<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.     

Hairy root induction and plant regeneration of medicinal plant Dracocephalum kotschyi

An efficient hairy root induction system for an important endangered medicinal plant, Dracocephalum kotschyi, was developed through Agrobacterium rhizogenes-mediated transformation by modifying the co-cultivation medium using five bacterial strains, A4, ATCC15834, LBA9402, MSU440, and A13 (MAFF-02-10266). A drastic increase in transformation frequency was observed when a Murashige and Skoog medium lacking NH₄NO₃ KH₂PO_4, KNO₃ and CaCl₂ was used, resulting in hairy root induction frequencies of 52.3 %, 69.6 %, 48.6 %, 89.0 %, and 80.0 % by A4, A13, LBA9402, MSU440, and ATCC15834 strains, respectively. For shoot induction, hairy roots and unorganized tumors induced by strain ATCC15834 were placed on an MS media supplemented with 0.1, 0.25, 0.5, and 1 mg/l BA plus 0.1 mg/l NAA. The high frequency of shoot regeneration and number of shoot were obtained in the medium containing 0.25 mg/l BA and 0.1 mg/l NAA. Root induction occurred from the base of regenerated shoots on the MS medium supplemented with 0.5 mg/l IBA after 10 days.     

An efficient in vitro system for somatic embryogenesis and podophyllotoxin production in Podophyllum hexandrum Royle

Podophyllum hexandrum Royle known as Indian mayapple is an important medicinal plant found only in higher altitudes (2,700 to 4,200 m) of the Himalayas. The highly valued anticancer drug Podophyllotoxin is obtained from the roots of this plant. Due to over exploitation, this endemic plant species is on the verge of extinction. In vitro culture for efficient regeneration and the production of podophyllotoxin is an important research priority for this plant. Hence, in the present study, an efficient plant regeneration system for mass multiplication through somatic embryogenesis was developed. We have screened P. hexandrum seeds collected from three different regions in the Himalayas to find their regenerative potentials. These variants showed variation in germination percentage as well as somatic embryogenic frequency. The seeds collected from the Milam area of Pithoragarh district showed better germination response (99.3 %) on Murashige and Skoog (MS) medium fortified with Gibberellic acid (GA₃ [5 mg/l]) and higher direct somatic embryogenic frequency (89.6 %). Maximum production of embryogenic callus (1.2 g fresh weight [FW]) was obtained when cotyledons containing the direct somatic embryo clusters were cultured in MS medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D [1.5 mg/l]) after 4 week of culture in complete darkness. In the present investigation, somatic embryogenesis was accomplished either by direct organogenesis or callus mediated pathways. The latter method resulted in a higher frequency of somatic embryo induction in hormone-free MS medium yielding 47.7 embryos/50 mg of embryogenic callus and subsequent germination in MS medium supplemented with GA₃ (5 mg/l). Seventy-nine percent of embryos attained complete maturity and germinated into normal plants with well-developed roots. Systematic histological analysis revealed the origin of somatic embryo and their ontogenesis. The higher level of podophyllotoxin (1.8 mg/g dry weight [DW]) was recorded in germinated somatic embryos when compared to field grown plants. The present system can be widely used for mass propagation, transgenic recovery, and podophyllotoxin production for commercial utilization.     

Embryogenesis,plant regeneration and cardiac glycoside determination in Digitalis ferruginea subsp. ferruginea L

The present study reports, for the first time, an efficient in vitro plant regeneration protocol for Digitalis ferruginea subsp. ferruginea L. (rusty foxglove). We have used different concentrations of gibberellic acid (GA₃) on Murashige and Skoog (MS) medium to assess the germination frequency of seeds. High frequency of germination was achieved on MS medium with 1.0 mg l^?1 GA₃. 6-Benzylaminopurine (BAP) combined with α-naphtaleneacetic acid (NAA) or 2, 4-dichlorophenoxy acetic acid (2, 4-D) in the induction MS medium induced both somatic embryogensis and shoot organogenesis. The highest percentage of callus growth (85 %) was obtained when hypocotyl explants were cultured on MS medium containing 0.5 mg l^?1 2, 4-D plus 1.0 mg l^?1 BAP. The maximum mean number of somatic embryos (7.3 ± 1.3 embryos) or shoots (12.0 ± 1.1 shoots) per callus was obtained when medium contained 0.25 mg l^?1 NAA plus 1.0 mg l^?1 BAP or 0.5 mg l^?1 NAA plus 2.0 mg l^?1 BAP. The regenerated shoots easily rooted on MS medium. Higher amounts of lanatoside C [13.2 ± 0.5 mg 100 g^?1 dry weight (dw)] and digoxin (2.93 ± 0.31 mg 100 g^?1 dw) accumulation were obtained when shoots were obtained by indirect regeneration. We also investigated derivatives of cardenolides, i.e., digitoxigenin (730 ± 180 mg 100 g^?1 dw), gitoxigenin (50 ± 20 mg 100 g^?1 dw) and digoxigenin (490 ± 170 mg 100 g^?1 dw) from natural samples.     

In vitro propagation and assessment of genetic stability of micropropagated Samanea saman (rain tree) using microsatellite markers

An efficient in vitro propagation of Samanea saman (rain tree) protocol has been successfully developed using nodal explants from a 20-year-old tree. Higher percentage (76 %) of explants produced up to five shoots per explant on Murashige and Skoog (MS) medium supplemented with 2 mg L^?1 6-benzyladenine (BA), 0.1 mg L^?1 gibberellic acid (GA₃) and 100 mg L^?1 casein hydrolysate after 3 weeks of culture. When explants were subcultured to fresh medium after harvesting first batch of shoots, more shoots could be generated (another eight shoots per explant). Shoot elongation was achieved (3 cm) when shoots were cultured on MS medium supplemented with 0.25 mg L^?1 BA and 0.75 mg L^?1 GA₃. In vitro generated shoots rooted on MS medium fortified with 0.75 mg L^?1 indole-3-butyric acid plus 0.1 % of activated charcoal. A higher percentage of explant response and shoots per explant were obtained on MS medium with BA and GA₃. Each responsive nodal explant yields an average of 15 rooted plants within a period of 10 weeks. Rooted plantlets were successfully acclimatized in green house with a survival rate of 90 %. Micropropagated plants were tested for genetic stability using simple sequence repeats (SSR) markers. Use of the 12 high-resolution SSR markers revealed the exact same genetic profile between the mother tree (donor) and micropropagated plants, suggesting the genetic fidelity of our micropropagation protocol. The same protocol was also used successfully in propagating a “Golden Rain Tree” although response of explant and efficiency of propagation was much lower. This protocol will be useful for germplasm preservation/large scale production of true-to-type clones of desirable genotypes.     

In vitro mass propagation and conservation of Nilgirianthus ciliatus through nodal explants: A globally endangered,high trade medicinal plant of Western Ghats

Nilgirianthus ciliatus is a globally endangered aromatic slender shrub of Western Ghats with extensive applications in Ayurveda. It is endangered due to its indiscriminate collection and overexploitation to meet the requirements of the pharmaceuticals. This study deals with the preservation of this endangered plant through in vitro nodal culture. Nodal explants were initially cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BA) or 6-furfurylaminopurine individually for primary shoot induction. For multiple shoot induction, primary shoots were transferred onto MS medium containing BA individually or in combination with auxins. Clusters of multiple shoots (up to 24.3) occurred with highest frequency (93.2%) on MS medium fortified with BA (3 mg l^?1) and indole-3-acetic acid (0.1 mg l^?1). In vitro regenerated plantlets were rooted on half-strength MS medium with maximum rooting frequency (82.2%) obtained in the presence of indole-3-butyric acid (1.0 mg l^?1). The rooted plantlets were acclimatized to soil with 100% survival rate. Results of this study allowed us to develop an efficient regeneration system that will permit to carry out restoration programmes of N. ciliatus in Western Ghats. In future, this protocol will be an invaluable tool to produce synthetic seeds for cryopreservation and long-term conservation.     

Direct and indirect shoot and bulblet regeneration from cultured leaf explants of Lilium pumilum,an endangered species

Alternative methods of in vitro cloning that involve both adventitious (direct) and callus intermediate (indirect) pathways were investigated for the endangered species Lilium pumilum. Plantlet regeneration was obtained from leaf explants, cultured on Murashige and Skoog (MS) basal medium supplemented with various combinations of auxins and cytokinins at different concentrations. About 30% of the explants directly formed adventitious shoots on MS medium containing 8.88 μM 6-benzyladenine (BA) and 2.69 μM α-naphthaleneacetic acid (NAA). For production of regenerable callus, callus formation followed by shoot induction was best when explants were initially cultured on MS medium supplemented with 9.05 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Regenerable calli were yellow or purple and readily regenerated shoots when subcultured onto MS medium containing 2.22 μM BA and 1.61 μM NAA. About 78% of the calli were able to produce adventitious shoots. Shoots were rooted on half-strength MS medium supplemented with 1.34 μM NAA and were successfully acclimatized to greenhouse conditions. This report describes an efficient method for the in vitro multiplication of whole plants from leaf explants of the endangered species L. pumilum.     

Somatic embryogenesis and synthetic seed production in Rhinacanthus nasutus (L.) Kurz.

Young healthy cotyledon and leaf explants of Rhinacanthus nasutus (L.) Kurz. were incubated on Murashige and Skoog (MS) medium supplemented with 1.0–5.0 mg/l 2, 4-dichlorophenoxyacetic acid (2,4-D) either alone or in combination with 0.3–1.5 mg/l indole-3-butyric acid (IBA). The optimum callus induction (100 %) was observed from cotyledon explants on MS medium supplemented with 4 mg/l 2, 4-D and 0.5 mg/l IBA. The friable, embryogenic callus when subcultured on half strength MS medium supplemented with IBA (3.0–5.0 mg/l) produced several somatic embryos at various stages of development (globular, heart, torpedo) after 45 days of culture. The highest frequency of callus embryogenesis was observed on ½MS medium supplemented with 4.0 mg/l IBA. Moreover, 47 % of incubated callus responded with a mean number of 16.3 somatic embryos per gram callus. For germination, somatic embryos at the torpedo stage were isolated and subcultured on ½MS medium supplemented with 0.5 mg/l each of 6-benzyladenine and indole-3-acetic acid. After 45 days of culture, plantlets developed with mean lengths of 3.8 cm. Somatic embryos at the torpedo stage were collected and suspended in a matrix of MS medium containing sodium alginate (3 % W/V), dropped into 100 mM calcium chloride (CaCl₂·2H₂O) solution for the production of synthetic seeds. Optimum growth ability of synthetic seed was obtained on MS medium supplemented with 0.2 mg/l gibberellic acid (GA₃). Well developed healthy plantlets derived from somatic embryos and synthetic seeds were hardened and successfully transplanted to soil.     

Effects of sodium nitroprusside on shoot multiplication and regeneration of Vanilla planifolia Andrews

Nitric oxide (NO) plays diverse roles in the growth and development of plants. The effects of a NO donor, sodium nitroprusside (SNP), on shoot multiplication and regeneration of Vanilla planifolia Andrews have been studied. Nodal segments of V. planifolia were used as explants to initiate shoots. The number of shoots per explant showed a significant increase in the presence of SNP and more than 93% of explants formed shoots. Supplementation of 10.0 μM SNP to Murashige and Skoog (MS) basal medium containing 1.0 mg/L 6-benzylaminopurine (BAP) produced the highest number of shoots per explant (10.33) after 60 d of culture. However, in this treatment, shoot length (3.76 cm) was less than in the other treatments, except for the plant growth regulator-free MS medium. MS medium containing only 1.0 mg/L BAP produced the highest shoot length (4.49 cm) with a mean number of 6.26 shoots per explant. These findings indicate that NO stimulated shoot development and may be considered as an intermediary of adventitious shoot regeneration, as has been suggested for other plant species.     

<Emphasis Type="Italic">In vitro</Emphasis> effects of GA<Subscript>3</Subscript> on morphogenesis of CIP potato explants and acclimatization of plantlets in field

Improvement of potato has been accomplished using conventional and non-conventional approaches coupled with numerous tissue culture procedures. The aim of the present study was to assess the efficacy of gibberellic acid (GA₃) on the morphogenesis of International Potato Center (CIP) potato explants and acclimatization of plantlets in the field. Nodal segments as an explant source (1–1.5 cm) were isolated from 31 CIP potato plantlets and were inoculated into Murashige and Skoog (MS) medium supplemented with 0.0 (control), 0.1, 0.5, or 1.0 mg L^?1of GA₃. The variation in growth parameters of the cultivars was then observed. The highest shoot induction occurred in MS medium containing 1.0 mg L^?1 GA₃ with an increase in the inter-nodal distance between nodes as compared to other treatments. Higher concentration (1.0 mg L^?1) of GA₃ significantly increased plant height and root length in the treated germplasm however; this concentration was inhibitory to the number of nodes and roots per plant. The number of leaves was significantly increased in plants receiving GA₃ treatment at lower concentration (0.1 mg L^?1). The 31 CIP genotypes were transplanted to the field and checked for yield quality traits. It was concluded from the results that GA₃ had significant effects on morphogenesis and was effective in the acclimatization of CIP potato plantlets in field.     

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.     

Optimization of in vitro and ex vitro regeneration and micromorphological studies in <Emphasis Type="Italic">Basella alba</Emphasis> L.

The optimum concentrations of the plant hormones for in vitro regeneration and subsequent effect of auxins on rooting (in vitro and ex vitro) of shoots of Basella alba L. have been investigated in present study. Nodal shoot segments were used as explants to initiate the cultures. The bud breaking from explants was observed within 1 week of incubation on agar gelled Murashige and Skoog’s (MS) medium. Multiple axillary shoots (7.30 ± 0.56 shoots per explant) were induced on MS medium supplemented with 2.0 mg/L 6-benzylaminopurine (BAP). The shoots were multiplied (maximum 17.10 ± 0.44 shoots per explant) on the same medium fortified with 0.5 mg/L each of BAP and Kin (Kinetin) +0.1 mg/L IAA. These shoots were excised and rooted in vitro (10.73 ± 0.92 roots per shoot) on half-strength MS medium augmented with 2.0 mg/L indole-3 butyric acid (IBA). Hundred percentage success rates have been achieved by ex vitro rooting of the in vitro regenerated shoots with IBA at 300 mg/L. The in vitro and ex vitro rooted shoots were acclimatized in greenhouse and subsequently transferred to the natural field conditions where 100 % survival rate was reported. The ex vitro rooting method was found more advantageous than in vitro rooting in terms of time, energy and survival percentage of B. alba. A comparative foliar micromorphological study of B. alba was conducted to understand the micromorphological changes in plants while shifting from in vitro to the in vivo conditions in terms of variations in stomatal index, venation pattern and vein density, and the arrangement of crystals. The study could help in understanding the response of in vitro raised plants towards in vivo environment.     

An efficient in vitro plant regeneration system for the medicinal plant Teucrium stocksianum Boiss.

An efficient and rapid plant regeneration system via direct organogenesis was established for Teucrium stocksianum Boiss. (Lamiaceae), an endangered and valuable medicinal plant. Hypocotyl explants excised from seedlings germinated in vitro were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of kinetin and indoleacetic acid (IAA) to induce shoot formation. Differentiation of multiple shoots was initiated within 3 weeks of culture. Optimal regeneration was achieved on medium containing 3 mg/l kinetin and 0.5 mg/l IAA. This particular medium composition significantly improved the production of multiple shoots directly from hypocotyl explants compared to other combinations of plant growth regulators. Root induction was achieved on half-strength MS medium containing indole-3-butyric acid. Rooted plantlets were successfully acclimatized, with a survival rate of 75–80%. The protocol developed in this study could be used for long-term in vitro conservation and mass propagation of this species.     

In vitro shoot regeneration from leaf explants of Echinops kebericho: an endangered endemic medicinal plant

Echinops kebericho is a critically endangered endemic medicinal plant of Ethiopia. It is threatened due to over harvesting of its roots for medicinal purposes and from poor seed viability. This study aimed to develop a protocol for in vitro shoot regeneration from leaf explants of E. kebericho. The seeds were sterilized using ethanol followed by Clorox or calcium hypochlorite. Shoots from the germinated seeds were cultured on Murashige and Skoog (MS) medium containing different concentrations of α-naphthalene acetic acid (NAA) and 6-benzyl amino purine (BAP). Young leaves were cultured on MS medium containing different concentrations of BAP and NAA for shoot regeneration. For shoot multiplication, shoots were excised and cultured on MS medium containing different concentrations of BAP or kinetin (KIN) and NAA. The highest mean number of initiated shoots (4.00 ± 0.57) with 100% shoot induction was obtained on medium containing 1.0 mg/L BAP and 0.2 mg/L NAA. The highest shoot regeneration (33%) and shoot number (2.13 ± 0.06) were obtained on MS medium containing 2.0 mg/L BAP and 0.5 mg/L NAA. Medium containing 1.0 mg/L KIN and 0.2 mg/L NAA produced the highest number of shoots (4.67 ± 0.33) per explant. This protocol can be used for genetic improvement and conservation of this endangered species.     

Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

In vitro propagation of an endemic and endangered medicinal plant <Emphasis Type="Italic">Notopterygium incisum</Emphasis>

An efficient system in vitro propagation for Notopterygium incisum Ting ex H. T. Chang, an endemic and endangered medicinal plant, was established to address increased demand and germplasm conservation goals. Optimum response in callus induction (CI) was observed on Murashige and Skoog (MS) medium supplemented with 1.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) and 0.2 mg/l 6-benzylaminopurine (BAP), which the induction rate and growth of callus were 84.44% and 0.67 g respectively. The highest shoot regeneration frequency (76.97%) and maximum number of shoots (3.6 shoots per callus) were achieved on MS medium with 1.5 mg/l BAP and 0.2 mg/l naphthalene acetic acid (NAA). Half-strength MS medium supplemented with 0.6 mg/l indole-3-butyric acid (IBA) was determined to be the best rooting medium, resulting in the maximum number of roots (18.6 roots per shoot) and the highest rooting frequency (92.28%). An approximate 83.8% survival rate among the regenerated plantlets was recorded after they were transplanted in the field at an altitude of 3200 m. An HPLC analysis showed that the content of two main chemical constituents, notopterol and isoimperatorin, in the rhizomes of 3-year-old regenerated plantlets was higher (3.84 mg/g and 4.05 mg/g, respectively) than that in commercially marketed crude drugs. This first report of complete regeneration in vitro could provide an alternative method for the rapid, large-scale production and conservation of this valuable, rare, and endangered medicinal plant.     

AgNO3 boosted high-frequency shoot regeneration in Vigna mungo (L.) Hepper

In order to further increase shoot regeneration frequency of Vigna mungo (L.) Hepper., the effects of AgNO₃ on this process was investigated in this study. The shoot tip and cotyledonary node explants were cultured on MS salts B5 Vitamins medium containing BA+TDZ+Ads+AgNO₃ for multiple shoot induction. AgNO₃ influenced the shoot bud formation and their subsequent proliferation. The best medium composition for multiple shoot induction was BA, TDZ combination with Ads and AgNO₃ in MSB5 medium. Maximum 39 shoots in cotyledonary node and 22 shoots in shoot tip were obtained per explants after 4 – 6 wk. of culture. Elongation and rooting were performed in GA₃ (0.6mg/l) and IBA (0.4mg/L) containing media respectively. The in vitro raised plantlets were acclimatized in green house and successfully transplanted to the field with a survival rate of 78%.     

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.     

Micropropagation of the Mongolian medicinal plant <Emphasis Type="Italic">Zygophyllum potaninii</Emphasis> via somatic embryogenesis

The Mongolian medicinal plant Zygophyllum potaninii has been assessed as an endangered species with regional status. We applied the somatic embryogenesis technique using aseptic in vitro germinants of the plant as an effective propagation technology. The seed germination rate in vitro was 16.5% after 2 weeks of culture. Embryonic calli (EC) and somatic embryos (SEs) were induced using the cotyledon or hypocotyl segments of the germinants. Calli were effectively induced on MS medium supplemented with 0.1 mg/L 2, 4-dichlorophenoxy acetic acid (2, 4-d) and 0.5 mg/L 6-benzylamino purine (BA). The callus was composed of pale yellow or pale green friable cells. SE formed from EC only on Murashige and Skoog medium (MS) with 0.5 mg/L abscisic acid (ABA). Other concentrations of ABA failed to induce SE formation. All SEs germinated in MS medium with different salt levels. However, normal plant conversion was achieved only on half-strength MS medium. The converted plantlets were effectively acclimatized in vitro in sand and transferred to a mixture of sand and perlite (1:1 v/v) in the greenhouse. After 8 weeks of culture, 55.4% of the plants survived. This is a first report of propagating the medicinal desert plant Z. potaninii via somatic embryogenesis and plant regeneration.     

Micropropagation of a fruit tree, Morus australis Poir. syn. M. acidosa Griff.

High frequency bud break and multiple shoots were induced in nodal explants collected between November to February from a 5 year old tree of Morus australis Poir syn. M. acidosa Griff. on Murashige and Skoog's medium supplemented with 6-benzylaminopurine (1.0 mg/1). Incorporation of gibberellic acid (0.3 mg/l) along with BAP (1.0 mg/l) not only induced faster bud break from nodal explants as well as from apical shoot buds, but it also enhanced the frequency of bud break. Nodal explants were more responsive than apical shoot buds. The shoots formed in vitro were multiplied further as nodal segments, and an average multiplication rate of 6-fold per subculture was established within 4–5 months. The shoots were successfully rooted on half-strength MS containing a combination of indole-3-acetic acid, indole-3-butyric acid and indole-3-propionic acid, each at 1.0 mg/1. The plantlets were successfully hardened off and established in natural soil.Abbreviations BAP 6-benzylaminopurine - GA₃ gibberellic acid - KN kinetin - IAA indole-3-acetic acid - IBA indole-3-butyric acid - IPA indole-3-propionic acid - MS Murashige and Skoog (1962) medium - NAA 1-naphthalene acetic acid