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 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.     

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 %.     

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 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.     

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.     

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.     

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 %.     

Modulation of in vitro morphogenesis in nodal segments of Salix tetrasperma Roxb. through the use of TDZ, different media types and culture regimes

A comparative performance of two different media formulations (woody plant medium (WPM) and Murashige and Skoog??s (MS) medium) for their ability to inflict in vitro shoot development in nodal segments of Salix tetrasperma Roxb. has been carried out. Thidiazuron (TDZ) in various concentrations was used as a supplement to the basal media. Media types, TDZ concentrations, exposure duration and culture regimes played an important role in affecting multiple shoot production. WPM supplemented with 2.5???M TDZ for 4?weeks exposure was found to be the best for maximum (4.53?±?0.27) shoots production in vitro. Transfer to a secondary medium consisting of 6-benzyladenine (1.0???M) and ??-naphthalene acetic acid (0.5???M) enhanced the multiplication rate and by the end of 12?weeks, 20.33?±?0.33 shoots with shoot length, 4.70?±?0.26?cm were produced on WPM. Rooting of the regenerated shoots was achieved on half strength basal media (either WPM or MS) containing 0.5???M indole-3-butyric acid. In all the experiments, different growth parameters were scored and WPM was found to be superior to MS medium. The regenerated plantlets were successfully acclimatized in the field with about 81?% survival.     

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.     

迷迭香离体培养快繁技术的研究

以茎段为起始外植体,进行迷迭香离体培养快繁技术的研究。结果表明:茎段在MS BA 0.2mg/L IBA 0.01 mg/L培养基上腋芽诱导分化效果最好,诱导率可达75%;诱导出的腋芽转入1/2 MS BA0.3 mg/L IBA 0.1 mg/L AC 1 g/L培养基中增殖与伸长效果最佳,每个腋芽的平均诱导芽数为3.5个,平均芽长为3.0 cm;在1/4 MS IBA 0.1 mg/L AC 1 g/L培养基中诱导生根,生根率达87%。     

An improvised in vitro vegetative propagation technique for <Emphasis Type="Italic">Bambusa tulda</Emphasis>: influence of season,sterilization and hormones

Season and concentration of sterilizing agents play a significant role for establishment of aseptic in vitro shoot cultures and sprouting of nodal explants from field growing culms of bamboo species. In the present investigation the nodal segment explants of Bambusa tulda Roxb collected in different seasons and treated with various concentrations of HgCl₂ showed significant variation in aseptic culture establishment and bud break. The rainy season (July–August) recorded with highest of 78% aseptic culture establishment whereas autumn recorded with lowest 46%. Summer and winter seasons emerged to be the best period, registering > 60% in vitro bud break. On the other hand, the autumn season had the lowest value for bud break, i.e. 42%. Among different doses of sterilizing agent tried, HgCl₂ 0.1% found to be suitable for maximum aseptic culture establishment (66%) as well as bud break (59%). However, among the interactions, summer season and the dose of 0.1% HgCl₂ exhibited maximum of 73% response for both aseptic culture establishment and bud break. MS medium (liquid) enriched with 5.0 µM BA + 5.0 µM Kn [Kinetin (N6-Furfuryladenine)] with additional supplementation of 100 µM glutamine + 0.1 µM IAA supported a maximum in vitro shoot multiplication of 4.75 fold. The proliferated shoots were successfully rooted on MS medium (liquid) supplemented 40 µM coumarin. The plantlets transferred to the polythene bags showed 98% survival.     

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.     

Plantlet regeneration of adult <Emphasis Type="Italic">Pinus massoniana</Emphasis> Lamb. trees using explants collected in March and thidiazuron in culture medium

A protocol for micropropagation using nodal explants from mature Pinus massoniana trees has been developed. Time of explant collection is crucial for the initial success of aseptic culture. Explants collected in early March gave the highest percentage of explant survival (64.5%) and shoot-forming percentage (52.3%). Thidiazuron (TDZ) concentration significantly influenced shoot formation; 4 μM TDZ was optimum, with 4.8 shoots produced per explant with a mean length of 7.1 cm after 120 days of culture. Regenerated shoots rooted for 60 days in basic medium with 1 μM NAA were ready for growth in pots. This is the first report on plantlet regeneration in vitro from mature trees of P. massoniana that provides a reliable method for propagating selected elites.     

Micropropagation of Vegetable Rennet (Withania coagulans [Stocks] Dunal)—A Critically Endangered Medicinal Plant

Withania coagulans (Stocks) Dunal (Solanaceae), popularly called vegetable rennet, is a critically endangered and highly valued medicinal plant. Overexploitation and reproductive failure forced the plant species toward the verge of complete extinction. We describe here the development of a simple, rapid, and cost effective in vitro micropropagation system for W. coagulans for mass-scale production of true-to-type plantlets using nodal shoot segments. Exactly 95.5 ± 0.34% explants responded within 8–10 days (d) and produced multiple shoot buds (4.1 ± 0.10 shoots of 2.95 ± 0.15 cm length) on 0.8% agar-gelled Murashige and Skoog's (MS) basal medium supplemented with 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), and additives (100 mg L^?1 L-ascorbic acid, 25 mg L^?1 each citric acid, adenine sulphate, and L-arginine). The shoots in cultures were multiplied by repeated transfer on MS medium with 4.44 μM BAP, 0.57 μM IAA, and additives. Further cultures were multiplied on a large-scale through the subculturing of shoot clumps differentiated in vitro, on MS medium supplemented with 1.11 μM BAP, 0.57 μM IAA, and additives. Maximum number (19.1 ± 0.28) of healthy (6.15 ± 0.25 cm) and viable shoots differentiated on this medium. The microshoots were rooted both in vitro and ex vitro. Exactly 67.3 ± 1.01% microshoots rooted in vitro within 25–30 d on agar-gelled half-strength MS salts supplemented with 29.52 μM indole-3-butyric acid (IBA) and 200 mg L^?1 of activated charcoal (AC). Alternatively, 73.8 ± 0.65% cloned shoots rooted on sterile soilrite (soilless compost and soil conditioner) under ex vitro conditions after pulse treatment with 2.46 mM IBA for 300 s. The clones of W. coagulans were hardened in a greenhouse within 40–45 d by slow and gradual exposure of plantlets from high relative humidity (RH; 70–80%) and low (26 ± 2°C) temperature to low RH (40–50%) and high (34 ± 2°C) temperature. The hardened plantlets were transferred to soil and stored in agro-net house with more than 90% survival rate. Replacement of pure and laboratory grade sucrose with commercial grade sugar, use of less expensive commercial grade agar-agar in culture medium, higher rate of shoot proliferation, single step ex vitro rooting, and hardening of plantlets in the greenhouse are advantageous features of the protocol. The micropropagation protocol defined here is reproducible, easy to follow, and would be helpful in large-scale restoration programs through true-to-type mass-multiplication of W. coagulans.     

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.     

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.     

In vitro clonal propagation of mature Tectona grandis through axillary bud proliferation

An in vitro clonal propagation procedure for mature Tectona grandis (teak) trees is described. Multiple shoots were induced from nodal segments through axillary bud proliferation. A shoot multiplication rate of 6.33 was achieved on Murashige and Skoog's (MS) medium supplemented with 10 μM 6-benzyladenine BA and 1 μM 1-naphthalene acetic acid (NAA) during every subculture cycle of 4 weeks. In vitro raised shoots could be successfully rooted (66.66%) on liquid MS medium supplemented with 15 μM NAA, with 1.60 roots per shoot, every 6 weeks of culture. In vitro hardening was carried out in sand soaked with half-strength MS medium (organic free). The plantlets were acclimatized first in a mist chamber and then in polybags in a mixture of soil, sand, and farmyard manure (1 : 1 : 1 v/v) in a shade house.     

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.     

Improvement of the tissue culture technique for <Emphasis Type="Italic">Melaleuca alternifolia</Emphasis>

Tea tree oil is extracted from the leaves and twigs of Melaleuca alternifolia (Maiden & Betche) Cheel, and it is widely used in medicines, food preservatives, cosmetics and health care products. Traditional propagation of M. alternifolia from seeds does not necessarily transfer the desired characteristics from their mother trees, the seedlings are not uniform, and the multiplication rate from cuttings is relatively low. For these reasons, it is necessary to develop tissue culture techniques for this species. This study showed that an efficient explant initiation medium for M. alternifolia was MS 1/2 + BA 0.6 mg L^?1 + NAA 0.1 mg L^?1 + sucrose 30 g L^?1, which yielded a 75.9 % initiation rate. An efficient multiplication medium was MS + BA 0.3 mg L^?1 + NAA 0.15 mg L^?1 + sucrose 30 g L^?1, which yielded a 4.3 multiplication rate and 3.2 cm shoot length. The rooting medium was MS 1/2 + IBA 0.1–0.25 mg L^?1 + sucrose 15 g L^?1, which yielded a 100 % rooting rate, 2.94–3.32 roots per individual and 1.36–1.44 cm root length. Local red-core soil was suitable as a transplant medium, and yielded 98 % survival. This study improved the tissue culture technique for mass-propagation of M. alternifolia, enabling the production of high quality plants for market.