Micropropagation of <Emphasis Type="Italic">Vitex agnus-castus</Emphasis>, (Verbenaceae)—a valuable medicinal plant

This study describes in vitro shoot induction and plant regeneration from a mature apical meristem and nodal explants of the endangered medicinal shrub Vitex agnus-castus. Multiple shoots were induced directly from the axis of nodal and apical meristem explants on Murashige and Skoog (MS) medium containing 3% sucrose and different concentrations (1.0, 1.5, 2.0, and 2.5 mg/l) of 6-benzyl aminopurine (BAP) in combination with Kinetin (Kin) and α-naphthalene acetic acid (NAA), both at 0.1 mg/l. BAP and Kinetin were used as supplements to MS basal medium, either individually or in combination with auxins. The optimal concentration of BAP for inducing bud break was found to be 2.0 mg/l when Kinetin was at 0.1 mg/l. Regeneration frequency was highest for both apical meristem and nodal explants (94.5% and 90.3%, respectively) when explants were cultured on MS medium supplemented with BAP (2.0 mg/l) and Kin (0.1 mg/l). A maximum of 7.7 ± 0.4 and 6.7 ± 0.2 shoots were obtained per explant for apical meristem and nodal explants, respectively. Regenerated shoots, transferred to MS medium supplemented with either 1.0 or 1.5 mg/l BAP combined with 0.1 mg/l GA₃, showed maximum elongation of 6.7 ± 0.4 and 6.0 ± 1.3 cm in apical meristem and nodal explants, respectively. In vitro regenerated shoots transferred to half-strength MS medium supplemented with 0.1 mg/l IBA induced 90.4% of the shoots to form roots after 30–35 d of culture. Up to 80% of the regenerated shoots were successfully established in soil in the greenhouse.     

High efficiency <Emphasis Type="Italic">in vitro</Emphasis> plant regeneration from epicotyl explants of Chinese peanut cultivars

An efficient organogenesis and micropropagation system was developed for in vitro plant regeneration of multiple cultivars of peanut (Arachis hypogaea). The system was used to regenerate plants from nine cultivars: Luhua no. 9, Luhua no. 13, Luhua no. 14, Fenghua no. 1, Fenghua no. 3, Fenghua no. 5, Huayu no. 23, Qinglan no. 2, and Baisha 1016. Epicotyl and embryo axis explants were cultured on Murashige and Skoog (MS) basal medium supplemented with 33.29–44.39 μM 6-benzyladenine (BAP) and 2.15–4.30 μM α-naphthaleneacetic acid (NAA). The highest rate of shoot formation was observed in epicotyl explants taken from 4-d-old seedlings (5.1 ± 1.4 shoots per explant). Optimum shoot development was observed in explants cultured on MS medium containing 4.48 μM BAP and 2.89 or 5.78 μM gibberellin (GA₃). Well-developed shoots (3–5 cm high) formed roots after 2 wk on MS medium containing 0–2.69 μM NAA. We observed that all multiple shoots formed at the site of epicotyl incision and at the upper end of each section, indicating physiological polarity of shoot formation. The maximum shoot induction rate for Luhua no. 9, Luhua no. 13, Luhua no. 14, Fenghua no. 1, Fenghua no. 3, Fenghua no. 5, Huayu no. 23, Qinglan no. 2, and Baisha 1016 was 60.0%, 83.3%, 80.7%, 91.5%, 86.0%, 59.7%, 75.0%, 67.3%, and 72.7%, respectively. This regeneration system will play a vital role in achieving the genetic improvement of Chinese peanut.     

Micropropagation of a tropical fruit tree Spondias mangifera Willd. through direct organogenesis

An efficient in vitro propagation is described for Spondias mangifera Willd., a medicinally important tree, using nodal explants obtained from 4-week-old seedlings. The frequency of shoot regeneration from seedling node was affected by various concentrations of BAP and successive transfer of mother explant. MS (Murashige and Skoog, Physiol Plant 15:473–497, 1962) medium supplemented with 1.0 mg l⁻¹ of 6-benzylaminopurine (BAP) was optimal for shoot multiplication. Upon this medium, highest number of shoots (about 10.6) per explants was obtained after fourth subculture of mother explants. Half-strength MS medium containing IAA (1.0 mg l⁻¹) was most effective for rooting of shoots. Regenerated plantlets were successfully acclimatized and transferred into soil with 80–90% survival rate. The regenerated plants were morphologically uniform and exhibited similar growth characteristics and vegetative morphology to the mother plants. This is the first report on micropropagation of S. mangifera, which can be applied for further genetic transformation assays and pharmaceutical purposes.     

An improved protocol for micropropagation of Mallotus repandus (Willd.) Müll.Arg.

Node and internode explants of Mallotus repandus were precultured on basal medium (BM: Murashige and Skoog (MS) medium with 3% sucrose and 0.55% Agargel) for 0–18 d before culture on shoot induction Medium (SIM: BM added with 4.44 μM of benzylaminopurine) for 4 wk. The cultures were subsequently transferred to BM for 4 wk for shoot elongation. Node explants precultured on BM for 14 d before incubation on SIM were at an optimum for shoot regeneration with the response rate of 95%, compared to a 21% response for the control without preculture. Internode explants precultured on BM for 16 d responded with an optimal shoot formation response rate of 69%, whereas the control response rate was 6%. The maximum shoot regeneration rates were 3.1 ± 0.3 and 2.7 ± 0.4 shoots/responding explant in node and internode explants, respectively. This study demonstrates for the first time that shoot organogenesis can be induced from internode explants of M. repandus. Furthermore, the results suggest that the explants need to acquire competence before shoot organogenesis. Rooting was obtained by incubation of regenerated shoots on half-strength MS with 10.74 μM of 1-naphthylacetic acid for a week before culture on half-strength MS for 4 wk. Regenerated plants were successfully transferred to soil.     

Micropropagation and slow growth conservation of cardamom (<Emphasis Type="Italic">Elettaria cardamomum</Emphasis> Maton)

Cardamom (Elettaria cardamomum Maton) has great commercial value as a spice crop in India. A one-step protocol for direct regeneration of plants and in vitro conservation by slow growth method has been developed. A maximum of 6.5 shoots/culture were obtained in 2 mo or 15.1 shoots/culture in 4 mo on Murashige and Skoog (Physiol Plant 15:473–497, 1962) medium (MS) + 5 μM benzylaminopurine gelled with 0.7% agar (micropropagation medium). Rooting also occurred simultaneously on the same medium. Using one shoot tip or nodal explant, about 30,375 plants can be regenerated in a year on the micropropagation medium. In vitro conservation by slow growth method was achieved on 1/2 MS (major salts) + 5 μM BAP + 0.7% agar (conservation medium); about 70% of the cultures survived up to 18 mo at 25 ± 2°C. Successful regrowth of plants on micropropagation medium was obtained by culturing nodal explants excised from 18-mo-old conserved plants. Some 96% of the plants survived the hardening treatment and grew normally in a greenhouse. If 24 cultures are conserved on the conservation medium, it is possible to regenerate at least 750 plants by using explants derived from 70% of the surviving shoots and culturing the same in micropropagation medium for 4 mo. These plants may be used for planting or as a source of explants for the next conservation cycle. On the basis of 20 random amplified polymorphic DNA and 13 inter-simple sequence repeat primers analyses, no significant reproducible variation was detected among the in vitro-conserved plants compared with the mother plants.     

An improved micropropagation of <Emphasis Type="Italic">Spilanthes acmella</Emphasis> L. through transverse thin cell layer culture

An efficient and improved in vitro propagation system for Spilanthes acmella L. using transverse thin cell layer (tTCL) culture system was established. The frequency of shoot regeneration from tTCL nodal segments was affected by concentrations of plant growth regulators and orientation of the explant. MS (Murashige and Skoog in Physiol Plant 15:473–497, 1962) medium with 5.0 mg dm⁻³ BAP was optimal for shoot regeneration. Upon this medium, the explant inoculated in the upright orientation exhibited a high frequency of shoot regeneration (about 97%), and the highest number of shoots (31.5) per explant. The intact node (1.0–1.5 cm) cultured on the same medium had significantly lower shoot multiplication ability with only 4.5 shoots per responsive explant. As compared to BAP alone, the combination of BAP and Kin or NAA did not have positive effects on shoot multiplication from tTCL nodal segments. Rooting of shoots was achieved on growth regulator free full-strength MS medium. Plantlets were transplanted into soil with 90–100% survival rate.     

Multiple Shoot Regeneration from Immature Embryo Explants of Papaya

A simple and rapid method for multiple shoot formation in vitro from immature embryo axis explants of Carica papaya L. cvs. Honey Dew, Washington and Co2 is described. Multiple shoot regeneration was achieved by culture of the explants on modified Murashige and Skoog (MS) medium supplemented either with thidiazuron (TDZ; 0.45–22.7 μM) or a combination of benzylaminopurine (BAP; 0.2 – 8.84 μM) and naphthalene acetic acid (NAA; 0.5 – 2.64 μM). Highest frequency of shoot regeneration occurred on medium supplemented either with 2.25 μM TDZ or a combination of BAP (4.4 μM) and NAA (0.5 μM). Composition of the basal media influenced the frequency of multiple shoot initiation. Stunted shoots regenerated at 4.5 μM and higher concentrations of TDZ. Such shoots could, however, be elongated by transfer to medium containing 5.7 μM GA₃. Rooting of the regenerated shoots was achieved in presence of indolebutyric acid (IBA; 4.92 – 19.68 μM), however, least response was in presence of 14.7 μM IBA. Rooted plants were hardened and transferred to pots. This revised version was published online in July 2006 with corrections to the Cover Date.     

Indirect regeneration of the Cancer bush (<Emphasis Type="Italic">Sutherlandia frutescens</Emphasis> L.) and detection of <Emphasis Type="SmallCaps">l</Emphasis>-canavanine in <Emphasis Type="Italic">in vitro</Emphasis> plantlets using NMR

The present study reports a simple protocol for indirect shoot organogenesis and plant regeneration of Sutherlandia using rachis and stem segments. Different concentrations (0.0–68.08 μmol l⁻¹) of thidiazuron (TDZ) were used for callus induction and shoot organogenesis. The highest percentage of callus formation (97.5%) and the highest percentage of explants forming shoots (88.8%) were obtained from rachis explants cultured onto Murashige and Skoog (MS) medium (Murashige and Skoog, Physiol. Plant. 15:473–495, 1962) supplemented with 45.41 μmol l⁻¹ TDZ. Scanning electron microscopy demonstrated the early development of adventitious shoots derived from callus cultures. Shoot clusters were further developed and grown in MS hormone-free medium. The presence of l-canavanine was determined by thin-layer chromatography and confirmed after column fractionation using silica gel and nuclear magnetic resonance spectroscopy. Individual shoots were rooted on different concentrations and combinations of MS salt strength and IBA. Half-strength MS salt medium supplemented with 24.6 μmol l⁻¹ IBA was optimal for root induction in which 78% of shoots were rooted. The in vitro plants were successfully acclimatized in a growth chamber with a 90% survival rate.     

Auxin pretreatment promotes regeneration of sugarcane (Saccharum spp. hybrids) midrib segment explants

We have developed a new, simple, quick and genotype-independent method for direct regeneration of sugarcane using novel midrib segment explants. Our protocol involves two steps: the pretreatment of starting material on MS (Murashige and Skoog (1962) Physiol Plant 15:473–497) medium containing 3.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D) for 8 days under continuous dark and subsequent transfer of the explants to MS medium augmented with 0.1 mg/l benzyladenine (BA) and 0.1 mg/l naphthaleneacetic acid (NAA) under light-dark conditions. On the regeneration medium, numerous globular structures appeared from the explants and subsequently differentiated into shoots. Regenerated shoots attained 2–5 cm height within 30 days of culture initiation and readily rooted on MS basal medium. Hardened plants were successfully established in the greenhouse. The regulation of sugarcane morphogenesis by auxin pretreatment is discussed.     

Plantlet regeneration from leaf derived callus of <Emphasis Type="Italic">Vanilla planifolia</Emphasis> Andr.

Vanilla planifolia is a tropical orchid mainly known for the aromatic flavor of its cured pods. Callus cultures were initiated from leaf and nodal explants of V. planifolia. Leaf explants showed better callus initiation than the nodal explants with callus biomass production maximal when cultured on Murashige and Skoog (MS) basal medium containing 4.52 mM 2,4-dichlorophenoxy acetic acid and 2.22 mM benzyladenine (BAP). Callus transferred to MS basal medium supplemented with 13.32 μM BAP, and 13.43 μM naphthaleneacetic acid (NAA) showed superior growth response and produced 14.0 ± 1.0 shoots with an average length of 3.6 ± 0.1 cm after 40 d. Subsequent transfer of the proliferated shootlets to MS basal medium supplemented with 8.88 μM BAP and 8.08 μM NAA produced 12.3 ± 0.14 plantlets with an average height of 3.6 cm ± 0.10 cm. All plantlets produced profuse rooting within 35–40 d after transfer to half-strength MS basal medium supplemented with NAA in combination with indole-3-acetic acid. Rooted plantlets were transferred for hardening, with 80% of the plantlets becoming successfully established in the field. Potentially, more than 100,000 plantlets could be produced within eight subcultures from callus obtained from leaf explant through the methods described here.     

Highly efficient plant regeneration and <Emphasis Type="Italic">in vitro</Emphasis> polyploid induction using hypocotyl explants from diploid mulberry (<Emphasis Type="Italic">Morus multicaulis</Emphasis> Poir.)

Efficient plant regeneration is essential for successful transformation and in vitro polyploidy induction in mulberry. A high frequency (80%) of plant regeneration from hypocotyls occurred under in vitro conditions in mulberry (Morus multicaulis Poir.). We identified three key factors for enhancing successful regeneration based on earlier work: (1) hypocotyl position, (2) the combination and concentration of growth regulators, and (3) the addition of AgNO₃. The highest frequency of shoot regeneration was achieved using hypocotyl segments, which are proximal to apical meristems, and the optimal culture conditions were Murashige and Skoog’s (MS) (Murashige and Skoog, 1962) basal medium supplemented with 3.0 mg l⁻¹ 6-benzylamino purine, 0.3 mg l⁻¹ indole-3-acetic acid, 0.1% polyvinypyrrolidone, and 1.0 mg/l silver nitrate (AgNO₃) under subdued light at 25 ± 2°C. Treating the shoots with 0.2% colchicine (dipping for 72 h) resulted in a 14% tetraploid frequency, whereas a 20% tetraploid frequency resulted from using a 0.25% colchicine (dripping for 5 d) treatment, as determined by chromosome number counts. The diploid plant chromosome number was 28 (2n = 2x = 28) and that of tetraploid plants was 56 (2n = 4x = 56). Regenerated shoots rooted easily in 8–10 d using half-strength basal MS medium with 0.5 mg l⁻¹ indole-3-butyric acid and were successfully established in the soil.     

High efficiency <Emphasis Type="Italic">In vitro</Emphasis> plant regeneration from cotyledon explants of <Emphasis Type="Italic">Incarvillea sinensis</Emphasis>

Summary A simple and effective procedure has been developed for plantlet regeneration from cotyledon-derived callus of the medicinally important herb and ornamental species, Incarvillea sinensis. An average of 18.4 adventitious shoots per explant were obtained from 100% cotyledon explants cultured on half-strength Murashige and Skoog (MS) medium containing 1.0 mg l⁻¹ 6-benzylaminopurine for 3 wk, followed by another 4 wk on hormone-free 1/2×MS medium. The cotyledon explants continued to expand and regenerate new shoots upon repeated subculturing onto fresh medium. Most regenerated shoots (66.9%) were rooted on 1/4×MS mediumcontaining 1.0 mg l⁻¹ indole-3-acetic acid, with an average of about 3.8 roots per shoot. Regenerated plants with well developed shoots and roots were successfully acclimatized in soil and were normal phenotypically.     

Improved plant regeneration in Capsicum annuum L. from nodal segments

Multiple shoots were induced by culturing nodal explants excised from 1-month-old aseptic seedlings of red pepper (Capsicum annuum L. cv. Pusa Jwala) on Murashige and Skoog (MS) medium supplemented with (0.1–10 μM) thidiazuron (TDZ). The rate of multiple shoot induction per explant was maximum (14.4 ± 0.06) on MS medium supplemented with 1.0 μM TDZ. Regenerated shoots were elongated well on growth regulator free MS medium. Adventitious roots were induced two weeks after transfer of elongated shoots to MS medium supplemented with auxins (IAA, IBA or NAA) in different concentrations. Optimum root formation frequency was obtained in medium containing 1.0 μM IBA. Ex-vitro rooting was also achieved by pulse treatment with 300 μM IBA for 10 min. Rooted shoots were transplanted in plastic pots containing garden soil (with 90 % survival rate), where they grew well and attained maturity. Regenerated plants were phenotypically and cytologically normal.     

Factors affecting plantlet regeneration from <Emphasis Type="Italic">in vitro</Emphasis> cultured immature embryos and cotyledons of <Emphasis Type="Italic">Prunus mume</Emphasis> “Xue mei”

Using immature embryos and cotyledons as explants, a successful system to culture immature embryos and induce direct regeneration from cotyledons was established for Prunus mume “Xuemei”. For immature embryo culture, a high frequency of plantlet formation (89.5%) from the embryonic axis was obtained using half-strength Murashige and Skoog (1/2 MS) medium supplemented with 13.2 μM 6-benzyladenine (BA) and 2.7 μM 1-naphthaleneacetic (NAA). Shoots formed directly from cotyledons with the embryo axis intact when explants were cultured on 1/2 MS medium containing 2.2 μM BA with different combinations of NAA (2.7, 5.4 μM) and indole-3-butyric acid (IBA) (0, 2.5, 5.0 μM). Better results were achieved when the embryonic axis was removed from the cotyledons and cultured on 1/2 MS medium supplement with 13.2 μM BA, 2.7 μM NAA or 2.2 μM BA, 2.2 μM thidiazuron (TDZ), and 2.7 μM NAA, respectively. Regenerated shoots were successfully rooted on 1/2 MS or Woody Plant medium (WPM) supplemented with 2.5–5.0 μM IBA. The effect of the embryonic axis, BA, and TDZ on cotyledon regeneration was investigated in detail. Rooted plantlets were transferred to soil successfully.     

<Emphasis Type="Italic">In vitro</Emphasis> adventitious shoot organogenesis and plant regeneration from seedling explants of <Emphasis Type="Italic">Albizia odoratissima</Emphasis> L.f. (Benth.)

Epicotyl, petiole, and cotyledon explants derived from 14-d-old seedlings of Albizia odoratissima were cultured on Murashige and Skoog (MS) basal medium supplemented with different concentrations of either 6-benzylaminopurine (BAP) solely or in combination with 0.5 μM naphthalene-3-acetic acid (NAA). The percentage of shoot regeneration and the number of shoots regenerated varied significantly depending on the type of explants used, the concentration of plant growth regulators, and the orientation of explants on the culture medium. The best response in terms of the percentage of shoot regeneration was obtained from epicotyls cultured horizontally on MS medium supplemented with 5 μM BAP, whereas the highest number of shoots per responding explant was recorded on medium containing 2.5 μM BAP and 0.5 μM NAA. Successful rooting was achieved by placing the microshoots onto MS medium containing 25 μM indole-3-butyric acid (IBA) for 24 h first, then transferring to the same medium without IBA. Of the various substrates tested, vermiculite was the best for plant acclimatization, as 75% of the plants survived and became established.     

An efficient protocol for large-scale plantlet production from male floral meristems of <Emphasis Type="Italic">Musa</Emphasis> spp. cultivars Virupakshi and Sirumalai

In vitro propagation has played a key role for obtaining large numbers of virus free, homogenous plants, and for breeding of plantains and bananas (Musa spp.). Explant sources utilized for banana micropropagation include suckers, shoot tips, and floral buds. The present study employed male floral meristems as explant material for micropropagation of hill banana ecotypes (AAB) ‘Virupakshi’ and ‘Sirumalai.’ Immature male floral buds were collected from healthy plants from hill banana growing areas. Exposure of explants to ethyl alcohol (70%, v/v) for 30 s, then mercuric chloride (0.1%, w/v) for 30 s, followed by three independent rinses of 5 min each in autoclaved, double-distilled water satisfactorily reduced the contamination. Male floral bud explants were cultured on Murashige and Skoog (MS) basal medium supplemented with different combinations of 6-benzylaminopurine (BAP), coconut water, naphthaleneacetic acid, gibberellic acid, and additional supplements. MS medium supplemented with 5 mg l⁻¹ BAP and coconut water (15%) was the most efficient media for shoot initiation and multiple shoot formation (15 shoots from a single part of a floral bud). The best response for shoot elongation was obtained using the combination of basal MS, 5 mg l⁻¹ BAP, 1 mg l⁻¹ naphthaleneacetic acid and 1.5 mg l⁻¹ gibberellic acid. Regenerated shoots were rooted in basal MS medium within 15–20 d. The rooted plantlets were transferred to a soil mixture and maintained at a temperature of 25 ± 2°C for 10 d and then at room temperature (30–32°C) for 2 wk, before transferring to a greenhouse. The regenerated plantlets showed 100% survival.     

In vitro Plant Regeneration of Melia azedarach L.: Shoot Organogenesis from Leaf Explants

In vitro regeneration of Melia azedarach L. was studied. Shoots were regenerated from calli initiated from leaflets of in vitro growing plants. The best medium for establishment of cultures was Murashige and Skoog (MS) medium with 4.44 μM benzylaminopurine (BAP) + 0.46 μM kinetin (KIN) + 16.29 μM adenine sulphate (ADE). Regenerated shoots were multiplied in MS + 0.44 μM BAP + 0.37 μM KIN + 3.26 μM ADE. Maximal rooting of 89 % was achieved by culture of regenerated shoots in MS + 12.26 μM indole-3-butyric acid for 3 d and subsequently in MS lacking growth regulators for 27 d. Rooted shoots were acclimatized and successfully transferred to soil. This revised version was published online in July 2006 with corrections to the Cover Date.     

Direct shoot bud induction and plant regeneration in <Emphasis Type="Italic">Capsicum frutescens</Emphasis> Mill.: influence of polyamines and polarity

Direct shoot bud induction and plant regeneration was achieved in Capsicum frutescens var. KTOC. Aseptically grown seedling explants devoid of roots, apical meristem and cotyledons were inoculated in an inverted position in medium comprising of Murashige and Skoog (Physiol Plant 15:472–497, 1962) basal medium supplemented with 2-(N-morpholine) ethanesulphonic acid buffer along with 2.28 μM indole-3-acetic acid, 10 μM silver nitrate and either of 13.31–89.77 μM benzyl adenine (BA), 9.29–23.23 μM kinetin, 0.91–9.12 μM zeatin, 2.46–9.84 μM 2-isopentenyl adenine. Profuse shoot bud induction was observed only in explants grown on a media supplemented with BA (26.63 μM) as a cytokinin source and 19.4 ± 4.2 shoot buds per explant was obtained in inverted mode under continuous light. Incorporation of polyamine inhibitors in the culture medium completely inhibited shoothoot bud induction. Incorporation of exogenous polyamines improved the induction of shoot buds under 24 h photoperiod. These buds were elongated in MS medium containing 2.8 μM gibberellic acid. Transfer of these shoots to hormone-free MS medium resulted in rooting and rooted plants were transferred to fields. This protocol can be efficiently used for mass propagation and presumably also for regeneration of genetically transformed C. frutescens.     

Somatic embryo productions by liquid shake culture of embryogenic calluses in <Emphasis Type="Italic">Vigna mungo</Emphasis> (L.) Hepper

The regeneration of plants via somatic embryogenesis liquid shake culture of embryogenic calluses was achieved in Vigna mungo (L.) Hepper (blackgram). The production of embryogenic callus was induced by seeding primary leaf explants of V. mungo onto Murashige and Skoog (MS) (Physiol Plant 15:473–497, 1962) medium supplemented (optimally) with 1.5 mg/l 2,4-dichloro-phenoxyacetic acid. The embryogenic callus was then transferred to liquid MS medium supplemented (optimally) with 0.25 mg/l 2,4-dichloro-phenoxyacetic acid. Globular, heart-shaped, and torpedo-shaped embryos developed in liquid culture. The optimal carbohydrate source for production of somatic embryos was 3% sucrose (compared to glucose, fructose, and maltose). l-Glutamine (20 mg/l) stimulated the production of all somatic embryo stages significantly. Torpedo-shaped embryos were transferred to MS (Physiol Plant 15:473–497, 1962) liquid medium containing 0.5 mg/l abscisic acid to induce the maturation of cotyledonary-stage embryos. Cotyledonary-stage embryos were transferred to 1/2-MS semi-solid basal medium for embryo conversion. Approximately 1–1.5% of the embryos developed into plants.     

Rapid clonal propagation of Vitex trifolia

This report describes in vitro shoot induction and plant regeneration from mature nodal explants of Vitex trifolia L. on Murashige and Skoog (MS) medium fortified with benzylaminopurine (BAP), kinetin (KN), thidiazuron (TDZ), adenine (ADE), and 2-isopentenyladenine (2-iP) (0.25 – 10.0 μM). Multiple shoots differentiated directly without callus mediation within 3 weeks when explants were cultured on medium supplemented with cytokinins. The maximum number of shoots (9 shoots per explant) was developed on a medium supplemented with 5.0 μM BAP. Shoot cultures was established repeatedly subculturing the original nodal explant on the same medium. Rooting of shoots was achieved on half strength MS medium supplemented with 0.5 μM naphthaleneacetic acid (NAA). Rooted plantlets transferred to pots containing autoclaved soil and vermiculite mixture (1:1) showed 90 % survival when transferred to outdoor.