rooted plantlets were also successfully transferred to potting mixture either in Rockwool plugs or bare rooted and maintained in propagation house conditions with ≥95% survival after 7 wk. These results indicate that micropropagation of M. pseudostygia is feasible for small to medium scale restoration purposes. The highest frequency of callus induction was from cultured zygotic embryos on basal medium with 5 μM α-naphthaleneacetic acid, whereas 2,4-dichlorophenoxacetic acid (2 or 5 μM) produced the largest callus sizes. A low frequency of shoot regeneration occurred in zygotic callus tissues in basal medium treatments containing cytokinin (kinetin or thidiazuron at 1 μM). A small proportion (<20%) of zygotic embryo callus explants from 2,4-dichlorophenoxyacetic acid treatments were found to be embryogenic, firstly developing embryo-like structures after 2 wk on basal medium (minus plant growth hormones), that continued to develop with approximately one in twenty germinating after a further 4 wk on basal medium to form small plantlets. Further optimisation is needed to improve somatic embryogenesis efficiency for mass propagation.     

Quantification of arabinogalactan proteins during in vitro morphogenesis induced by β-d-glucosyl Yariv reagent in Centaurium erythraea root culture

Arabinogalactan proteins (AGPs) are a family of highly glycosylated cell surface proteins located at the plasma membrane and plant cell wall. AGPs play important roles in plant growth and development. Yariv phenylglycoside (βGlcY), synthetic red-brown dye that specifically binds and precipitates AGPs, has been used for detection and quantification of AGPs in plant tissue. Graded concentrations of βGlcY (0–75 μM) were used to investigate the effect of this synthetic dye on induction of in vitro morphogenesis in Centaurium erythraea root culture on two nutrient media: ½MS and ½MS + IBA 1.0 μM. Regeneration of C. erythraea shoots on root explants was stimulated on both media supplemented with 25 μM βGlcY after 8 weeks in culture. Quantification of AGPs in different tissues of C. erythraea was determinate with single radial diffusion method. This work emphasizes clear effect of βGlcY on induction of morphogenesis in vitro in C. erythraea root culture.     

Organogenesis of Phaseolus angularis L.: high efficiency of adventitious shoot regeneration from etiolated seedlings in the presence of N6-benzylaminopurine and thidiazuron

A step-wise procedure for the regeneration of fertile plants by organogenesis from cultures of the economically important Phaseolus angularis L., cultivars: KS-6, KS-7 and KS-8 using etiolated seedlings was established. Pre-culture of 5-day old seedling explants with MS (Murashige and Skoog (1962) Physiol Plant 15:473–493) + B₅-vitamins (Gamborg et al. (1968) Exp Cell Res 50:151–158) liquid medium containing either 5.0 μM TDZ or 5.0 μM BAP under dark condition was essential for organogenesis. Bud growth and shoot multiplication were stimulated by reducing the BAP concentrations from 5.0 to 2.5 μM after 3 weeks. The maximum frequency of shoot induction was 65.2% (33.8 ± 2.54 shoots/explant) in cultivar KS-8 followed by KS-7 34.6% (23.4 ± 1.91 shoots/explant) and KS-6 30.6% (21.2 ± 2.28 shoots/explant). The multiplied buds elongated after transferring to solid MSB₅ medium supplemented with 4.0 μM GA₃, 12.5 μM AgNO₃ and 0.4 μM IBA. Up to 98% rooting efficiency of was obtained when the shoots were pulse-treated with liquid medium containing 4.5 μM IBA for 10 min. The rooted plantlets were transferred to pots in the greenhouse, where they grew, mature, flowered and bared pod normally. The efficient shoot bud induction capability was found to be cultivar dependent. All the three cultivars tested formed multiple shoots. This efficient and rapid regeneration system may also be helpful for Agrobacterium- or particle gun-mediated transformation for this important legume crop.     

Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting

An efficient and improved method for in vitro propagation of mature tree of Dalbergia sissoo, an ecologically and commercially important timber yielding species, has been developed through axillary shoot proliferation. Bud breaking occurred from nodal shoot segments derived from rejuvenated shoots produced during early spring from a 20–25-year-old lopped tree, on MS medium containing 8.88 μM benzylaminopurine (BAP). Multiple shoots differentiated (20–21shoots/node) on re-culture of explants on half-strength agar gelled amended MS medium with a combination of 2.22 μM of BAP and 0.002 μM of thidiazuron (TDZ) with 1.0 mM each of Ca(NO₃)₂, K₂SO₄, KCl, and NH₄(SO₄)₂. The maximum shoot multiplication (29–30 shoots/node) was achieved on subculturing in the above mentioned but liquid medium. Furthermore, the problem of shoot tip necrosis and defoliation observed on solid medium were overcome by the use of liquid medium. Ex vitro rooting was achieved on soilrite after basal treatment of microshoots with 984 μM of indole-3-butyric acid (IBA) for 2 min. About 90 % microshoots were rooted on soilrite within 2–3 weeks under the greenhouse conditions. From 20 nodal shoot segments, about 435 hardened plants were acclimatized and transplanted. This is the first report for rapid in vitro propagation of mature trees of D. sissoo on liquid medium followed by ex vitro rooting.     

Light-induced production of antidepressant compounds in etiolated shoot cultures of Hypericum hookerianum Wight & Arn. (Hypericaceae)

Hypericum hookerianum is a lesser known ethnomedicinal plant having wound healing, antitumor and anti-HSV-1 properties. Isolated nodes of in vitro shoots sub-cultured in the dark for 4 weeks on half strength Murashige and Skoog medium solidified with Gelzan (1.5 g l^?1), and supplemented with 2.325 μM kinetin produced 8.0 ± 0.40 etiolated shoots of 5.0 ± 0.62 cm length at 74 % efficiency versus 9.2 ± 0.6 healthy shoots of 4.4 ± 0.5 cm obtained from nodes in light at 96 % efficiency. Low concentrations of hypericin were found in wild plant [0.35 ± 0.09 mg g^?1 dry weight (DW)] and control green shoot cultures (0.91 ± 0.03 mg g^?1 DW). Etiolated shoots exposed to a 12 h photoperiod (50 μmol m^?2 s^?1) through 1–25 days turned red incrementally due to synthesis and accumulation of 0.1–3.83 mg g^?1 DW hypericin in sub-epidermal cortical cells of the stem and varied shaped cells of the distorted mesophyll. Flavonoid and anthocyanin concentrations of the etiolated shoots subjected to the 12 h photoperiod were 3–5 fold higher than the control shoot cultures while total chlorophylls [1.97 ± 0.05 mg g^?1 fresh weight (FW)] of the light exposed shoots were significantly less compared to the control (2.86 ± 0.18 mg g^?1 FW) and natural plant (6.82 ± 0.29 mg g^?1 FW). HPLC analysis of shoot extracts revealed the presence of 0.14 ± 0.03, 0.16 ± 0.02 and 1.45 ± 0.16 mg g^?1 DW hyperforin in wild plant, control shoot cultures and etiolated shoot cultures illuminated for 25 days, respectively. Despite a reasonable presence in etiolated shoots (0.61 ± 0.15 g^?1 FW), total phenols did not increase significantly during illumination. The results indicate light induced synthesis of anti-depressant phenolic derivatives (hypericin, hyperforin and flavonoids) in etiolated shoot cultures of H. hookerianum.     

Rapid in vitro multiplication and ex vitro establishment of Caribbean copper plant (Euphorbia cotinifolia L.): an important medicinal shrub

An efficient micropropagation protocol was developed for E. cotinifolia by utilizing mature nodal segments for axillary shoot proliferation. The nodal explants from a 2-year-old plant were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations (0.5, 2.5, 5.0, 7.5 and 10.0 μM) of 6-benzyladenine (BA), Kinetin and 2-isopentenyl adenine singly as well as in combination with α-naphthalene acetic acid (NAA) or Indole-3-butyric acid (IBA) (0.1, 0.5 and 1.0 μM). The highest regeneration frequency (92 %) with multiple shoots (13.0 ± 1.15) and shoot length (4.23 ± 0.14 cm) was achieved on MS medium supplemented with 5.0 μM BA and 0.1 μM NAA after 8 weeks of culture. Further experiments were performed to test the effects of medium type, medium strength, pH and subculture passages on shoot induction and proliferation. An enhancement in average number of shoots (16.6 ± 0.45) per explant was obtained after four subculture passages. Micro shoots exhibited in vitro rooting on half strength MS medium containing 2.5 μM Indole-3-butyric acid (IBA) after 4 weeks of culture. The in vitro raised healthy plantlets with well-developed roots and shoots were successfully acclimatized in plastic cups containing sterile soilrite for 8 weeks under culture room conditions (150 PPFD) prior to field transfer. Through the acclimatization period (0–56 days), photosynthetic pigments (Chlorophyll a, b and Carotenoid content) decreased during the initial 2 weeks followed by significant increase during the successive period (21–56 days) of acclimatization. At the same time, all the tested antioxidant enzymes (SOD, CAT, APX and GR) exhibited an increasing trend throughout the acclimatization period. The culture room acclimatized plantlets were successfully established in earthen pots containing garden soil in greenhouse with 70 % survival rate.