Induction of somatic embryogenesis and analysis of genetic fidelity of in vitro<Emphasis Type="Italic">-</Emphasis>derived plantlets of <Emphasis Type="Italic">Bambusa nutans</Emphasis> Wall., using AFLP markers

Bambusa nutans Wall., is an evergreen, perennial, and multipurpose bamboo having strong culms, which are largely used for construction, scaffolding, craft purposes, pulp, and paper industry. Multiple shoots from nodal segments (3–4 cm) of young branches of mature culms were established in Murashige and Skoog (1962) (MS) medium supplemented with various concentrations of 6-benzylaminopurine (BAP) (1.0–6.0 mg l⁻¹) or in combination with α-naphthaleneacetic acid (NAA) (0.5–1.0 mg l⁻¹) or kinetin (Kn) (1.0–2.0 mg l⁻¹). February–March and December were found to be the best seasons for culture establishments. Maximum shoots were achieved on MS medium fortified with BAP (2.0 mg l⁻¹). Embryogenic callus (slightly greenish compact, globular, and slow growing) was initiated from the base of severed sprouted buds in 2–3 subsequent subcultures on MS medium supplemented with 2,4-dichlorophenoxy acetic acid (2,4-D) (5.0 mg l⁻¹) under dark incubations. Maturation and germination of well-organized somatic embryos was achieved on MS medium containing BAP and 2,4-D (1.0 mg l⁻¹ each) with 20.0 mg l⁻¹ ascorbic acid. Full-strength MS medium supplemented with 2% glucose favored further development of proliferated somatic embryos into plantlets. Genetic variations of field-established B. nutans plants regenerated through tissue cultures were assessed by amplified fragment length polymorphism (AFLP) analysis using 6 primer combinations. Four hundred and seven scorable fragments were amplified, of which 402 (98.8%) have recorded conservation at various morphogenetic stages leading to plantlets regeneration, therefore, revealed a high level of genetic stability.     

An efficient micropropagation system for Celastrus paniculatus Willd.: a vulnerable medicinal plant

A micropropagation protocol was developed for Celastrus paniculatus, a vulnerable medicinal plant. Cultures were initiated from nodal explants collected from young shoots of a 12-year-old plant in MS basal medium. An average of five shoots were produced in MS medium supplemented with 1.5 mg l⁻¹ benzyl adenine (BA) and 0.1 mg l⁻¹ naphthalene acetic acid (NAA) after two subculture cycles with a 30-day interval. Continuous subculture in the same medium for three more cycles resulted in reduction of the number of multiple shoots (2 or 3 shoots), vitrification of the shoots, and callus formation. Vitrification of cultures could be overcome by the use of MS medium supplemented with lower concentrations of BA (0.05 mg l⁻¹) and NAA (0.01 mg l⁻¹). Among the various rooting trials, ex vitro rooting of shoots with simultaneous hardening was most efficient. The method standardized in the present study is simple, as it eliminated separate steps for in vitro rooting and hardening. Qualitative chemical similarity of the tissue culture regenerants with the mother plant was confirmed using high performance thin-layer chromatographic (HPTLC) profiling.     

Micropropagation of <Emphasis Type="Italic">Salvadora</Emphasis><Emphasis Type="Italic">persica</Emphasis>-a tree of arid horticulture and forestry

A micropropagation method for Jaal (Salvadora persica)—a tree of arid horticulture and forestry has been developed. Nodal segments of fresh shoot sprouts originated from axillary buds obtained from a plant around 35–40 years old lopped plant were used as explants for establishment of in vitro cultures. Surface-sterilized explants produced optimum number of shoots through activation of axillary buds on Murashige and Skoog’s (MS) medium containing 8.88 μM BA (6-benzyladenine) + additives (25 mgl⁻¹ each of adenine sulphate, arginine, citric acid, 50 mgl⁻¹ ascorbic acid). The shoot multiplication was influenced by the successive transfer of the mother explants for 4–5 passages. The maximum number (23.1 ± 0.73 shoots per explant) of shoots were regenerated on MS supplemented with 1.11 μM BA + 1.16 μM Kn (Kinetin) + 0.54 μM NAA (α-naphthalene acetic acid). About 90% shoots pulse-treated with a combination of 2460.27 μM Indole-3-butyric acid (IBA) + 494.56 μM NOA (2-naphthoxy acetic acid) were rooted ex vitro on soilrite within 15–18 days. Over 80% cloned plantlets were hardened successfully in a green house and transferred to polybag/pots.     

Regeneration of <Emphasis Type="Italic">Melia volkensii</Emphasis> Gürke (Meliaceae) through direct somatic embryogenesis

Experiments were conducted to study plant regeneration through direct somatic embryogenesis using mature zygotic embryo and cotyledonary explants from seeds of Melia volkensii stored for <3 and >12 months. Explants were cultured on Murashige and Skoog (MS) medium supplemented with BAP, NAA and 2,4-D (0.5, 1.0 and 2.0 mg l⁻¹) alone, and BAP (0.5, 1.0, 2.0 and 4.0 mg l⁻¹) in combination with 2,4-D or NAA (0.2 and 0.5 mg l⁻¹). After 4 weeks in culture, up to 60% of cotyledonary explants from the seeds stored for <3 months produced direct somatic embryos on BAP (0.5–4.0 mg l⁻¹) in combination with 2,4-D (0.2 mg l⁻¹). The number of somatic embryos ranged from 5 to 14 per explant in BAP (0.5 mg l⁻¹) and 2,4-D (0.2 mg l⁻¹) combination. Only 20% of cotyledonary explants from seeds stored for >12 months produced somatic embryos. Mature zygotic embryos failed to produce any somatic embryos. Subcultures of somatic embryos from cotyledonary explants of seeds stored for <3 months formed clusters of shootlets on semi solid MS and 1/2 MS media. After 6 weeks of subculture on multiplication MS media augmented with BAP (0.5 mg l⁻¹) and IAA (0.2 mg l⁻¹), 70% of the shoot tips formed 4–7 shoots per explant. Up to 33% of the multiplied shoots were rooted in MS medium supplemented with 2.0 mg l⁻¹ IBA. Plantlets developed normally into seedlings in the greenhouse.     

In vitro clonal propagation of Gardenia latifolia Ait.: a toy making woody tree

An efficient, in vitro clonal propagation protocol has been established for Gardenia latifolia Ait. using mature nodal explants on Murashige and Skoog (MS) medium fortified with cytokinins (BA/Kn/2-iP) (1.0–5.0 mg l^?1) in combination with auxin IAA (0.5 mg l^?1). Maximum bud break (87 %) with shoot number (7.2 ± 0.26) observed on MS medium supplemented with BA (4.0 mg l^?1) and IAA (0.5 mg l^?1). Maximum number of shoots (30 ± 0.46) with shoot length of (0.9 ± 0.03 cm) observed on MS medium supplemented with BA (2.0 mg l^?1), Kn (2.0 mg l^?1) and IAA (0.5 mg l^?1). Further elongation of shoots (3.5 ± 0.06 cm) was achieved on MS medium supplemented with BA (1.0 mg l^?1) and IAA (0.1 mg l^?1). About 70 % of root induction occurred in half-strength MS medium supplemented with IBA (4.0 mg l^?1) in 4–6 weeks. Further elongation of roots with average length (9.0 cm) was achieved in culture bottles containing vermiculite and ¼ strength MS salts. After their partial hardening in these bottles for 30 days they were transferred to pots containing a mixture of soil and vermicompost (1:1) for acclimatization. The acclimatized plantlets were established in the field successfully with 85 % survival rate.     

Efficient in vitro regeneration of <Emphasis Type="Italic">Leucaena leucocephala</Emphasis> using immature zygotic embryos as explants

A method for efficient in vitro regeneration of Leucaena leucocephala cv. K636 was developed using immature zygotic embryos as the explant. Multiple shoot induction was achieved by culturing the explants on half-strength Murashige and Skoog (MS) medium supplemented with thidiazuron (TDZ) (0.03 to 1.5 μM). The maximum number of shoots per explants was achieved in 0.26 μM TDZ-supplemented media. TDZ concentration significantly influenced the induction of multiple shoots as well as the shoot-length. TDZ at 0.35 μM or higher concentrations resulted in abnormal stunted shoots. Full-strength MS media devoid of any plant growth regulator were used for shoot elongation. In vitro root induction was achieved in half-strength MS media supplemented either with 0.54 μM 1-naphthaleneacetic acid (NAA) or with 14.76 μM indole-3-butyric acid (IBA) in combination with 0.23 μM kinetin (Kn). Media supplemented with 14.76 μM IBA in combination with 0.23 μM Kn induced twofold–threefold more roots than media supplemented with 0.54 μM NAA. However, the average root length was lower in 14.76 μM IBA in combination with 0.23 μM Kn than in 0.54 μM NAA. Regenerated plants were maintained under normal condition after hardening. Plants, which were rooted on media supplemented with 14.76 μM IBA in combination with 0.23 μM Kn showed higher survival rate during hardening than those rooted on 0.54 μM NAA supplemented media.     

Thidiazuron-induced somatic embryos,their multiplication,maturation, and conversion in <Emphasis Type="Italic">Cinnamomum pauciflorum</Emphasis> Nees (Lauraceae)

Thidiazuron (TDZ) induced somatic embryogenesis from immature zygotic embryos in Cinnamomum pauciflorum Nees while 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzylaminopurine (BA) or picloram only induced callus and/or adventitious buds. The highest induction frequency for somatic embryogenesis was achieved with MS medium (Murashige and Skoog in Physiol Plant 15:473–497 1962) supplemented with 2.5 μM TDZ using torpedo-shaped embryos (3–5 mm in length) as explants. In addition, induction medium was supplemented with 0.8 g l⁻¹ casein, 0.4 g l⁻¹ glutamine, and 10 g l⁻¹ sucrose. Somatic embryos (SEs) initiated from root tips or hypocotyls without callus formation. SEs were maintained and multiplied via secondary somatic embryogenesis. Embryo maintenance medium was similar to induction medium except that TDZ was reduced to 0.5 μM. Secondary embryogenesis was enhanced by supplementation of 5 g l⁻¹ activated charcoal in the culture. The best medium for embryo maturation was MS medium containing 30 g l⁻¹ sucrose and 5 g l⁻¹ Phytagel without plant growth regulators. A typical mature SE consisted of two large cotyledons and a short embryo proper. Approximately 82% of selected mature SEs were able to germinate and 63% could convert into plantlets on germination medium that was composed of half strength MS medium salts, 10 g l⁻¹ sucrose, 3 g l⁻¹ Phytagel, and 5 g l⁻¹ activated charcoal.     

Direct plant regeneration from nodal explants of <Emphasis Type="Italic">Balanites aegyptiaca</Emphasis> L. (Del.): a valuable medicinal tree

This report describes in vitro shoot induction and plant regeneration from nodal segments of Balanites aegyptiaca on Murashige and Skoog (MS) medium fortified with 6-benzyladenine (BA), thidiazuron (TDZ) and kinetin (Kin) (0.5–20.0 μM). MS medium supplemented with BA (12.5 μM) was the most effective in inducing bud break and growth and also in initiating multiple shoot proliferation. However, the optimal level of TDZ supplementation to the culture medium was 5.0 μM. Shoot cultures were established by repeatedly subculturing the original nodal explants on the same medium. Highest number of shoots (11.5 ± 0.7) and shoot length (5.0 ± 0.2 cm) were achieved when cultures were subcultured on MS medium supplemented with 12.5 μM BA and 1.0 μM α-naphthalene acetic acid (NAA). The shoots regenerated from TDZ supplemented medium when subcultured to hormone free MS basal medium considerably increased the rate of shoot multiplication and shoot length by the end of fifth subculture. Rooting of the shoots was achieved on MS medium augmented with 1.0 μM indole-3-butyric acid (IBA) plus 0.5% activated charcoal followed by their transfer to half strength MS basal medium. The in vitro raised plantlets with well developed shoots and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse with 70% survival rate. The results of this study provide the first successful report on in vitro direct plant regeneration of B. aegyptiaca.     

An efficient method for clonal propagation and in vitro establishment of softwood shoots from epicormic buds of teak (Tectona grandis L.)

Softwood shoots were produced from 40 cm long stem segments placed horizontally in flat trays containing sterilized sand under natural light or shade conditions for subsequent rooting and micropropagation studies in teak (Tectona grandis L.). Higher number of shoots (6.17) per log was produced under natural light as compared to shade conditions. Forcing was also better in natural light as compared to shade in terms of shoot length, number of nodes or leaves. For rooting, 2–4 cm long softwood shoots were excised and treated with either indole-3-butyric acid (IBA) or α-naphthyl acetic acid (NAA) at 0, 1000, 2000 or 3000 μmol·L^–1 each or with combinations (1000 + 1000, 2000 + 2000 or 3000 + 3000 μmol·L^–1) and then placed in flat trays containing autoclaved sand at 25 ± 2ºC in 16 h photoperiod at 35 µmol·m^–2·s^–1. After 28 days, softwood cuttings treated with IBA + NAA (3000 + 3000 μmol·L–1) had highest rooting percentage (89.3%) with 5.5 mean roots. Shoot apex and nodal explants of softwood cuttings were pretreated with 0.1% (w/v) ascorbic acid, boric acid, activated charcoal, citric acid, glutamine or polyvinylpolypyrollidone (PVP) for 24 h to remove phenolic compounds before surface disinfestation. Glutamine (Gl) and PVP were equally effective resulting in 60% establishment of shoot apices on MS medium supplemented with 10 μmol·L–1 6-benzylaminopurine (BAP) + 5 μmol·L–1 NAA. Using shoot apices, highest (42.80) number of multiple shoots with 54.33 mm shoot length were obtained on MS + BAP (8.8 μmol·L–1) + IBA (2 μmol·L–1) after 45 days. Shoots were successfully rooted and acclimatized to greenhouse     

In vitro propagation of a medicinal plant: Tripterygium wilfordii Hook f.

In this study a reliable protocol was developed for the establishment of commercial in vitro cultures of Tripterygium wilfordii Hook f.. Juvenile shoots from one-year-old elite plants were used as the source of explants. New axillary shoots were obtained after 30 days of culture on a MS medium supplemented with BAP (2.0 mg·L^–1) and NAA (0.1 mg·L^–1). The optimal multiplication medium was a modified MS medium supplemented with BAP (1.0 mg·L^–1) and NAA (0.1 mg·L^–1). This yielded a multiplication rate of 2.4 for each subculture. Slightly more than 92% of shoots rooted when cultured on a modified MS medium containing IBA (0.2 mg·L^–1) and acti-vated charcoal (0.5 mg·L^–1). Activated charcoal promoted both a strong and a high rooting rate during the rooting phase. Plantlets were transferred to pots for a short acclimatization stage in a greenhouse where 95% of the plantlets survived. This highly reproduci-ble procedure can be adopted for large-scale propagation of T. wilfordii.     

In vitro plant regeneration system for <Emphasis Type="Italic">Cassia siamea</Emphasis> Lam., a leguminous tree of economic importance

A method for rapid in vitro propagation of Cassia siamea Lam. using cotyledonary node explants, excised from 14-day old aseptic seedlings, has been established. Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzyladenine (BA), kinetin (Kn) and thidiazuron (TDZ) singly or in combination with auxins was used for regeneration studies. Among the single treatment of three cytokinins BA at 1.0 μM was found to be optimum for direct shoot regeneration as it induced an average of 8.20 ± 0.66 shoots per explant. The regeneration frequency further enhanced with the application of auxin along with optimal BA concentration. The highest frequency for shoot regeneration (90%), the maximum number of shoots per explant (12.20 ± 0.73) and the maximum shoot length (6.40 ± 0.07) cm were obtained on the medium consisted of MS + 1.0 μM BA + 0.5 μM NAA. Successful in vitro rooting was induced from cut end of the microshoots when placed on half-strength MS + IBA (2.5 μM). The regenerated shoots with well developed root system were successfully acclimatized and established in pots containing sterilized garden soil and garden manure (1:1) and grown under greenhouse conditions with 85% survival rate.     

Clonal propagation ofGmelina arborea Roxb. byIn vitro culture

In vitro propagation technique ofGmelina arborea multipurpose and a fast growing tree species was studied. Nodal segment including axillary bud was used as a explant. They were cultured on MS media containing various concentrations (0–10 mg/l) of BAP alone or in combination with 0.002 mg/l of IBA. Nodal segments showed axillary bud proliferation in almost all media tested. MS media containing 0.22 mg/l of BAP alone and 2 mg/l of BAP in combination with 0.002 mg/l of IBA were effective for inducing multiple shoots and shoot elongation. MS medium supplemented with 0.02 mg/l of NAA and 1 mg/l of IBA gave the best result for rooting. The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the green house. Adventitious shoots production from stem explants that were taken from regenerated plantletin vitro was also discussed. Stem segments were tested for their morphogenetic potential on MS media with various combinations and concentrations of BAP, zeatin and TDZ. Successfull result was obtained on MS media supplemented with 2 mg/l of BAP and 1 mg/l of zeatin or supplemented with 0.5 mg/l of BAP and 0.5 mg/l of TDZ. The shoots obtained on MS media containing 2 mg/l of BAP and 1 mg/l of zeatin rooted on MS media containing 0.02 mg/l of NAA and 1 mg/l of IBA, and plantlets were successfully obtained. A part of this paper was presented at the 109th Annual Meeting of the Japanese Forest Society (1998).     

In vitro clonal propagation of <Emphasis Type="Italic">Balanites aegyptiaca</Emphasis> (L.) Del

In vitro propagation technique of Balanites aegyptiaca, a multipurpose woody tree was studied. Nodal segments including axillary bud from mature tree were used as an explant and their morphogenetic potential was tested on MS media with various concentrations (2.5–15.0 μM) of 6-benzyladenine (BA), Kinetin, and Thidiazuron alone or in combination with different concentrations (0.5–2.5 μM) of α-naphthalene acetic acid (NAA). Nodal segments showed axillary bud proliferation in almost all media tried. MS medium containing 12.5 μM BA alone was effective for inducing multiple shoots (5.0 ± 0.22) with an average shoot length (3.7 ± 0.26 cm) in 67% of cultures. A better shoot differentiation and elongation was achieved in a combined treatment of BA (12.5 μM) and NAA (1.0 μM). Half strength MS medium supplemented with Indole-3-butyric acid (IBA) gave the best result for rooting. The maximum frequency of root formation (68%), number of roots (5.3 ± 0.32) and root length (4.1 ± 0.38 cm) was obtained on half strength MS medium containing 1.0 μM IBA. The regenerated plantlets were potted and acclimatized successfully in a growth chamber and then moved to the greenhouse.     

Micropropagation of red sanders (Pterocarpus santalinus L.) using mature nodal explants

In vitro clonal multiplication of Pterocarpus santalinus L. was achieved using mature nodal explants of a 10-year-old elite quality tree. Combinations of serial transfer technique and incorporation of antioxidants (250 mg/l L-ascorbic acid and 50 mg/l citric acid) into the culture medium helped to minimize medium browning and improve explant survival during shoot sprouting. About 70% of explants were sprouted on Murashige and Skoog (MS) liquid medium containing 4.4 μM 6-benzyladenine (BA). The explant harvest period also influenced the bud break and shoot sprouting in nodal explants. The combination of 4.4 μM BA and 2.2 μM thidiazuron (TDZ) was found to be the most suitable growth regulator for obtaining the highest percentage of nodal segment sprouting (74%–75%), the number of secondary shoots per primary shoot (two or three), the shoot length (5–6 cm), the number of new nodal segments generated per active explant (four or five), and the multiplication coefficient (3.5) within 6 weeks. Repeated subculturing of nodal explants obtained from shoot cultures enabled continuous production of healthy axillary shoots. At the end of the sixth passage, about 90% of nodal explants produced five or six healthy green shoots, each being about 6.6 cm long with six or seven nodes. Multiplication coefficient was also increased from the first subculture (5.4) to the sixth subculture (8.3). The best rooting response was achieved on solidified half-strength MS medium supplemented with 4.9 μM indole-3-butyric acid (IBA). About 70% of the micropropagated plantlets were established successfully in 20-cm pots containing a mixture of soil and farmyard manure (4 : 1 ratio) and formed new leaflets.     

Establishment of an in vitro plant regeneration protocol for Casuarina cunninghamiana Miq. via indirect organogenesis

An in vitro plant regeneration protocol via indirect organogenesis from morphogenetic callus was established for Casuarina cunninghamiana Miq. Effects of plant growth regulator NAA (naphthaleneacetic acid) and BAP (6-benzylaminopurine), sucrose and AgNO₃ on callus induction, adventitious bud differentiation and shoot development were examined. Explants used were epicotyl fragments from 45-day-old seedlings. The largest callus (4.29 mm in diameter) was obtained after 1 month on a basic culture medium consisting of Murashige and Skoog ? macro- and full strength micro- elements, Nitsch and Nitsch vitamins, supplemented with 0.54 μM NAA, 3.30 μM BAP, and 30 g L⁻¹ sucrose. The calli were subcultured in the same medium above for 2 months. They were then cultured for another 2 months for adventitious bud differentiation and shoot development. The highest mean adventitious bud differentiation, number of shoots formed per callus and number of shoots ≥2 cm long per callus (47.50%, 27.38 and 4.75, respectively) were achieved on the above medium modified with NAA at 0.27 μM and supplemented with AgNO₃ 1 mg L⁻¹. Shoots were successfully rooted without plant growth regulator and the rooted plantlets survived and grew normally. This protocol for in vitro plant regeneration provides a tool not only for vegetative propagation but also for plant genetic transformation and gene function studies of C. cunninghamiana.     

Early recognition of graft compatibility in <Emphasis Type="Italic">Uapaca kirkiana</Emphasis> Müell Arg. clones,provenances and species

Examination of callus micro-grafts in Uapaca kirkiana Müell Arg. was carried out with the objective of determining early signs of graft compatibility. Leaves from U. kirkiana, U. nitida and Jatropha curcas trees were used for callus induction. Two pieces of callus were co-cultured on Murashige and Skoog (MS) medium with different supplements. Co-cultured calli were embedded in paraffin wax and dissected. The specimens were stained in safranin and fast green before viewing under a light microscope. Results showed that MS medium with 0.1 mg l⁻¹ thidiazuron (TDZ) and 0.5 mg l⁻¹ naphthaleneacetic acid (NAA) or 1.0 mg l⁻¹ dichlorophenoxyacetic acid (2,4-D) and 0.5 mg l⁻¹ NAA was effective for callus induction. There were no necrotic layers at the unions within U. kirkiana clones and provenances, but a differential growth (irregularity) between U. kirkiana and U. nitida co-cultured calli. Phenol deposits were observed at the union interfaces of U. kirkiana combinations and were high on calli derived from mature trees. Phenol deposits were absent at the union of J. curcas heterografts. Necrotic layers developed at the unions of U. kirkiana and J. curcas micro-grafts and indicating an outright graft incompatibility. Accumulation of phenol deposits at the union interfaces inhibited graft compatibility in many U. kirkiana combinations. Callus fusion technique can be used to identify partners with an outright graft incompatibility, especially for distant related plant species.     

Micropropagation and callus culture of <Emphasis Type="Italic">Saussurea laniceps</Emphasis>, an alpine medicinal plant

Cottonhead windhairdaisy (Saussurea laniceps Hand.-Mazz.) is one of the most famous and important medicinal herbs in China. Illegal collection from wild populations is increasingly threatening the present environment of S. laniceps. Establishment of an efficient method for micropropagation is the best way to change its endangered situation. When mature seeds of S. laniceps were cultured on hormone-free MS medium, plantlets were formed from germinated seeds in 7–10 d. Then 0.5 cm × 0.5 cm leaf explants were transplanted to MS medium supplemented with 1-naphthalene-acetic acid (NAA)/2,4-D and benzyladenine (BA)/KT and callus was achieved 10 d after transfer. Shoot bud regeneration occurred from callus cultured on MS medium supplemented with different growth regulators 20 d after culturing. The regeneration percentages varied with the different components of plant growth regulators. The percent regeneration from callus pretreated at low temperature of 5°C increased significantly compared with those incubated at 23/20°C directly. Optimal regeneration was observed with explants on media supplemented with 1.5 mg&#8226;L^–1 BA plus 0.2 mg&#8226;L^-1 NAA. In the presence of 0.2 mg&#8226;L^–1 NAA in half-strength MS, 78% of the shoots formed roots. Plantlets from explants showed 63% survival after acclimatization.     

In vitro morphogenic response and metal accumulation in <Emphasis Type="Italic">Albizia lebbeck</Emphasis> (L.) cultures grown under metal stress

An efficient regeneration protocol for rapid mass propagation and uptake of heavy metals in Albizia lebbeck (L.), a fast growing, medicinally as well as economically important timber yielding tree was developed. Nodal segments derived from a 20-year-old tree were cultured on MS (Murashige and Skoog) medium supplemented with 10 μM 6-Benzyladenine (BA) and 1 μM α-Naphthalene acetic acid (NAA) showed optimum shoot regeneration frequency (76.6%), number of shoots (23.2 ± 0.28) per explant and shoot length (2.86 ± 0.08 cm) after 10 weeks of culture. After standardizing a reliable protocol for micropropagation, effects of ZnSO4 (0.06–0.48 mM), CuSO₄ (0.02–0.2 mM) and CdCl₂ (0.0001–0.001 mM) on shoot morphogenesis were also assessed. The regenerated shoots maintained on maintenance medium (MS + 10.0 μM BA + 1.0 μM NAA) containing ZnSO₄ (0.06 mM) showed maximum response in terms of shoot number (24.5 ± 0.83) and length (5.9 ± 0.05 cm) after 10 weeks of culture. Proline content showed an increasing trend while chlorophyll (a and b) content exhibited decreasing trend with an increased metal concentrations compared to MM cultures, and maximum increase in proline and decrease in chlorophyll content was recorded in cultures grown on Cd-enriched medium. Best rooting was accomplished on half strength MS medium with 2.0 μM IBA and ZnSO₄ (0.06 mM). The plantlets thus obtained were successfully hardened and transferred to greenhouse with 75% survival rate and exhibited normal morphological characteristics compared to donor plant.     

Direct shoot organogenesis from leaf explants of Embelia ribes Burm. f.: a vulnerable medicinal plant

An efficient system was developed for direct plant regeneration from in vitro-derived leaf explants of Embelia ribes Burm. f., a vulnerable medicinal woody climber of the Western Ghats of India. The in vitro procedure involved three steps that included induction of shoot initials from leaf tissue, regeneration and elongation of shoots from the shoot initials, and rooting of shoots. The induction of shoot initials was achieved on Murashige and Skoog (MS) solid medium supplemented with different concentrations of thidiazuron (TDZ). The best medium for shoot induction was MS with 0.272 μM TDZ. Numerous shoot primordia developed within 2–3 weeks on the leaf margin as well as on the midrib region, without any callus phase. In the second step, the shoot clumps separated from the leaf explant on transfer to MS basal medium, resulting in the differentiation of 90% of the shoot initials into well-developed shoots. The 2- to 3-cm-long shoots rooted on half-strength MS basal medium supplemented with 4.90 μM indole-3-butyric acid (IBA) and 3% (w/v) sucrose in the third stage. The rooted plants could be established in soil with 70% success. This protocol could be utilized for in vitro propagation and conservation of this important threatened medicinal plant.     

Influence of cytokinins,basal media and pH on adventitious shoot regeneration from excised root cultures of <Emphasis Type="Italic">Albizia lebbeck</Emphasis>

A highly reproducible and efficient in vitro shoot regeneration system was developed in a potential medicinal plant, Albizia lebbeck using root explants. Root explants from 15 day-old-aseptic seedlings 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 (Kn), 2-Isopentenyl adenine (2-iP) singly as well as in combination with α-Naphthalene acetic acid (NAA) (0.1, 0.5, 1.0, 1.5 and 2.0 μM). The highest rate of shoot multiplication (16.0 ± 1.87 for the average shoot number and 5.16 ± 0.38 cm for shoot length) was achieved on MS medium supplemented with 7.5 μM BA and 0.5 μM NAA. The effects of medium type, medium strength, pH and subculture on shoot induction and proliferation were also tested. An average of 21.6±2.87 shoots per explants could be obtained following this protocol. Rooting was achieved on microshoots using half strength MS medium with 2.0 μM Indole-3-butyric acid (IBA) after four weeks of culture. The in vitro raised healthy plantlets were successfully established in earthen pots containing garden soil and grown in greenhouse with >80% survival rate.