In vitro regeneration of clonally uniform plants of <Emphasis Type="Italic">Crataeva magna</Emphasis>: a high value medicinal tree by axillary branching method

Crataeva magna (Lour.) DC (synonym C. nurvala) is a high-value Indian medicinal tree. The multiple uses of C. magna have resulted in its over-exploitation. Erratic seed germination combined with destructive harvesting and habitat destruction in the form of deforestation has added to the enormity of the problem. Therefore, the need for conservation of this plant is vital. Development of an efficient micropropagation protocol will play a significant role in meeting the requirement of quality planting material for commercial cultivation thereby conserving the species in its natural habitat. In the present study, shoot multiplication was achieved by culturing single node segments derived from a field grown tree on Murashige and Skoog’s (MS) medium supplemented with 2.66 μM N6-benzyladenine, 1.39 μM Kinetin (Kn), 0.57 μM indole-3-acetic acid (IAA), 3% sucrose and 0.2% gelrite. 96% rooting was achieved within 22 days by culturing the in vitro formed shoots on half strength MS medium with 11.42 μM IAA, 9.8 μM indole-3-butyric acid, 0.46 μM Kn and 198.25 μM phloroglucinol. Following a simple hardening procedure involving sequential transfer of plants to a greenhouse, polyhouse, and shade net, the tissue-cultured plants were transferred to the field where the survival rate was 100%. To this date 500 plants have been produced. Inter simple sequence repeat analysis has confirmed genetic uniformity of the tissue-cultured plants.     

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.     

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.     

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.     

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.     

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.     

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.     

Rapid clonal multiplication of a woody tree, <Emphasis Type="Italic">Vitex negundo</Emphasis> L. through axillary shoots proliferation

An efficient and improved shoot regeneration technique for the micropropagation of Vitex negundo, an aromatic and medicinal shrub through in vitro culture of nodal segments with axillary buds, is described. Thidiazuron (TDZ) used at 1.0 μM was the most effective in inducing bud break and growth, and also in initiating multiple shoot proliferation at the rate of 25 microshoots per nodal explant with axillary buds, after 4 weeks of culture. By repeated subculturing of nodal explants, a high-frequency multiplication rate was established. Optimum shoot multiplication and elongation was achieved when TDZ exposed explants were subcultured on Murashige and Skoog (MS) media containing a combination of 1.0 μM 6-benzyladenine (BA) and 0.5 μM α-naphthalene acetic acid (NAA). Efficient rooting was achieved directly in soilrite when basal portion of the shoots were treated with 500 μM indole-3-butyric acid for 10 min which was the most effective in inducing roots, as 97% of the microshoots produced roots. Plantlets went through a hardening phase in a controlled plant growth chamber, prior to ex-vitro transfer. Micropropagated plants grew well, attained maturity and flowered. No phenotypical differences for morphogenesis were observed among the regenerants.     

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.     

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     

Micropropagation of Vitex negundo L. and Random Amplified Polymorphic DNA Analysis of Micropropagated Plants

A micropropagation protocol was established for a medicinal plant Vitex negundo. Genetic stability of micropropagated plants was investigated. Multiple shoots were induced from nodal explants cultured on Murashige and Skoog (MS) medium with 0.53 μM naphthalene acetic acid (NAA) and 11.0 μM benzyl aminopurine (BAP) along with additives (ascorbic acid, 283.9 μM; citric acid, 130.1 μM; and arginine, 143.6 μM). Shoots were further multiplied by repeated transfer of the mother explant. The shoots were further multiplied on MS medium + 0.57 μM indole-3-acetic acid (IAA) and 6.6 μM BAP. The micropropagated shoots were pulse treated with 122.5 μM indole-3-butyric acid (IBA), in liquid MS medium and then transferred to autoclaved soilrite. These rooted ex vitro. Shoots were also rooted in vitro on a half-strength MS medium + 2.45 μM IBA. The survival rate of in vitro rooted plantlets was poor during hardening compared to ex vitro rooted plantlets. About 95% of the ex vitro rooted, hardened plantlets survived in the field. Genetic stability of micropropagated plants was tested by using 25 random amplified polymorphic DNA primers. The cloned plants exhibited no variation in banding pattern in comparison with the mother plant.     

Micropropagation of mature Terminalia catappa (Indian Almond), a medicinally important forest tree

We report an efficient in vitro propagation method for Terminalia catappa using nodal segments of a 15-year-old mature tree. The nodal segments were cultured on MS medium supplemented with 6-benzyladenine (BA; 0.5–3.0 mg l⁻¹) or Kinetin (Kn; 0.5–3.0 mg l⁻¹) for bud breaking and multiple shoot induction. About 85% of the explant responded (2.8 ± 0.41 shoots per node with 2.7 ± 0.14 cm length) within 15 days of inoculation in Murashige and Skoog medium fortified with 2.0 mg l⁻¹ of BA. Further shoot multiplication was achieved by repeated transfer of mother explants and subculturing of in vitro-produced shoots on medium supplemented with various concentrations of BA (0.25–1.5 mg l⁻¹) or Kn (0.25–1.5 mg l⁻¹) or on their combinations. Optimal number of shoots and shoot length were recorded on MS medium supplemented with 0.25 mg l⁻¹ of BA and 0.25 mg l⁻¹ of Kn. The multiplied shoots were used for ex vitro rooting after treatment for 4 min with indole-3-butyric acid (IBA; 50–500 mg l⁻¹) or α-naphthalene acetic acid (NAA; 50–500 mg l⁻¹). About 80% of the shoots treated with 200 mg l⁻¹ of IBA produced ex vitro roots with an average of 2.8 roots per shoot. Nearly 75% of these plantlets could be acclimatized within 5 weeks and successfully established in the field. This is the first report on micropropagation of T. catappa, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

Micropropagation of an endangered Indian sandalwood (Santalum album L.)

Santalum album is known as East Indian sandalwood. It is the most economically important tree harvested for heartwood oil, and India is among the chief exporters of sandalwood and its products. Multiple shoots were induced from nodal shoot segments derived from a 50- to 60-year-old candidate plus tree (CPT) on Murashige and Skoog (MS) medium supplemented with 0.53 μM α-naphthaleneacetic acid (NAA) and 11.09 μM 6-benzylaminopurine (BA). In vitro differentiated shoots were multiplied on MS medium with 0.53 μM NAA, 4.44 μM BA, and additives: 283.93 μM ascorbic acid, 118.10 μM citric acid, 104.04 μM cystine, 342.24 μM glutamine, and 10% (v/v) coconut milk. New shoots were harvested repeatedly for up to three subculture passages on fresh medium at 4-week intervals. Microshoots treated with 98.4 μM indole-3-butyric acid (IBA) for 48 h produced roots on growth-regulator-free, quarter-strength MS basal salts medium with vitamin B₅ and 2% sucrose. In vitro root induction was achieved from microshoots pulsed with 1230 μM IBA for 30 min in soilrite rooting medium. The percentage of rooting in soilrite was higher than that for agar medium, and in vitro raised plants were established in the field and showed normal growth.     

In vitro propagation of the African mahogany <Emphasis Type="Italic">Khaya senegalensis</Emphasis>

A protocol was developed for shoot proliferation and plantlet formation of Khaya senegalensis, an important medicinal and timber plantation species introduced to Australia and southern Asia from western and central Africa. We assessed effects of the plant growth regulators, benzyladenine, kinetin, naphthalene acetic acid and gibberellic acid, on shoot proliferation and subsequent plantlet conversion. Shoot proliferation over four passages was higher in media containing benzyladenine than in media containing other growth regulators, and optimal proliferation from seed of three different sources was consistently obtained in medium containing 4.4 μM benzyladenine. Shoots from this medium were converted to plantlets at high frequencies (76–90%) after treatment with 19.6 μM indole-3-butyric acid, and almost all plantlets were successfully acclimatized to nursery conditions. These methods provide the means for establishing in vitro and ex vitro clone banks of juvenile K. senegalensis trees for field selection of desired genotypes and tropical plantation establishment.     

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.     

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.     

In vitro plantlet regeneration from Australian Red Cedar (Toona ciliata,Meliaceae)

In vitro regeneration of complete plants from nodal single-bud segments of 2-year-old Australian Cedar (Toona ciliata) trees were obtained under defined nutritional and environmental conditions. Explants were dissected from plants obtained by germination of seeds and growth in pots in a greenhouse. The best medium for shoot regeneration was that of Murashige and Skoog at 1/4 strength with 3% sucrose (1/4 MS), supplemented with 0.1 mg/l IBA and 0.5 mg/l BAP. Rooting of regenerated shoots was observed in MS medium with 0.1 mg/l IBA. Using mature tree material was more difficult. Forced flushing was used to induce shoot development on branches of a 10-year-old tree. Nodal segments of these epicormic shoots formed shoots in vitro on 1/4 MS + 0.01 mg/l IBA + 5 mg/l BAP, but rooting was never observed.     

Micropropagation of Anogeissus latifolia (Roxb. Ex DC.) Wall,ex Guill. & Perr.- A Tree of Fragile Ecosystems

Abstract

A micropropagation process was developed for Anogeissus latifolia-a tree of fragile ecosystems. Multiple shoots were regenerated from cotyledonary node and epicotyl explants on Murashige and Skoog's (MS) medium containing 0.1 mgl^-1 indole-3-acetic acid (IAA) + 1.5 mgl ^-l 6-benzylaminopurine (BAP) + additives (25 mgl ^-l each of adenine sulphate, L-arginine, ascorbic-, citric acids and 1.0 mM L-asparagine) + 200 μM Fe-EDTA (ferric-ethylenediaminetetraacetic acid) salt. The shoots differentiated in vitro were subcultured and repeatedly transferred onto fresh medium but with 1.0 mgl^-1 of BAP to achieve 4-5 fold rate of shoot multiplication. After every 4th week of culture, from each culture bottle 8-10 shoots could be harvested for rooting. The shoots produced in culture were rooted in vitro on half strength MS medium with 1.0 mgl ^-l either of IBA (indolebutyric acid) or NAA (naphthaleneacetic acid). The shoots were pulse treated with combination (100 mgl ^-l each) of IBA and NOA (2-naphthoxy acetic acid) rooted ex vitro. The ex vitro root induction method is highly efficient and plantlets so generated could be acclimatized and pot transferred. The process developed can be used for large scale production of plants of A.     

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).     

Contribution of trees to soil carbon sequestration under agroforestry systems in the West African Sahel

Consequent to recent recognition of agricultural soils as carbon (C) sinks, agroforestry practices in the West African Sahel (WAS) region have received attention for their C sequestration potential. This study was undertaken in the Ségou region of Mali that represents the WAS, to examine the extent of C sequestration, especially in soils, in agroforestry systems. Five land-use systems were selected in farmers’ fields [two traditional parkland systems, two improved agroforestry systems (live fence and fodder bank), and a so-called abandoned land]. Soil samples taken from three depths (0–10 cm, 10–40 cm, and 40–100 cm) were fractionated into three size classes (2,000–250 μm, 250–53 μm, and <53 μm) and their C contents determined. Whole-soil C contents, g kg⁻¹ soil, across three depths ranged from 1.33–4.69 in the parklands, 1.11–4.42 in live fence, 1.87–2.30 in fodder bank, and 3.69–5.30 in abandoned land; and they correlated positively with silt + clay content. Using the ¹³C isotopic ratio as an indicator of relative contribution of trees (C3 plants) and crops (C4 plants) to soil C, more tree-origin C was found in larger particle size and surface soil and indicated that long-term tree presence promoted storage of protected C in deeper soil. Existing long-standing agroforestry practices of the region such as the parklands seemed to have little advantage for sequestering additional C, whereas improved agroforestry practices such as live fence and fodder bank introduced in treeless croplands seemed to be advantageous.