Highest Shoot Regeneration Frequency Research Articles

Thidiazuron- and Zeatin-induced High-frequency Shoot Regeneration from Leaf and Shoot-tip Explants of Strawberry

Strawberry (Fragaria ×ananassa cv. Camarosa) was evaluated to determine a high-frequency shoot regeneration response for leaf and shoot-tip explants. For direct organogenesis from strawberry leaves, combinations of moderate concentrations of thidiazuron [TDZ (0, 2, and 4 μm)] and 6-benzylaminopurine [BAP (0, 4, and 9 μm)] added into medium containing Murashige and Skoog (MS) basal salts were compared. The most shoots regenerated per leaf explant were observed with 4-μm TDZ. Regeneration from shoot tips was evaluated with 0-, 2-, 4-, 8-, and 16-μm zeatin, kinetin, or 6-α,α-dimethylallylamino purine (2ip) tested individually. Optimum shoot proliferation was achieved from shoot-tip explants on medium containing 4-μm zeatin. Rooting was best without cytokinins in the medium; however, adequate rooting was obtained on the 4-μm zeatin treatment as well.

Lipid peroxidation, H<sub>2</sub>O<sub>2</sub> content, and antioxidants during acclimatization of Abrus precatorius to ex vitro conditions

An efficient, rapid, and reproducible plant regeneration protocol was successfully developed for Abrus precatorius L. using mature nodal explants excised from a 5-year-old field grown plant. The highest shoot regeneration frequency (87 %) with maximum number of multiple shoots (15.0) and shoot length (4.8 cm) were recorded on Murashige and Skoog (MS) medium amended with 2.5 μM thidiazuron, 120 mg dm−3 polyvinylpyrrolidone, and 0.5 μM α-naphthalene acetic acid. The best treatment for maximum root (4.0) induction was half strength MS medium supplemented with 1.5 μM indole-3-butyric acid. The in vitro plantlets with well-developed shoots and roots were successfully transferred into plastic cups with Soilrite and acclimatized in a culture room under photon flux density (PFD) of 150 μmol m−2 s−1, thereafter transferred to a greenhouse with PFD of 300 μmol m−2 s−1, and finally to a field with 70 % survival rate. During the acclimatization period (0–49 d), leaf chlorophyll and carotenoid content increased whereas malondialdehyde and H2O2 content decreased probably due to increasing activities of antioxidant enzymes (catalase, superoxide dismutase, glutathione reductase, and ascorbate peroxidase). Our work suggests that micropropagated plants developed an antioxidant enzymatic protective system to avoid oxidative stress during establishment under ex vitro environment.

Direct Organogenesis in Punica granatum L. cv. Kandhari Kabuli from Hypocotyl Explants

A high efficiency plant regeneration protocol has been developed for Punica granatum L. cv. Kandhari Kabuli using hypocotyl explants. Hypocotyl explants (about 0.5–1.0 cm in size) were procured from 15 to 20-day-old in vitro germinated seedlings. The highest frequency of shoot regeneration (68.21 %) along with 3.18 average number of shoots per explant was obtained via direct organogenesis from hypocotyl explants cultured on Murashige and Skoog’s medium supplemented with 2.0 mg/l 6-benzyl amino purine and 1.5 mg/l α-naphthalene acetic acid. High percentage root regeneration (85.62 %) in in vitro developed shoots was obtained on half strength Murashige and Skoog’s medium supplemented with 0.10 mg/l α-naphthalene acetic acid and 0.02 % activated charcoal. Pomegranate plantlets were able to regenerate within 6 weeks and well rooted plantlets were successfully acclimatized in plastic cups containing autoclaved peat moss. They were weaned in a growth room and finally moved to a greenhouse. This is the first report on direct organogenesis in P. granatum L. cv. Kandhari Kabuli using hypocotyl explants. This regeneration protocol will be very useful for undertaking genetic manipulation providing a value addition to pomegranate propagation in future.

In vitro organogenesis from leaf and transverse thin cell layer derived callus cultures of Talinum triangulare (Jacq.) Willd.

Talinum triangulare is an important medicinal herb used traditionally in the treatment of various diseases. The present study was intended to develop a rapid and efficient protocol for indirect organogenesis from leaf discs and transverse thin cell layer (tTCL) of internodal explants of T. triangulare. Best callusing response (100 %) was observed with tTCL explants on Murashige and Skoog (MS) medium supplemented with 4.44 μM 6-benzyl amino purine and 5.37 μM α-naphthalene acetic acid (NAA). High frequency shoot regeneration (96.67 %) was obtained from tTCL derived calli on MS medium supplemented with a combination of 0.45 μM thidiazuron and 0.27 μM NAA, by producing 9.20 ± 0.35 shoots with a shoot length of 2.74 ± 0.03 cm. In vitro rooting of the microshoots was recorded on half-strength MS medium containing 2.46 μM indole-3-butyric acid by eliciting 15.20 ± 0.27 roots with a length of 4.25 ± 0.11 cm. The rooted shoots were acclimatized on garden soil, sand and coco pith (1:1:3 v/v) planting substrate. The plantlets were successfully established under field conditions with 100 % survival rate. The hardened plants exhibited homogeneity and no observable morphological variations were detected among the regenerants and the mother plants of T. triangulare.

In vitro propagation of Chirita moonii Gardn. (Gesneriaceae), a potential ornamental plant endemic to Sri Lanka

SummaryAn efficient and rapid regeneration protocol via multiple shoot formation was developed for the endemic herb, Chirita moonii Gardn., which has potentially high ornamental value. Nodal and leaf explants were cultured on Murashige and Skoog (MS) medium supplemented with different concentrations of 6-benzylaminopurine (BAP), thidiazuron (TDZ), or indole-3-acetic acid (IAA), alone or in various combinations. Regenerated shoots were cultured on MS medium supplemented with different concentrations of BAP for shoot multiplication. Micro-shoots were established on full-strength or half-strength MS medium supplemented with different concentrations of 1-naphthaleneacetic acid (NAA) for root induction. The highest rate of shoot induction from nodal explants of C. moonii (8.3 shoots per nodal explant) occurred on basal MS medium supplemented with 0.5 mg l–1 TDZ, and the highest frequency of shoot regeneration was 100% on the same medium. The highest rate of shoot multiplication (12.5 shoots per shoot explant) was observed on MS medium supplemented with 3.0 mg l–1 BAP. The highest rate of shoot induction from leaf explants (22.4 shoots per explant) was observed on MS medium supplemented with 0.5 mg l–1 TDZ. The highest frequency of root induction (95%), the highest mean number of roots per shoot (8.3 roots per shoot), and the maximum mean root length (5.6 cm) were observed on half-strength MS medium supplemented with 0.5 mg l–1 NAA. Plants were acclimatised, with a 91% survival rate, in a 1:1:1:1 (v/v/v/v) mix of sand:compost (leaf mould):charcoal:top soil in 6 cm 12 cm plastic pots in a net-house.

Protocol optimization and evaluation of rice varieties response to <i>in vitro</i> regeneration

Understanding the control of related molecular mechanisms is impossible without efficient and reproducible regeneration and stable genetic transformation. The key factors for enhancing successful regeneration are genotypes, tissue source of explants, combination and concentration of growth regulators, and culture conditions. In the present study different methods of regeneration were tested in a set of 12 rice accession representing indica, japonica, aromatic and wild groups. The highest frequency of shoot regeneration was achieved using mesocotyls derived from in vitro grown seeds cultured on Murashige and Skoogs, basal medium without growth regulators, when cultured on medium supplemented with Benzyl Adenine (BA) 0.5 mg/l under subdued light at 25°C ± 2°C. Under these conditions mesocotyls (Plurality of meristems) produced 3 to 4 tiller shoots in primary culture. One seed/One single mesocotyls segment produced over 5 to 9 shoots, arising primarily through direct organogenesis after 3 weeks of culture. Through callusing phase different rice cultivars produced different percentage of callusing however Basmati 370 gave high percentage in response to callussing, organogenesis and root formation. In between these two methods, the direct shoot regeneration gave 80% - 100% result besides the wild species in respect with their genotype whereas the indirect organogenesis gave only 10% - 30% of plantlets. So the direct multiplication from mesocotyls is an efficient method for plantlet regeneration of rice cultivars through in vitro culture. Tissue culture derived plants when evaluated with rice markers no variability is found. For regeneration genotypes and its trigger ing factors like medium composition, culture conditions and type of explants are playing a combined role.

High Frequency Shoots Regeneration from Cotyledon Explants of Teasle Gourd Via Organogenesis

 Abstract—Organogenic callus induction and high frequency shoot regeneration were achieved from cotyledon explants of teasle gourd. About 94.8% of cotyledon explants derived from 18 day old cotyledon produced green, compact nodular organogenic callus in MS medium containing BA 1 mgl-1 after 51 days with 95% shoot induction. The elongated shoots rooted in MS medium supplemented with IBA 0.3 mgl-1. Rooted plants were acclimatized in growth room and subsequently established in soil with 100% survivability. This protocol produced an average of 19.8 shoots cotyledon-1 explant in 22.2 days of culturing.

Protuberance-mediated high frequency of adventitious shoot regeneration from sweetpotato leaf explants [ Ipomoea batatas (L.) Lam.

In this report, a novel, reproducible and efficient protocol for protuberance-mediated adventitious shoot regeneration has been established for sweetpotato [Ipomoea batatas (L.) Lam.]. The leaf explants were cultured on a modified Murashige and Skoog (MS) medium containing various concentrations (0.5, 1.0, 2.0, 5.0 or 10 μM) of Thidiazuron (TDZ). The adventitious shoots were developed from the base of leaf explants on an MS medium supplemented with 2.0, 5.0 or 10.0 μM TDZ. The responsive explants were transferred to a freshly modified MS medium containing TDZ. Green-colored, oval, or cone-shaped shoot-forming protuberances were developed from explants. It appeared that the frequency of protuberance development depended on the concentration of TDZ. However, this did not further improve, when the concentration of TDZ increased from 5.0 μM to 10.0 μM. Adventitious shoot buds were developed from protuberances, and the histology of protuberances revealed a direct origin of shoot buds. In vitro raised shoots were rooted either on a modified MS medium alone or on a modified MS medium containing indole-3-butyric acid (IBA). The regenerated plants were acclimatized and successfully established in a greenhouse.

English

  Seed germination, seedling growth and in vitro regeneration studies were conducted on H. floribunda(Apocynaceae), a potential reforestation candidate. The seeds germinated readily with 96.0 ± 2.5% germination in 10 days. The seedlings exhibited a slow growth rate, attaining a shoot height of 35.5 ± 4.46 cm and a leaf area of 23.86 ± 3.89 cm2 in 12 weeks. Murashige and Skoog (MS) medium supplemented with various concentrations of 2, 4-dichlorophenoxyacetic acid, (2, 4-D) alone or combined with Benzyladenine induced callus formation in leaf, internode and node explants. Massive callus production was generally observed with explants cultured on MS media supplemented with 4.5 µM 2, 4-D alone or combined with 2.2 µM BA. The highest frequency of shoot regeneration was induced by a combination of 4.5 µM 2, 4-D and 2.2 µM BA; no complete plantlets were however produced.   Key words: Callus, germination, H. floribunda, plant regeneration, seedling growth.

Actions for ex situ conservation of Gloriosa superba L. - an endangered ornamental cum medicinal plant

Factors affecting in vitro propagation and microtuberization were evaluated for Gloriosa superba L., an endangered ornamental cum medicinal plant having limited reproductive capacity. Surface sterilization of tuber explants with 0.1% mercuric chloride (HgCl2) for 5 min eliminated the contamination effectively with highest survival rate. Among the various combinations used, Murashige and Skoog (MS) medium with 2.0 mg L−1 6-benzylaminopurine (BAP) + 0.5 mg L−1 α-naphthalene acetic acid (NAA) containing 3% sucrose with 16-h photoperiod exhibited the greatest in vitro tuberization (3.2) with the highest shoot regeneration frequency (90%). The longest tuber regeneration occurred on MS media containing 4% sucrose. Transfer of in vitro-regenerated shoots to half-strength MS medium with 1.0 mg L−1 indole-3-butyric acid (IBA) + 0.5 mg L−1 NAA showed maximum root induction (66.6%). The in vitro-grown plantlets were successfully acclimatized and transplanted to sterilized soil and sand mixture (3:1) in the glasshouse with 70% survival. The colchicine content was determined in the tubers of ex vitro plants by HPLC using the same retention time (1.5 min) as that of the standard colchicine. This revealed that the micropropagation protocol developed by us for rapid mass production could be used as raw material for colchicine extraction and provides a basis for germplasm conservation and genetic improvement of G. superba.

In vitro micropropagation of Dracaena sanderiana Sander ex Mast: An important indoor ornamental plant

A protocol has been developed for in vitro plant regeneration from a nodal explant of Dracaena sanderiana Sander ex Mast. Nodal explant showed high callus induction potentiality on MS medium supplemented with 6.78μM 2,4-dichlorophenoxyacetic acid (2,4-D) followed by 46.5μM chlorophenoxy acetic acid (CPA). The highest frequency of shoot regeneration (85%) and number of shoots per explant (5.6) were obtained on medium supplemented with 7.84μM N6-benzylaminopurine (BA). Rooting was high on MS solid compared to liquid medium when added with 7.38μM indole-3-butyric acid (IBA). Fifty percent of the roots were also directly rooted as microcuttings on soil rite, sand and peat mixture (1:1:1). In vitro and ex vitro raised plantlets were used for acclimatization. More than 90% of the plantlets was successfully acclimatized and established in plastic pots. Ex vitro transferred plantlets were normal without any phenotypic aberrations.

Role of TDZ in the Quick Regeneration of Multiple Shoots from Nodal Explant of Vitex trifolia L.—an Important Medicinal Plant

The effect of thidiazuron (TDZ) has been investigated in shoot multiplication for a simple, efficient, rapid, and commercially applicable regeneration protocol of an important medicinal plant, Vitex trifolia. Multiple shoots were induced in nodal explants obtained from a mature tree on Murashige and Skoog (MS) medium supplemented with TDZ in various concentrations (0.5, 1.0, 2.5, 5.0, 7.5, or 10.0μM). Prolonged exposure of the culture to TDZ had an adverse affect. To avoid this, the cultures were transferred to TDZ-free MS medium or MS medium fortified with various concentrations of 6-benzyladenine (BA) alone or in combination with α-naphthalene acetic acid (NAA) to enhance multiplication, proliferation, and elongation of induced shoots. Optimum shoot multiplication and elongation was achieved when TDZ-exposed explants were repeatedly subcultured on MS media containing a combination of 1.0μM BA and 0.5μM NAA. The highest shoot regeneration frequency (90%) and maximum number (22.3 ± 0.2) of shoots per explant with shoot length of (5.2 ± 0.2cm) was recorded on MS medium fortified with 5.0μM TDZ. In vitro rooting of isolated shoots was achieved best in half-strength MS medium containing 0.5μM NAA. Properly rooted plantlets were successfully hardened off and acclimatized in thermocol cups containing sterile Soilrite. These plantlets were then transferred to pots containing different potting substrate; percentage survival of the plantlets was highest in vermiculite/garden soil mixture (1:1) and successfully transfer to greenhouse under sunlight.

The Effect of Sodium Hypochlorite Solutions on the Viability and In Vitro Regeneration Capacity of the Tissue

Although there are many chemicals used for sterilization purposes such as ethanol, hydrogen peroxide, bromine water, mercuric chloride, silver nitrate and antibiotics, sodium hypochlorite solutions have been most widely used. Since NaOCl has a strong oxidizing property which makes it highly reactive with amino acids, nucleic acids, amines, and amides, it is highly effective against all kinds of bacteria, fungi, and viruses. The general reaction between amino acids and NaOCl produces the respective aldehyde, NH4Cl and CO2. Thus, during the sterilization process, direct contact of the tissue with NaOCl depending on it’s concentration, application period and temperature may have a hazardous effect on the health of the tissue. In plant tissue culture studies, high-frequency shoot regeneration which is highly affected by tissue health, is a prerequisite for an efficient gene transformation system and a clonal propagation of plants. The most important treatment prior to culture initiation is the sterilization of the tissue. In plant tissue culture, elimination of microorganisms from the tissue has great importance. From one hand, sterilization process aims to eliminate all microorganisms that can easily grow on the tissue; on the other hand, it should guarantee the tissue’s viability and regeneration capacity. This review has focused on the effects of concentration, application period and temperature of NaOCl using for eliminating microorganisms on the viability and in vitro regeneration capacity of the tissue.

Micropropagation of Rosa damascena Mill. through transverse thin cell layer culture

A basic factor underlying the success of large‐scale micropropagation and genetic transformation of any plant species is regeneration. In order to regenerate propagules of Rosa damascena Mill. on a large scale, an efficient and improved in vitro propagation system has been established using transverse thin cell layer culture (tTCL). By optimizing the position of the tissue and applying an improved selection procedure, in vitro shoots were elongated in 8 weeks of culture. Modified Murashige and Skoog (1962)(MS) medium fortified with 4.0 mg l‐1 6‐benzylaminopurine (BAP) and 0.4mg l‐1 anaphthalene acetic acid (NAA) gave optimal shoot regeneration. The explant was inoculated on this medium in the upright position and exhibited a high frequency of shoot regeneration (~96.66%), and it also gave the highest number of shoots (22.33/explant). The horizontally placed explant on an average 7.66 shoots/explant. Our experiments indicate that explant orientation strongly influences the organogenesis response. The frequency of shoot initiation and the number of multiple shoots produced from each explant were significantly dependent on the plant source, concentration of plant growth regulators and the orientation of the explants and contributed significantly to in vitro regeneration. Rooting of well developed shoots was achieved on hormone free ¼ strength MS medium with 4% sucrose.

An Efficient and Reproducible Method for in vitro Clonal Multiplication of Rauvolfia tetraphylla L. and Evaluation of Genetic Stability using DNA-Based Markers

An efficient protocol is described for the rapid in vitro clonal propagation of an endangered medicinal plant, Rauvolfia tetraphylla L., through high frequency shoot induction from nodal explants collected from young shoots of a field grown plant. Effects of growth regulators [6-benzyladenine (BA), kinetin (Kin) 2iP, or α-naphthalene acetic acid (NAA)], carbohydrates, different medium [Murashige and Skoog (MS), Woody Plant Medium (WPM), Gamborg medium (B5), Linsmier and Skoog medium (LS)], and various pH levels on in vitro morphogenesis were investigated. The highest frequency of shoot regeneration (90 %) and maximum number of shoot (35.4 ± 2.3) per explant were observed on WPM medium supplemented with 7.5 μM BA, 2.5 μM NAA, and 30 g/l sucrose at pH 5.8. Well-developed shoots, 4-5 cm in length, were successfully rooted ex vitro at 90 % by a 30-min pulse treatment with 150 μM IBA prior to their transfer in planting substrates. The survival rate of transplantation reached 90 % when transferred to field condition. Genetic stability of micropropagated plantlets was assessed and compared with mother plant using Random Amplified Polymorphic DNA and Inter Simple Sequence Repeats markers. No variation was observed in DNA fingerprinting patterns among the micropropagated plants, which were similar to that of the donor plant illustrating their genetic uniformity and clonal fidelity. This confirms that clonal propagation of this plant using axillary shoot buds can be used for commercial exploitation of the selected genotype where a high degree of fidelity is an essential prerequisite. The work contributed to a better in vitro regeneration and clonal mass multiplication of R. tetraphylla and to develop a strategy for the germplasm conservation of this endangered medicinal plant.

High frequency shoot regeneration of dark blue statice (Limonium sinuatum L. Mill) by combining phytohormones with adenine in in vitro culture

High frequency shoot regeneration of dark blue statice (Limonium sinuatum L. Mill) by combining phytohormones with adenine in in vitro culture

Combination of thidiazuron and 2-isopentenyladenine promotes highly efficient adventitious shoot regeneration from cotyledons of mature sunflower (Helianthus annuus L.) seeds

Genetic improvement of sunflower (Helianthus annuus L.) through the use of biotechnological tools requires a reliable in vitro shoot regeneration system. Tissue culture protocols reported to date for sunflower suffer from low efficiency, poor reproducibility, genotype dependence and a tendency for flowering in vitro. The present study describes an efficient protocol system for shoot regeneration via direct adventitious shoot organogenesis from cotyledons of mature seeds of sunflower. About 169 media combinations comprising 12 different growth regulator combinations in various concentrations were assessed for induction of shoots from cotyledons derived from mature seeds and also from seedling tissues of 2–20-day-old seedlings. Appearance of shoots from seedling tissues was sporadic and the frequency of shoot regeneration was low. Cotyledon explants from mature seeds were consistent with regard to frequency of adventitious shoot regeneration and number of shoots per explant. A high frequency (93.86 %) of adventitious shoot regeneration was obtained within 2 weeks of culture initiation on Murashige and Skoog (MS) medium supplemented with 9.84 μM 2-isopentenyladenine (2-iP), 2.85 μM indole-3-acetic acid (IAA) and 0.45 μM thidiazuron (TDZ). Use of 2-iP in the shoot induction and elongation media prevented precocious flowering. Statistical analysis revealed significant effects of explant orientation, age of seedlings, and genotype on adventitious organogenesis. Maximum shoot regeneration was obtained when cotyledons from 0 and 1-day-old seedlings were placed with their adaxial surface in contact with the medium surface. The protocol developed was tested on 42 genotypes and found to be applicable to a wide range of genotypes. Histological studies indicated that the shoots originated predominantly through adventive organogenesis from the sub-epidermal and cortical regions.

High frequency shoot regeneration and plant establishment of Rauvolfia serpentina: An endangered medicinal plant

An efficient method was developed for high frequency shoot regeneration and plant establishment from nodal segment explants of Rauvolfia serpentina. Among the three different cytokinins (6-benzyladenine (BA), kinetin (Kin) and 2-isopentenyl adenine (2-iP)) tested as supplements to Murashige and Skoog (MS) medium, BA at 5.0 μM concentration was found effective in inducing multiple shoots. Effects of different medium (Murashige and Skoog (MS), Gamborg medium (B5), Schenk and Hildebrandt (SH) and Woody Plant Medium (WPM)) on in vitro regeneration were also evaluated. The highest frequency of shoot regeneration (90%) and maximum number of shoot (45.4 ± 3.2 per explants) with an average shoot length of 4.7 ± 0.21 cm were standardized on WPM supplemented with 5.0 μM BA along with 1.0 μM α-naphthalene acetic acid (NAA). In vitro regenerated micro-shoots rooted best on WPM supplemented with 1.0 μM NAA. Plantlets with well-developed shoot and roots, following acclimation in the greenhouse, were successfully established in field condition, and 90% of plants were survived. The regenerated plants were morphologically normal and did not show any immediate detectable phenotypic variation. The method developed can be successfully employed for large-scale multiplication and long-term in vitro conservation of R. serpentina. Key words: Nodal explants, plant growth regulators, in vitro, plant regeneration, micro-propagation.

Thidiazuron‐Induced Tissue Culture Regeneration from Quartered‐Seed Explants of Arachis Paraguariensis

ABSTRACTArachis paraguariensis Chodat & Hassl. is a potential source of novel genes for the genetic improvement of cultivated peanut (Arachis hypogaea L.) because some of its accessions show high levels of resistance to early leaf spot caused by Cercospora arachidicola Hori. In this study, induction of high frequency shoot regeneration from quartered‐seed explants was accomplished for six plant introductions of A. paraguariensis under continuous light on Murashige and Skoog (MS) medium containing 4.4 mg L−1 thidiazuron (TDZ) in combination with 2.2 mg L−1 6‐γ‐γ‐(dimethylallylamino)‐purine (2ip). Recovery of a moderately high number of plantlets per quarter seed cultured was also achieved on medium containing 4.4 mg L−1 thidiazuron in combination with 1.1 to 4.4 mg L−1 6‐benzylaminopurine (BAP) with bud formation occurring as early as 1 wk after culture initiation. There were no differences in seed production or in early leaf spot incidence between plants of two genotypes of A. paraguariensis derived from seeds vs. in vitro tissue culture derived plants; however, cultivated peanut cv. Florunner had a higher incidence of early leaf spot.

In vitro regeneration from internodal explants of bitter melon (Momordica charantia L.) via indirect organogenesis

Organogenic callus induction and high frequency shoot regeneration were achieved from internodal explants of bitter melon. About 97.5% of internodal explants derived from 30 day old in vivo grown plants produced green, compact nodular organogenic callus in Murashige and Skoog (MS) plus Gamborg et al. (1968) (B 5 ) medium containing 5.0 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 2.0 μM thidiazuron (TDZ) after two successive transfers at 11 days interval. Adventitious shoots were produced from organogenic callus when it was transferred to MS medium supplemented with 4.0 μM TDZ, 1.5 μM 2,4-D and 0.07 mM L-glutamine with shoot induction frequency of 96.5% and regeneration of adventitious shoots from callus (48 shoots per explant). Shoot proliferation occurred when callus with emerging shoots was transferred in the same medium at an interval of 15 days. The regenerated shoots were elongated on the same medium. The elongated shoots were rooted in MS medium supplemented with 3.0 μM indole 3-butyric acid (IBA). Rooted plants were acclimatized in green-house and subsequently established in soil with a survival rate of 95%. This protocol yielded an average of 48 shoots per internodal explant after 80 days of culture. Keywords: Adventitious shoots, growth regulators, hardening, organogenic callus, Momordica charantia