Physiological,elemental, and stable isotope responses of the organs of mungbean to reduced atmospheric pressure

The effects of hypobaric conditions on stable isotope and mineral element concentrations during the germination of mungbean [Vigna radiata (Linn.) Wilczek] were evaluated. Mungbean seeds were cultured in lower atmospheric pressure (60 kPa) and normal air pressure (101 kPa) conditions, respectively. Oxygen and carbon dioxide partial pressures were maintained at 21 and 0.04 kPa, respectively. At 60 kPa, the fresh weight (FW) and dry weight (DW) of plants significantly increased by 5.41 and 9.62%, respectively, compared to those at 101 kPa after culturing for 7 d. Twelve mineral elements were compared among three organs (leaf, stem, and root) from seedlings grown under hypobaric and normal atmospheric conditions. This showed that lower air pressure generally improved element accumulation in the plant. A significantly lower value of δ ¹³C was observed at 60 kPa compared to that at 101 kPa. In addition, a significant increase in δ ¹⁵N value was detected in three different organs of plants grown under 60 kPa. Our survey provides a foundation for future field and laboratory studies on the influence of air pressure on plants, particularly in terms of stable isotope and mineral elements.     

Rapid in vitro production of true-to-type plants of Pogostemon heyneaus through dedieferentiated axillary buds

Summary Rapid propagation of Pogostemon heyneanus Benth. (Lamiaceae) was accomplished through culture of node explants on Murashige and Skoog (MS) medium containing N ⁶-benzyladenine (BA). Random amplified polymorphic DNA (RAPD) and gas chromatographic (GC) analysis of in vitro-derived progenies were used to determine the true-to-type nature of in vitro-derived plantlets. At the optimum level of BA (2.22μM), the axillary buds underwent a degree of dedifferentiation to become small globular green masses from which a mean of 17.1 shoots were developed within 40d. Retaining the culture without subenlture enhanced the number of shoots (>30 shoots). Inereased callus proliferation was observed at higher concentrations of BA in concomitance with a reduction in number of shoots. However, prolonged culture without subculture (more than 60d) initiated 25–30 shoot buds from the callus. Culture of node segments excised from in vitro shoots on fresh medium with optimal BA (2.22μM) exhibited a similar response, but with an increase of shoots (mean of 26.3 shoots per node) within 40d. Subeulture of shoot clumps on half-strength MS basal medium resulted in elongation (more than 4cm) of most of the shoots along with the development of new shoots. Shoots developed were rooted most successfully on half-strength MS medium with 4.9 μM indole-3-butyric acid (IBA). Plantlets derived from the best rooting medium established in small cups exhibited 95% survival. Plantlets successfully established in field conditions exhibited morphological characteristies identical to the source plant. The RAPD profile of the in vitro-derived plants and source plant, using 10 random primers, was similar. The gas chromatogram of the extracted oils from in vitro-derived plants and the source plant showed similar patterns.     

Effect of plant growth regulators on regeneration of plantlets from bud cultures ofCymbopogon nardus L. and the detection of essential oils from thein vitro plantlets

Cymbopogon nardus L. could be propagated via tissue culture using axillary buds as explants. The aseptic bud explants obtained using double sterilization methods produced stunted abnormal multiple shoots when they were cultured on Murashige and Skoog medium (MS) supplemented with 1.0 mg L^-1 or 2.0 mg L^-1 benzyladenine (BA). Stunted shoots that cultured on MS + 1.0 mg L^-1 BA + 1.0 mg L^-1 N⁶-isopentenyl-adenine (2iP) could induce elongation of shoots from about 60% of the stunted shoots. Normal multiple shoots could be induced at the highest (19.7 shoots per bud) from the bud explants within six weeks when cultured on proliferation medium consisted of MS supplemented with 0.3 mg L^-1 BA and 0.1 mg L^-1 indole-3-butyric acid (IBA). The separated individual shoot produced roots when transferred to basic MS solid medium. The essential oils that were contained in the mature plants namely citronellal, geraniol and citronellol were also found in thein vitro C. nardus plantlets. Citronellal was the main essential oil component in the matured plants while geraniol was the main component in thein vitro plantlets.     

Vegetative propagation of adult Eucalyptus grandis X urophylla and comparison of growth between micropropagated plantlets and rooted cuttings

Methods for the production of micropropagated plantlets and rooted cuttings were developed and used to vegetatively multiply adult Eucalyptus grandis X urophylla. Rooting success was less than 5% when cuttings excised from twigs of 3-year-old trees were used. The rooted cuttings were grown in the greenhouse as explant- or cutting-donors and maintained at a height of 30 to 100 cm by trimming back periodically. Good rooting success (95%) of cuttings was obtained for epicormic shoots produced from donor plants after trimming 5 times. Explants of both apical and axillary buds taken from the donor plants produced multiple shoots when cultured in vitro. In vitro multiple shoot production was optimal on MS medium containing 0.1 mg/l BA and 0.01 mg/l NAA averaging 13.7 shoots per explant in a 40-day culture period. Shoot elongation was accelerated on a modified MS medium containing half strength potassium nitrate and sucrose. Elongated shoots excised at approximately 1.5 cm in length were successfully rooted on media with NAA or IBA concentrations ranging from 0.1 to 10 mg/l. Root formation was optimal on medium consisting of full strength MS basal macro elements and vitamins, half strength micro elements, 1% sucrose and supplemented with 0.3 mg/l IBA. In the field test, no significant differences were found in tree height and DBH between micropropagated plantlets and rooted cuttings at 1 and 3 years old, with the exception at 2 years old. A considerable difference arose between the 2 types of vegetative propagules in physiological response to flowering, caused by dissimilar degrees of rejuvenation.Abbreviations BA Benzyl-Aminopurine - NAA Naphthalene Acetic Acid - IBA Indole-3-Butyric Acid - MS medium Murashige and Skoog's medium - DBH Diameter at Breast Height     

Micropropagation of Terminalia arjuna Roxb. from cotyledonary nodes

Cotyledonary node explants excised from 21 day old seedlings of T. arjuna produced multiple shoots when cultured on full strength MS or modified MS (1/2 strength major salts and Fe-EDTA) medium supplemented with different concentrations (0.1-1.0 mg/l) of BAP. Maximum 8.9 shoots/explant could be recorded after 30 days of inoculation on modified MS medium supplemented with BAP (0.5 mg/l). A proliferating shoot culture was established by reculturing the original cotyledonary nodes (2-3 times) on shoot multiplication medium after each harvest of the newly formed shoots. Shoots (each having 2-3 nodes/shoot) thus obtained were also used as a source of nodal explant that gave rise to 1-2 shoots when cultured on modified MS+BAP (0.5 mg/l) medium. Thus, 45-55 shoots could be obtained after 60 days of culture initiation from a single cotyledonary node. About 88% shoots rooted well after 15 hr pulse treatment with IBA (1 mg/l) in liquid MS medium followed by transfer to modified MS medium without IBA. About 80% of these plantlets were successfully acclimatized in plastic pots containing sand and soil mixture and 70% plantlets transferred in the field those survived even after 6 months of transplantation.     

Efficient regeneration and antioxidative enzyme activities in <Emphasis Type="Italic">Brassica rapa</Emphasis> var. <Emphasis Type="Italic">turnip</Emphasis>

The regeneration potential and antioxidative enzyme activities of economically important Brassica rapa var. turnip were evaluated. Calli were induced from leaf explants of seed-derived plantlets on Murashige and Skoog (MS) medium incorporated with different concentrations of various plant growth regulators (PGRs). The highest leaf explant response (83%) was recorded for 2.0 mg l⁻¹ benzyladenine (BA) and 1.0 mg l⁻¹ α-naphthaleneacetic acid (NAA). Subsequent subculturing of callus after 3 weeks of culture, on medium with similar compositions of PGRs, induced shoot organogenesis. The highest shoot induction response (83%) was recorded for 5.0 mg l⁻¹ BA after 5 weeks of transfer. However, 7.8 shoots/explant were recorded for 2.0 mg l⁻¹ BA. The transferring of shoots to elongation medium resulted in 5.1-cm-long shoots on 10 mg l⁻¹ of gibberellic acid (GA₃). Rooted plantlets were obtained on MS medium containing different concentrations of indole butyric acid (IBA). The determination of activities of antioxidative enzymes (superoxide dismutase [SOD], ascorbate peroxidase [APX], catalase [CAT], glutathione peroxidase [GPX], and peroxidase [POD]) revealed involvement of these enzymes in callus formation and differentiation. All of the activities were interlinked with each other and played significant roles in the scavenging of toxic free radicals. This study will help in the advancement of a regeneration protocol for B. rapa var. turnip and the understanding of the functions of antioxidative enzymes in plant differentiation.     

An improved system for the <Emphasis Type="Italic">in vitro</Emphasis> regeneration of <Emphasis Type="Italic">Thapsia garganica</Emphasis> via direct organogenesis – influence of auxins and cytokinins

An improved protocol for mass multiplication directly from leaf material of Thapsia garganicawas developed. Using factorial experimentation, auxins (NAA, IAA, 2,4-D) and cytokinin (BA, kinetin) combinations at 0–2 mg l⁻¹ added to Murashige and Skoog (MS) medium with 30 g l⁻¹ sucrose and 8 g l⁻¹ agar (pH 5.8) were tested for their effect on direct regeneration on leaflet and petiole explants. Of the media tested, the 0.5:1.5 NAA:BA medium was comparable for direct shoot organogenesis to the 2 mg l⁻¹ kinetin supplemented medium. However, when shoots were multiplied on these media, the 2 mg l⁻¹ kinetin without auxins was most effective as it kept the percentage of callus-derived plantlets to 3% and the number of hyperhydric shoots were minimal at a frequency of 2% compared to 25% on the 0.5:1.5 NAA:BA medium. The 2 mg l⁻¹ kinetin medium induced adventitious bud formation in 36% of the explants after 30 days. When the cultures were transferred to the same medium for multiplication, an average of six shoots (4.3 cm) were derived from each shoot base. Other combinations resulted in callus formation that either preceded shoot production or occurred together with adventitious shoot induction; whereas the 2 mg l⁻¹ medium resulted solely in adventitious buds that readily converted and elongated to shoots. On the 0.5:1.5 NAA:BA medium which tended to induce hyperhydric shoots in culture, agar (0.8, [w/v]) (15% hyperhydric plantlets) was more useful in maintaining a high health status in regenerating plantlets than gellan gum (Gelrite^®; 0.25, [w/v]) (60% hyperhydric plantlets). Although rooting in vitro was difficult, 58% of the propagules were successfully acclimatized when plants were exposed to fungicidal solutions as pre- and post-acclimatization treatments. A comprehensive protocol that allows for a reduction in mortality due to damping-off diseases during ex vitro transplantation of the in vitro-derived T. garganica plantlets is reported. The acclimatization procedure presented here is potentially suited to other umbelliferous species where fungal rots hamper ex vitro establishment.     

High frequency plant regeneration following abnormal shoot organogenesis in the medicinal tree Hovenia dulcis

An efficient plant regeneration protocol for shoot organogenesis from Hovenia dulcis callus cultures was established. Induction of organogenic callus was achieved on Murashige and Skoog (MS) medium supplemented with 4.65 μM kinetin and 4.5 μM 2,4-dichlorophenoxyacetic acid (2,4-D). Further differentiation of organogenic callus into primordia, shoot-like structures, and plantlets was achieved on MS medium supplemented with 0.23 μM gibberellic acid (GA₃) and 0.46 μM kinetin. Numerous abnormal shoots developed upon transfer of callus to MS medium containing cytokinins, and these failed to grow further into whole plantlets. However, transfer of ‘abnormal’ shoots to a fresh MS medium lacking cytokinins resulted in growth of normal shoots. Elongated shoots subsequently were rooted in basal MS medium, and whole plantlets were established in a soil mix. Analysis of regenerated plants using random amplified polymorphic DNA (RAPD) confirmed the genetic stability of these regenerant plantlets.     

In vitro shoot multiplication and plantlet regeneration from nodal explants of <Emphasis Type="Italic">Cassia angustifolia </Emphasis>(Vahl.): a medicinal plant

An efficient, rapid and reproducible plant regeneration protocol was successfully developed for Cassia angustifolia using nodal explants excised from 14-day-old aseptic seedlings. Of the two cytokinins, 6-benzyladenine (BA) and thidiazuron (TDZ) evaluated as supplements to Murashige and Skoog (MS) medium, TDZ at an optimal concentration of 5.0 μM was effective in inducing multiple shoots. The highest rate of shoot multiplication was achieved on MS medium supplemented with 5.0 μM TDZ and 1.0 μM indole-3-acetic acid (IAA) at pH 5.8. The regenerated shoots when subcultured on hormone free MS medium considerably increased the rate of shoot multiplication and shoot length by end of fourth subculture passage. Rooting was achieved on the isolated shoots using MS medium with 60 μM indole -3- butyric acid (IBA) and 1% activated charcoal for 1 week and subsequently transferring the shootlets to half strength MS liquid media without IBA and activated charcoal. The in vitro raised plantlets with well-developed shoot and roots were successfully established in earthen pots containing garden soil and grown in greenhouse.     

Micropropagation of Pistachio from mature trees

Multiple shoots were induced from nodal segments of mature trees of Pistacia vera L. on Murashige and Skoog (MS) medium supplemented with benzylaminopurine (BA). Maximum shoot production was obtained from shoot tips taken from in vitro proliferated shoots when cultured on solidified MS medium containing 8.8 μM BA. The multiplication rate was 20 microshoots per explant on the 30th day. Rooting of microshoots was achieved in MS medium supplemented with indole butyric acid (IBA). Rooted plantlets reassumed independent growth after a short period of acclimatisation. Stable regenerated plants were established in the greenhouse. This revised version was published online in June 2006 with corrections to the Cover Date.     

Plantlet regeneration from fascicular buds of seedling shoot apices of <Emphasis Type="Italic">Pinus roxburghii</Emphasis> Sarg

Shoot apices of Pinus roxburghii Sarg were cultured on Murashige and Skoog’s medium (MS) supplemented with cytokinins [6-benzyladenine (BA), kinetin and N-benzyl-9-(2-tetrahydropyranyl) adenine (BPA)] alone and in combination with auxin, α-napthaleneacetic acid (NAA). Of the three cytokinins tested at varying concentrations, medium supplemented with 10 μM BA was found optimal in respect of explant responsiveness (97.22 %) and average number of buds induced per explant (7.42). The concentration of cytokinins in the induction medium had a profound effect on rate of elongation of induced buds on MS basal medium containing 0.5 % activated charcoal. Further, shoots induced on lower concentrations of BA increased up to 2.4 times in length in 4 weeks. Decapitation of the explant enhanced the rate of axillary bud elongation. Proliferating shoot cultures were established by sub-culturing the axillary shoots on MS supplemented with 10 μM BA. Shoots 2–3 cm in length were suitable for culturing as more buds were induced on them compared to longer or shorter shoots. Root primordia were induced on 70.83 % shoots when transferred to 1/2 MS medium supplemented with 5.0 μM NAA. Elongation of root primordia (60 %) was achieved in liquid 1/2 MS basal medium. The plantlets were successfully transferred to soil after hardening; the time period from initiation of shoot buds to transplantation being 20–22 weeks.     

Plant regeneration from phyllode explants of Acacia crassicarpa via organogenesis

In this paper we report the establishment of Acacia crassicarpa regeneration through organogenesis. We used phyllode (leaf) explants excised from 60-day-old in vitro seedlings for green compact nodule induction and, tested Murashige and Skoog (MS) media supplemented with various concentrations of 1-phenyl-3-(thiadiazol-5-yl) urea (thidiazuron) (TDZ) and α-naphthaleneacetic acid (NAA). Under the optimized condition, green compact nodules and adventitious shoots were induced in 10 and 40 days, respectively, on the medium containing a combination of 0.5 mg l⁻¹ TDZ and 0.5 mg l⁻¹ NAA. This medium also yielded the highest rate (56%) of adventitious shoots forming from the nodules. Efficient shoot elongation was achieved by transferring the clusters of adventitious shoots to medium containing 0.1 mg l⁻¹ TDZ within 2 months. The elongated adventitious shoots were rooted at a rate of 96.5% on half-strength MS medium with 0.5 mg l⁻¹ 3-indolebutyric acid (IBA) in 1 month. Rooted plantlets were hardened and successfully established in soil with an 80% survival rate. To our knowledge, this is the first report describing a detailed protocol for regeneration through organogenesis using phyllodes as explants for A. crassicarpa.     

Liquid culture for efficient micropropagation of Wasabia Japonica (MIQ.) matsumura

Summary An efficient protocol for in vitro propagation of the valuable medicinal plant, Wasabia japonica (Miq.) Matsumura is described through shoot tip proliferation and direct regeneration. Multiple shoots were induced from shoort tips cultured on Murashige and Skoog (MS) semi-solid medium containing various concentrations (0.5–50 μM) of N⁶-benzyladenine (BA), thidiazuron, kinetin, and zeatin. A comparison was made on shoot multiplication between semi-solid and liquid culture media. Well-developed shoots were obtained using full-strength MS semi-solid medium containing 5.0 μM BA. However, the greatest shoot proliferation was achieved on either full- or half-strength MS liquid media supplemented with 5.0 μM BA for 4 wk (15.3±0.9 and 15.0±0.7 shoots per explant, respectively), and on half-strength MS liquid medium for 6 wk (25.8±1.3 shoots per explant) in culture. In contrast, the maximum number of shoots per explant on full-strength MS semi-solid medium was achieved with either 5.0 μM BA (10.4±0.6 shoots per explant) or 10.0 μM kinetin (10.9±0.8 shoots per explant). Fresh weight of explants and length of shoots derived from full-strength MS liquid medium (1055±77 mg and 34.2±1.0 mm, respectively) were significantly higher than those derived from full-strength MS semisolid medium (437.6±17.3 mg and 15.4±0.7 mm, respectively). Quarter-strength MS liquid medium had no significant difference in shoot proliferation when compared to quarter-strength MS semi-solid medium. Elongated shoots were separated and rooted on half-strength MS semi-solid media fortified with 1-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or indole-3-acetic acid (IAA) ranging from 0.1 to 10.0 μM. Root formation was greatest with IBA when compared with IAA and NAA. One hundred percent of shoots were rooted on half-strength MS medium with 5.0 μM IBA, while vigorous roots were obtained with 10.0 μM IBA. Micropropagated plantlets were successfully established in soil with 95% survival rate after heardening.     

Micropropagation of Spilanthes acmella Murr.

Multiple shoots of Spilanthes acmella Murr. were induced from hypocotyl segments obtained from 1-week-old seedlings on Murashige and Skoog's (MS) medium containing benzyladenine (BA), isopentenyl adenine, and naphthaleneacetic acid (NAA). High frequency shoot proliferation (95 %) and maximum number of shoots per explant (10 ± 0.6) were recorded with 0.5 mg dm^–3 BA in combination with 0.1 mg dm^–3 NAA. A proliferation was achieved by repeatedly subculturing the nodal segments on shoot multiplication medium. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing indole-3-butyric acid (1.0 mg dm^–3). 95 % of the plantlets were successfully acclimatized and established in soil. Transplanted plantlets showed normal flowering without any morphological variation.     

An improved micropropagation of <Emphasis Type="Italic">Eclipta alba</Emphasis> by in vitro priming with chlorocholine chloride

An efficient method of micropropagation for Eclipta alba from young nodal axils of shoot tip explants has been developed by giving special attention to ‘priming’ in vitro plantlets in view of increasing their hardening ability after transplantation ex vitro. Among 3 cytokinins—BAP, kinetin and TDZ, BAP was found most effective in inducing and proliferating adventitious shoots. The highest frequency of responding explants (100%) and maximum number of shoots (23.0) per explant were obtained after 60 days culture on MS medium containing 8.8 μM BAP. Cent percent shoots developed roots directly from shoot base when transferred to growth regulator-free MS medium. For priming E. alba microshoots, 6.3 μM of chlorocholine chloride (CCC) was found most effective. The major changes observed in 30 days old treated shoots were, production of increased number of root, elevation of chlorophyll level in leaves and increase in plant biomass. Furthermore, arrested undesirable shoot elongation made the plants sturdier and more suitable for acclimatization. The primed micropropagated E. alba plants were healthy and survived by higher frequency (100%) in soil in comparison to the non-treated plants (84% survival).     

In vitro Micropropagation of a Medicinal Plant Species Sophora flavescens

A micro-propagating system based on the young stem node segments of Sophora flavescens Ait. (Fabaceae) was established. Murashige and Skoog (MS) basal medium supplemented with 8.88 μM 6-benzyladenine (BA) plus 2.69 μM α-naphthalene acetic acid (NAA) and that with only 5.37 μM NAA were found the best in promoting proliferation of shoots and induction of root, respectively. Percentages of shoot induction and number of shoot per explant were up to 93.4 % and 4.2 and rooting rate to 82.4 %, respectively. The segments of the regenerated shoots could be continuously induced to reproduce new shoots through subculture on the same medium in 30-d intervals and still kept this activity after being subcultured for 6 generations. After the regenerated plantlets were transplanted to field, they grew well, showing no any visible abnormalities. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sodium nitroprusside promotes multiplication and regeneration of <Emphasis Type="Italic">Malus hupehensis</Emphasis> in vitro plantlets

The effects of sodium nitroprusside (SNP) on the multiplication, regeneration and rooting of Malus hupehensis Rehd. var. pinyiensis Jiang in tissue culture have been investigated. The results showed that the multiplication of plantlets was promoted significantly by applying 20 μM SNP to the Murashige and Skoog (MS) medium containing 2.0 μM 6-benzylaminopurine (BA) and 1.0 μM zeatin (ZT). Multiplication of plantlets from the 1st subculture was more sensitive to SNP than that from the 4th or 7th subculture. The differentiation and regeneration of adventitious shoots from leaves or cotyledons increased significantly when 20–30 μM SNP was supplied to the medium MS containing 25 μM BA, 2.5 μM α-naphthaleneacetic acid (NAA) and 2.5 μM ZT. Adventitious shoots regeneration frequency from cotyledons was higher than that from leaves at the presence of SNP. The rooting of plantlets was promoted by SNP significantly and the best result for rooting was achieved in the half-strength MS medium containing 75 μM SNP. In addition, adventitious roots without callus distributed at the base of shoots when SNP was supplied.     

Somatic embryogenesis and organogenesis of Agave victoriae-reginae: Considerations for its conservation

Agave victoriae-reginae somatic embryos were produced through a callus phase from seedling stem segments cultured on MS medium. The optimal treatment was MS medium with 2.26 M 2,4-D. Multiple shoot regeneration was induced from axillary buds from stem segments cultured on MS medium with 2.2–4.4 M BA. Effect of MS and modified MS medium with 50% macronutrient concentration, both containing 2.2 M BA and sucrose at the following concentrations, 20, 30, 45 and 60 g l^–1, resulted in inconsistent multiple shoot formation. Shoots and somatic embryos formed by this indirect pathway could have a multicellular origin, which might lead to genetic variation. The direct development of pre-existent buds occurred on MS basal medium and increased in the presence of BA; this might be a pathway for the rescue of genotypes of endangered species. Embryos and shoots developed and grew roots on MS medium. Complete plantlets were obtained on MS basal medium. A total of 92% per cent of the plantlets survived and grew when transferred to the greenhouse. Agave micropropagation could supply the commercial plant demand, diminishing the gathering of seeds and plants of this endangered species from the wild.     

Micropropagation in Peanut (Arachis hypogaea L.)

Multiple shoots in Arachis hypogaea L. could be induced from the de-embryonated cotyledons (DC), embryo-axes (EA) and mature whole seeds (MWS) in MS medium supplemented with different levels of benzylaminopurine (BAP). DC was the most suitable explant with 57.9 % induction and more than 40 shoots per explant in 31.6 % of cases. Though EA and MWS had high percent induction at or above 30 mg dm^–3 BAP, only 10 – 14 shoots per explant were observed. In DC, multiple shoots were confined to the proximal end and in EA they originated from the axillary bud region. Histological studies on DC confirmed the origin of shoots from the region of attachment with the embryo. Shoots could be rooted in MS medium containing 2 g dm^–3 charcoal and 200 mg dm^–3 casein hydrolysate. Sixty percent of the rooted plantlets could be established in the field.     

Micropropagation of Bixa orellana using phytohormones and triacontanol

An efficient micropropagation protocol for annatto (Bixa orellana L.) was achieved using nodal shoot tip explants. Shoot buds were obtained on the Murashige and Skoog (MS) medium supplemented with various concentrations and combinations of indole-3-acetic acid (IAA), N⁶-benzyladenine (BA) and triacontanol (TRIA). Maximum of 213 shoot buds along with 18 primary shoots were produced on MS medium containing 0.05 μM IAA, 8.87 μM BA, and 11.2 μM TRIA. The primary shoots elongated best on MS medium containing 6.66 μM BA and 2.45 μM indole-3-butyric acid (IBA). The regenerated shoots rooted best on MS medium supplemented with 4.9 μM IBA. The in vitro rooted plantlets were hardened and establishment rate under field conditions was 70 to 80 %.