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 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.     

Shoot multiplication and plant regeneration in <Emphasis Type="Italic">Caragana fruticosa</Emphasis> (Pall.) Besser

Different nutrient media can affect in vitro culturing protocols, and experimentation under varied growth conditions is valuable in plants where in vitro methods are in preliminary stages. We carried out the first in vitro propagation studies for the endangered species Caragana fruticosa (Fabaceae). We evaluated various nutrient media for their impact on shoot elongation and axillary bud proliferation using different concentrations of 6-benzylaminopurine (BA) and α-naphthaleneacetic acid (NAA). Shoot elongation was evaluated based on adventitious shoot primary culture and subculture regeneration from Caragana seedlings. Our goal was to improve both micropropagation and regeneration in C. fruticosa. MS nutrient media was superior to 1/2MS macronutrients, DKW, QL, and WPM for shoot elongation and axillary shoot proliferation. Shoots grown on 1/2MS and WPM exhibited some chlorosis, and shoots on QL produced larger leavers than plants growing on normal medium. The shoot proliferation coefficient on MS media supplemented with 2.22 μM BA and 0.44 μM BA + 2.69 μM NAA was significantly higher than that with other treatments in the primary culture. Shoots on 2.22 μM BA showed a higher proliferation coefficient (3.17) than others in the subculture. Shoots were rooted on 1/2MS medium with the addition of different concentrations of NAA. The optimal concentration for rooting was 0.27 μM NAA (74%). Roots exhibited many stout and long root hairs. Survivl of established plantlets was 82% at 30 days after transfer to soil. Plants established in the green house showed normal growth and displayed no apparent morphological differences compared to stock plants.     

Stimulation of in vitro organogenesis from epicotyl explants and successive micropropagation round in Cassia angustifolia Vahl.: an important source of sennosides

This report describes a protocol for the in vitro shoot induction and plant regeneration from epicotyl explants of Cassia angustifolia on Murashige and Skoog (MS) medium supplemented with 6-benzyladenine (BA), Kinetin and 2-iP (0.5–10.0 μM). MS medium supplemented with BA (5.0 μM) was the most effective in inducing adventitious shoots and growth. The highest rate of shoot multiplication was achieved on MS medium supplemented with BA (5.0 μM) and Indole-3-acetic acid (IAA, 1.0 μM). The nodal segments excised from the shoots regenerated from BA (5.0 μM) and IAA (1.0 μM) were used as explants for next three round of micropropagation. The number of shoots significantly increased at successive round of micropropagation. For rooting, MS medium supplemented with 2.0 μM indole-3-butyric acid proved to be better than that supplemented with IAA or α-naphthalene acetic acid. The in vitro raised plantlets with well developed shoot and roots were successfully established in earthen pots containing garden soil and were grown in greenhouse. About 52 plants (85 %) survived out of 60 plants transferred in garden soil.     

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.     

In vitro regeneration and the antioxidant enzymatic system on acclimatization of micropropagated Vitex trifolia L.

The objective of the study was to develop an in vitro shoot regeneration protocol by utilising shoot tips explant from Vitex trifolia L. Shoot tip explants obtained from a 3-year old plant was cultured on Murashige and Skoog (MS) medium supplemented with various concentrations (1.0, 2.5, 5.0, 7.5 or 10.0 µM) of thidiazuron (TDZ). The optimal level of TDZ supplementation to the culture medium was 5.0 μM for 15 d induction period. The highest number of shoots (22.2 ± 0.1) and shoot length (5.1 ± 0.1 cm) were achieved when TDZ-exposed explants were sub-cultured on MS medium containing 6-benzyladenine (1.0 µM) and 0.5 µM α-naphthalene acetic acid (NAA) after 8 weeks of culture. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5 µM NAA. During the acclimatization period, changes in activities of antioxidant enzymes were observed. Superoxide dismutase activity increased reaching maximum at 28th day after transplantation. Likewise, an upregulation of the catalase, ascorbate peroxidase and glutathione reductase enzyme activities were also observed. These observed changes reflected the ability of plants in developing an antioxidant enzymatic defence system aiding in survival against oxidative stress and in reducing release of free radicals. Plantlets were successfully hardened off and acclimatized in earthen pots containing garden soil with a survival rate of 90 %.     

Non-aerated liquid culture promotes shoot organogenesis in <Emphasis Type="Italic">Eucalyptus globulus</Emphasis> Labill

Eucalyptus is very recalcitrant to in vitro culture. In this research, an efficient shoot organogenesis system was developed using 60-day-old plants of Eucalyptus globulus grown in vitro and non-aerated liquid medium to improve shoot proliferation. Cultures were initiated with hypocotyls and leaf segments from plantlets cultivated on semisolid ½ MS modified medium supplemented with 4.44 µM 6-Benzyladenine (BA) and 16.1 µM 1-Naphthaleneacetic acid (NAA). Calli were transferred to shoot induction medium, with either 0.5 or 2.7 µM NAA. Shoot multiplication was carried out on 4.44 µM BA + 0.5 µM NAA medium, and semisolid and non-aerated liquid systems were compared for improving shoot proliferation. Rooting of adventitious shoots was evaluated on medium containing NAA or Indole-3-butyric acid -IBA (5 and 16 µM). Callogenesis was obtained from both types of explants, although shoot formation was only obtained from leaf-derived calli. Shoot proliferation on 4.44 µM BA + 0.5 µM NAA resulted in the most shoots/callus. Non-aerated liquid medium was more efficient in promoting shoot multiplication (53.5 shoots/callus) than was semisolid medium (28.5 shoots/callus). Levels of phenolic compounds were significantly reduced in the shoots cultivated in liquid medium. Efficient rooting (76%) was obtained using 16 µM IBA.     

An efficient in vitro process for recurrent production of cloned plants of Vitex negundo L

An efficient method for cultivation of Vitex negundo L. through axillary shoot (collected from micropropagated plants) proliferation has been successfully developed, which can be employed at a commercial scale. Axillary shoot induction was most successful using nodal explants for propagation on Murashige and Skoog (MS) medium supplemented with various concentrations of single cytokinin or in various combination with auxins. We obtained the maximum percentage (97.6 ± 1.45) response with highest number (16.4 ± 0.60) of shoots per explant on MS medium supplemented with 6-benzyladenine (BA) and ??-naphthalene acetic acid (NAA) at concentrations of 5.0 and 0.5 ??M, respectively. Shoot regeneration frequency was optimized by manipulating pH and using various media. MS medium and pH 5.8 was found to be the optimum for maximum regeneration. Nodal explants from in vitro regenerated microshoots too developed shoots, thus making the process recurrent. Shootlets with 4?C5 nodes were utilized for in vitro rooting, and best response was evaluated on MS medium supplemented with 1.0 ??M indole-3-butyric acid (IBA). The well-developed micropropagated plants were acclimatized (97%) successfully within 2 weeks in soilrite and planted ex vitro in normal garden soil, where they grew well without any morphological and genetic variations. The present regeneration process not only favoured the rapid multiplication but also expressed the regeneration capability of micropropagated plants.     

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.     

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.     

Establishment of a high-frequency regeneration system in <Emphasis Type="Italic">Cerasus humilis</Emphasis>, an important economic shrub

Cerasus humilis is a species of small, perennial, drought-resistant and multipurpose deciduous shrub grown in arid and semi-arid conditions in northern China. In this study, an efficient protocol for the rapid micropropagation of C. humilis has been standardized using stem and/or leaf explants. Direct multiple shoot induction was observed when the stem explants were cultured on Murashige and Skoog (MS) medium supplemented with different plant growth regulators. The highest shoot induction was obtained when stem explants from adult trees were cultured on MS medium supplemented with 2.0 mg L^?1 6-benzyladenine (6-BA) and 0.9 mg L^?1 α-naphthaleneacetic acid (NAA). The leaf and stem explants cultured on MS medium with 1.0 mg L^?1 6-BA and 0.6 mg L^?1 NAA, and 0.5 mg L^?1 6-BA and 0.8 mg L^?1 NAA, respectively, produced the highest induction frequency of callus. Maximum proliferation of callus was observed on MS medium containing a combination of 0.5 mg L^?1 6-BA with 0.6 mg L^?1 2,4-dichlorophenoxyacetic acid (2,4-d). Optimal shoots differentiated from callus were obtained on MS medium supplemented with 5.0mg L^?1 6-BA and 0.9 mg L^?1 NAA. In vitro rooting was achieved on half-strength (1/2) MS medium containing 0.5 mg L^?1 NAA. Rooted plantlets were hardened under control conditions and successfully acclimatized under field conditions.     

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.     

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.     

Micropropagation of <Emphasis Type="Italic">Eucalyptus grandis</Emphasis> × <Emphasis Type="Italic">E. urophylla</Emphasis> AEC 224 clone

Genetic transformation systems require protocols that allow regenerating transgenic plants from transformed tissues. This study aimed to establish a protocol for indirect organogenesis in leaf explants of a Eucalyptus grandis  ×  E. urophylla AEC 224 clone. During callogenesis stage, several concentrations of NAA and then NAA or 2,4-D combined with TDZ were tested in JADS culture medium for 30 days, followed by subculture of the explants in the regeneration medium, containing 5.0 µM BA and 0.5 µM NAA for another 30 days. In these media, the explant oxidation rate was high (95 %). Thus, in order to reduce oxidation, different culture media were compared: WPM, MS, JADS and modified QL, followed by explant transfer onto regeneration medium. The highest percentage of regeneration and the lowest oxidation rate were achieved on WPM medium. Then, NAA and 2,4-D were tested in combination with TDZ and also TDZ and BA combined with NAA in WPM medium. The most efficient culture media in terms of shoot regeneration were WPM supplemented with 0.25 µM TDZ and 0.1 µM NAA during 30 days for callus induction and then with 5.0 µM BA and 0.5 µM NAA for another 30 days. This protocol yielded a regeneration rate of 43 %, with a low oxidation of tissues. A rooting experiment was conducted using half strength MS medium and comparing three concentrations of IBA (2.46, 4.90 and 7.35 µM). The highest rooting percentage (35 %) was obtained on medium containing 2.46 µM IBA. Once the shoots were rooted, acclimatization in a greenhouse was not challenging and plant survival reached 100 %.     

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.     

Direct shoot bud regeneration from shoot tip explants of <Emphasis Type="Italic">Enicostema axillare</Emphasis>: an important medicinal plant

An efficient protocol has been developed for in vitro propagation of Enicostema axillare using shoot tip explants. The shoot tip explants were cultured on MS medium supplemented with various combinations of (BAP, KIN) and (NAA/IAA & IBA) in different concentrations between 0.5 and 2.0 mg/l for multiple shoot bud induction. The highest percent of (98.51 %) was observed at 1.0 mg/l BAP in combination with 0.2 mg/l KIN while maximum number of shoot buds (8.41 shoots/explant) was noticed on MS medium containing 1.0 mg/l BAP and 0.2 mg/l KIN combination. The highest frequency (90.82 %) of multiple shoot bud regeneration was observed at 1.0 mg/l BAP and 0.5 mg/l IBA with 15.12 ± 2.12 shoots/explants. The regenerated multiple shoots were transferred to half-strength MS medium augmented with different concentration of 0.5–2.5 mg/l IBA for rooting. Among the different concentrations of IBA tested, maximum percentage of rooting (100 %) was observed in MS medium augmented with 1.5 mg/l IBA. The rooted plantlets were successfully transferred into plastic cups containing soil and sand in the ratio of 1:1. Subsequently established in the field conditions with 90 % of survival rate. The protocol developed can be utilized for both large scale plant production and conservation of germplasm of this species. The described method can be successfully employed for large-scale multiplication and in vitro conservation as well as production of secondary metabolites of E. axillare.     

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.     

金银花优良无性系离体快繁研究

本试验以金银花优良无性系苍源1#为外植体,采用离体快繁技术建立其组培快繁体系,以期能在短期内进行大量扩繁。研究结果表明：茎段是较适合的外植体材料,在MS＋BA 0.5 mg/L（单位下同）＋IBA 0.5培养基上能较好地诱导生芽,建立无菌系;在MS＋BA 0.1～0.5＋NAA 0.1～0.3的培养基上能有效增殖,增殖系数可达6.5;生根培养基以MS＋IBA2.0为宜,生根率可达93%。     

Shoot proliferation and callus regeneration from nodular buds of Drepanostachyum luodianense

This research reports on an efficient shoot proliferation and callus regeneration system for bamboo.Young, semi-lignified branches with one lateral bud from Drepanostachyum luodianense(Yi et R. S. Wang) Keng f.were used as explants. Disinfection with 0.1% HgCl_2 for 8 min was the optimum treatment and the best medium for bud initiation was Murashige and Skoog(MS) medium containing 3.0 mg L~(-1)6-benzyladenine(BA). Multiple shoots were induced from nodal shoot segments on MS medium containing 5.0 mg L~(-1) BA, 0.5 mg L~(-1) kinetin(Kin), and 1.0 mg L~(-1) naphthaleneacetic acid(NAA). The highest frequency of callus formation(65.6%) was on MS medium containing 4.0 mg L~(-1)2,4-dichlorophenoxyacetic acid(2, 4-D), 0.5 mg L~(-1) NAA, and 0.1 mg L~(-1) thidiazuron(TDZ). The optimum medium for callus proliferation was MS medium with 4 mg L~(-1)2,4-D, 0.5 mg L~(-1) TDZ and 0.5 mg L~(-1) NAA, and the optimum hormone combination was 4 mg L~(-1) BA ? 0.5 mg L~(-1) NAA for callus redifferentiation(up to 85.6%). A 100% rooting was achieved on MS medium supplemented with 2.0 mg L~(-1) NAA and 0.5 mg L~(-1)3-indole butyric acid(IBA). Rooted plantlets were acclimatized in a greenhouse in humus soil ? perlite(1:1) substrate. These micropropagated callus induction and regeneration systems for bamboo will be useful for genetic engineering and multiplication.     

In vitro plant regeneration from cotyledon-derived callus cultures of leguminous tree Gleditsia caspica Desf.

An in vitro regeneration system was developed using organogenic callus derived from in vitro grown cotyledonary explants of Gleditsia caspica Desf., an important leguminous tree. Murashige and Skoog (MS) basal medium augmented with 0.2 g L^?1 myo-inositol and various concentrations of either 2,4-dichlorophenoxyacetic acid (2,4-D), naphthaleneacetic acid, or indole-3-butyric acid (IBA) alone as well as combined with cytokinins was used for callus induction. The highest frequency of organogenic yellowish-white and nodular callus (93 %) was obtained from explants grown on medium supplemented with 13.5 μM 2,4-D and 4.4 μM benzyladenine (BA). The yellowish-white and nodular callus when transferred to MS medium supplemented with BA (2.2–17.7 μM) or kinetin (KT; 2.3–18.8 μM) solely or in combination with 2.3 μM 2,4-D produced several microshoots after 5 weeks culture. The calli cultured on MS medium with 4.4 μM BA singly showed superior growth response and produced both maximum shoot regeneration (94 %) and the highest mean number (4.3) of microshoots per callus. Transfer of regenerated microshoots onto modified MS basal medium fortified with 5.8 μM gibberellic acid and 4.4 μM BA resulted in the maximum number of internodes per shoot and the highest shoot elongation after a period of 6 weeks. Optimum rooting of 90 %, an average 6.1 roots per shoot, and a mean root length of 3.6 cm was observed when half-strength MS medium was supplemented with 9.8 μM IBA and 0.92 μM KT. The regenerated healthy plants with well-developed shoots and roots showed a survival rate of 77 % after acclimatization and transplanting to garden soil for a 10-week hardening period under ex vitro conditions.