In Vitro Morphogenesis and Plantlet Regeneration in Tinospora cordifolia (Willd.) Miers Ex Hook. F. & Thoms from Cultured Leaf Discs

Tinospora cordifolia (Willd.) Miers ex Hook F & Thoms (Family: Menispermaceae), commonly known as heart-leaved Moonseed plant in English and Giloy in Hindi, is one of the important National Medicinal Plant Board (NMPB) listed medicinal plants. It is usually employed in conventional ayurvedic treatment. In present investigation, an efficient and reproducible plant regeneration system was developed through direct and indirect organogenesis from cultured nodal segments excised from one-year-old greenhouse-grown plant. An average 6-8 initiation days was recorded for callus initiation for inoculated nodal segments on MS medium supplemented with 3.0 mgl-1 2, 4-D with 100 percent callus induction competence with an average fresh weight of 12.00 g. Maximum shoot proliferation (98%), shoot numbers (5.16±0.13) with greater length (5.88±0.04 cm) and leaf numbers (6.16±0.13) were achieved on MS medium enriched with 1.5 mgl-1 BAP in combination with 2.0 mgl-1 Kn after 40 days of inoculation. During the shoot multiplication, leaching of phenolics was observed frequently and to evade this, two adjuvants viz., ascorbic acid and activated charcoal were employed. Addition of 100 mgl-1 ascorbic acid in combination with 5.0 gl-1 activated charcoal with optimized plant growth regulators to MS medium reduced phenol exudation ultimately leading to much healthier plant and decreased leaf defoliation. The highest (84%) response of root induction, number of roots (4.16±0.30) with higher root length (5.20±0.008 cm) was observed on half- strength MS medium augmented with 1.0 mgl-1 IBA after 45 days of transfer in rooting medium. Among diverse potting mixture combinations, maximum survival (85%) with maximum plant height (16.16±0.13cm) was attained in combination of cocopeat: vermiculite (1:1) under greenhouse conditions after five weeks of hardening. Plants after acclimatization survived well in nature. Although the traits were not scored quantitatively, regenerated plantlets appeared phenotypically normal and as its mother plants.

Dendrocalamus asper (giant bamboo) is a clumping type of bamboo belonging to the Poaceae family. Due to its economic and environmental value, demand for this species has increased tremendously. Conventional propagation methods have limitations due to low seed viability and the lack of healthy clumps. Therefore, an in vitro mass propagation protocol was developed to provide healthy plants for large-scale plantations. Seeds were used as the explant and they were surface sterilized and cultured on MS medium free of growth regulators. Nodal segments taken from in vitro germinated seedlings were used for shoot initiation. The best medium for shoot induction (MS medium supplemented with 0.0–2.5 mg/L BAP), best medium for multiple shoot induction (MS medium supplemented with 0.0–5.0 mg/L BAP), effect of shoot cluster size (shoot clusters containing 1–4 shoots) and effect of physical state of the medium (semisolid and liquid media) on multiple shoot induction were determined using shoots per node, mean shoot length and mean number of leaves per shoot after 6 weeks of incubation. Elongated shoots were transferred into a rooting medium and the best medium for root induction (MS medium supplemented with 0.0–5.0 mg/L IBA and IAA) and effect of cluster size (shoot clusters containing 1–4 shoots) on rooting were determined using the number of roots and root length after 6 weeks of incubation. All the cultures were maintained under a 16-hour photoperiod. Well-developed plantlets were transferred to coir pellets and after four weeks transferred into different potting mixtures containing different combinations of sand, compost and coir dust. Unless otherwise mentioned there were at least twenty replicates in all treatments. The highest mean number of shoots per node (16.87±0.52), mean shoot length (4.12±0.27 cm) and mean number of leaves per shoot (4.80±0.33) were observed in the MS medium supplemented with 1.0 mg/L BAP. The MS medium supplemented with 2.0 mg/L BAP was the best for multiple shoot induction, shoot cluster with 3 shoots was the best cluster size and the liquid medium had a better effect on shoot multiplication. The half-strength MS medium supplemented with 2.0 mg/L IBA was the best for in vitro root induction with the highest mean number of roots (7.15±0.77) and a mean root length of 10.79±1.11 cm. Shoot clusters with 3 shoots was the best cluster size for root induction. A sand: compost: coir dust (1:1:1) mixture was the best potting mixture giving 100% survival. These findings provide a reliable micropropagation protocol for D. asper, which holds great promise for meeting the growing demand for bamboo resources and promoting sustainable bamboo cultivation.

An efficient protocol for in vitro regeneration of Strychnos potatorum L. f. through indirect organogenesis was developed using mature leaf disc explants. The explants were selected from twenty years old tree. Cultured on Murashige and Skoog’s medium supplemented with 2,4-dichloro phenoxy acetic acid (2,4-D) was used for the induction of organogenic calli. A pale yellow coloured friable callus developed at 2 mg/L-1 2,4-D where showed higher morphogenic potential. Further de-novo shoots formation and multiplication in optimal concentrations of cytokinins with different combinations of auxins. An average mean shoots number (14.2±0.42) and shoots height (3.5±0.27cm) was obtained from piece of calli in 6-benzylaminopurine (BAP) 1 mg/L-1 with 0.5 mg/L-1 α-naphthalene acetic acid (NAA). BAP in combinations with NAA was found to be superior to shoot development than other combination used. The well developed shoots were transferred to root induction medium for effective rooting. The rooting medium consisted of in half-strength MS medium augmented with 0.5 mg/L-1 NAA was used. The maximal mean number of roots per shoots 6.5±0.53 was recorded in 4 weeks. The rooted plantlets were removed from the culture tubes, gently washed with sterile double distilled water to remove agar adhering on the plant surface. The regenerated plants were transferred to plastic cups filled with a potting mixture containing sterile sand and soilrite (1:1 ratio) and allowed to grow in the greenhouse. The slowly acclimatized plants were further transferred to substrate containing red soil and farmyard manure (1:1) for effective hardening. 75% survival rate was recorded. This protocol can be useful for the ex-situ conservation and rehabilitation of this vulnerable medicinal tree S. potatorum.
 Keywords: Indirect organogenesis, friable callus, shoot development, 6-benzylaminopurine, α-naphthalene acetic acid

Anthurium is one of the most demanded pot plants in the European market. The effect of different pH (5.7 and 6) and electrical conductivity (EC) of MS culture medium (MS, 1/2 MS, and 1/4 MS) on growth parameters of Anthurium andreanum 'Tropical' was studied. An experiment was conducted on a factorial basis in a completely randomized design with three replications, including eight plant lets for each treatment. Leaf and root number, plant fresh weight, plant leaf area, total chlorophyll, chlorophyll a (Chl a) and chlorophyll b (Chl b) contents were significantly affected by EC of the culture medium. The largest number of leaves and roots were produced in 1/2 and 1/4 concentrations of MS medium, respectively. Full concentration MS medium resulted in the highest plant fresh weight, plant leaf area, total chlorophyll, Chl a, and Chl b content. Plant fresh weight was also significantly affected by pH of the culture medium. The interaction effect of pH and EC of medium significantly affected the carotenoid content. Plant lets grown on 1/4 MS medium had the highest and lowest carotenoid content at pH 5.7 and 6, respectively. No significant difference was observed in plant height, root or leaf number, leaf area, total chlorophyll, Chl a, or Chl b content in response to medium pH. In general, MS and 1/2 MS culture medium at pH 5.7 showed the most beneficial effects on growth parameters of anthurium 'Tropical' plant lets.

The objective of this study was to induce a rapid as well as prolific shoot regeneration protocol for micropropagation and RAPD analysis of Guizotia abyssinica Cass. which is an important herbaceous plant of immense industrial value via direct and indirect organogenesis from apical bud, axillary bud, leaf and internode explants. Best seed germination was obtained on cotton irrigated with liquid MS medium. Out of the four explants used, apical bud proved to be the best in terms of shoot regeneration and multiplication. Best shoot multiplication was obtained from apical bud, axillary bud and leaf explants on MS medium supplemented with 2.22μM BAP+2.85μM IAA. Whereas supplementation of MS medium with 2.22μM BAP+28.55μM IAA produced maximum number of shoots from internode explants. BAP (0.44μM) in combination with Kn (0.46μM) proved suitable for maximum mean shoot length. Moreover, culturing the regenerated shoots on half-strength liquid MS medium supplemented with NAA (2.68μM) induced maximum rooting from elongated shoots (direct and indirect regeneration). The plantlets were established in plastic cups containing vermiculite, soil, sand and farm yard manure and then successfully transferred to field with 97.33% survival. Analysis of RAPD recognized 197 different amplification products and showed the presence of somaclonal variation in the plantlets arising from direct regeneration as well as from indirect regeneration. The protocol developed in this study is suitable for propagation of quality planting material for commercialization, germplasm conservation and for future genetic improvement studies.

Micropropagation, micromorphological evaluation and genetic homogeneity validation of Cucumis collosus (Rottl.) Cogn.: A wild progenitor of melon

Background: Apple (Malus × domestica Borkh.) is one of the most important fruit crops in the world. Traditionally, vegetative propagation methods (including cutting, budding, and layering) are time-consuming (about three years), with low production rates and low success in obtaining virus-free plants. Objective: The present study was planned to investigate the in vitro propagation of apple (M. domestica) cultivars from nodal segments. Methodology: The Murashige and Skoog (MS) medium supplemented with sucrose and different concentrations of plant growth regulators (PGRs) were used for shoot proliferation and root induction. The optimal concentrations of PGRs in the MS medium were assessed. The effect of full and half-strength MS medium on root induction was investigated. Results: Examination of the effects of MS medium supplemented with various concentrations of indole-3-acetic acid (IAA) and kinetin revealed that the significantly highest shoot response was recorded for the ‘Princess’ cultivar with maximum shoot proliferation rate (65.25%), shoot number per explant (2.57), shoot length (7.28cm), and leaf number per shoot (6.15) after four weeks of culture. The root induction in micro shoots of three apple cultivars was observed after 20 days of culturing. The strength MS medium (full and half) containing 1.5 mg/L IAA significantly affected (at P<0.05, chi-square test) root induction in all three apple cultivars, especially rooting rate. However, there was no significant difference in root number and root length per micro shoot among the apple cultivars. Among the cultivars, significantly the highest rooting rate(48.30%), root number (6.25), and root length (4.15cm) were recorded for cultivar ‘Princess’ on full-strength MS medium. Conclusion: PGR combination of IAA (1.0 mg/L) and kinetin (3.0 mg/L) was found to be the best for shoot proliferation. The shoot responses were found to increase with an increase in kinetin concentration combined with IAA at 1.00 mg/L.

The present study develops a protocol for rapid in vitro micropropagation of a critically endangered and floriculturally most important epiphytic orchid, Dendrobium primulinum Lindl. through the culture of small shoot tip explants (0.3 to 0.5mm) derived from in vitro grown seedlings. The shoot tip explants cultured on solidified Murashige and Skoog (MS) basal medium and MS medium alone or supplemented with combination of various concentrations of growth regulators; α-naphthalene acetic acid (NAA) and 6-benzylaminopurine (BAP), produced shoots and multiple shoots. The maximum numbers of rootless healthy shoots were observed on MS medium fortified with BAP 1.5 mgl -1 with an average value of 4.5 shoots per culture where shoot multiplication was initiated after 5 weeks of culture of shoot tip. Among the different tested combination, MS medium with BAP (1.5 mg l -1 ) and NAA (0.5 mg l -1 ) were most effective for the shoot multiplication. MS medium supplemented with various concentrations of rooting hormones viz. NAA, IAA and IBA showed positive response in development of roots, except NAA 0.5 mgl -1 . The rooting was observed after 3 weeks of culture of shoot tip. The various concentrations of IAA and IBA were found to be effective hormone for rooting of D. primulinum in comparison to NAA. The best rooting response was observed on MS medium with exogenous supply of IAA 0.5 mgl -1 . The well developed in vitro rooted plantlets were hardened successfully in the potting mixture containing cocopeat and sphagnum moss in the ratio of 2:1. Nearly 70% of plantlets survived.

The present investigation was carried out to develop an efficient protocol for in vitro propagation of Brucea mollis Wall. ex Kurz, an important medicinal plant, confined to only Karbi Anglong district of Assam. The plant is extensively used to cure malaria. Due to several natural factors like inefficient pollination, seed dispersal mechanisms and anthropogenic activities, the plant is facing extinction. Through indirect organogenesis from leaf and internode explants on MS (Murashige and Skoog) media, callus induction was achieved at a maximum (100%). However, the minimum time (7–15 days) was observed for induction of callus on B5 as compared to MS media. The color and texture of the callus varied depending on the type of explants as well as the concentrations of growth regulators used in both the media. Best response for percentage of callus induction (100%) was obtained from the leaf explants on MS media, when the media was supplemented with the four combinations of growth regulators, i.e., (i) BAP (8.88 μM) + NAA (1.61 μM), (ii)BAP (8.88 μM) + NAA (2.68 μM), (iii) BAP (8.88 μM) + 2,4-D (2.27 μM) and (iv) BAP (8.88 μM) + 2,4-D (4.54 μM). While in the case of internode explants, maximum (100%) percentage for callus induction was obtained with BAP (8.88 μM) + NAA (1.61 μM) and BAP (8.88 μM) + NAA (2.68 μM). Multiple shoot regeneration was achieved from the callus of all the three types of explants when transferred to shoot regeneration media (both MS and B5 media) supplemented with BAP, NAA, Kinetin and IBA at different concentrations and combinations. The maximum percentage of shoot regeneration (100%) from the calli of all explant types were obtained with the combination of BAP, NAA and IBA. However, the best response in terms of minimum number of days taken to regenerate shoots and maximum shoot length was obtained using the leaf explants at BAP (8.88 μM) + NAA (2.68 μM) + Kinetin (13.95 μM) on MS media. The regenerated shoots were cultured for rooting on half strength media supplemented with IBA and NAA singly, and the highest rooting percentage was achieved when MS media was supplemented with 14.76 μM IBA. About 60% of the plantlets survived during acclimatization and were successfully transferred to the field. The regeneration protocols standardized for B. mollis could be found most effective for mass propagation of the medicinally important plant as well as its conservation.

Explants were taken from field grown plants in 2010. The shoot apical meristem of different sizes was cultured on Murashige and skoog’s (MS) medium supplemented with different concentrations and combinations of 6-benzylamino-purine (BAP), kinetin (Kin) and α- naphthaleneacetic acid (NAA) either alone or in combination with each other under different temperature conditions ranging from 23 to 27°C. Shoot formation response from shoot apical meristem showed that MS medium containing 1.0 mg/l BAP showed best response for shoot formation. For shoot multiplication, MS medium containing 1.0 mg/l BAP + 0.25 mg/l kin provided the best multiplication response which was 8 shoot per culture vial within 21.6 days after inoculation into shoot multiplication medium. Shoot formation and multiplication response was also affected by temperature variations. The best results were obtained at 27°C ± 1°C. By increase or decrease in temperature, the rate of in vitro response was also decreased. For rooting of well developed in vitro shoots MS medium supplemented with 1.0 mg/l Indole-3- butyric acid (IBA)+ 0.5 mg/l NAA showed 3.6 roots per plant after 6.8 days of inoculation into rooting medium with an average root length of 2.4 cm. 100% hardening response was obtained in Peat moss after 21 days of transplantation in glass house. The experiments were designed in completely randomized pattern. Key words: Murashige and skoog’s medium, proliferation, banana, micro propagation system.

Explants were taken from field grown plants in 2010. The shoot apical meristem of different sizes was cultured on Murashige and skoog’s (MS) medium supplemented with different concentrations and combinations of 6-benzylamino-purine (BAP), kinetin (Kin) and α- naphthaleneacetic acid (NAA) either alone or in combination with each other under different temperature conditions ranging from 23 to 27°C. Shoot formation response from shoot apical meristem showed that MS medium containing 1.0 mg/l BAP showed best response for shoot formation. For shoot multiplication, MS medium containing 1.0 mg/l BAP + 0.25 mg/l kin provided the best multiplication response which was 8 shoot per culture vial within 21.6 days after inoculation into shoot multiplication medium. Shoot formation and multiplication response was also affected by temperature variations. The best results were obtained at 27°C ± 1°C. By increase or decrease in temperature, the rate of in vitro response was also decreased. For rooting of well developed in vitro shoots MS medium supplemented with 1.0 mg/l Indole-3- butyric acid (IBA)+ 0.5 mg/l NAA showed 3.6 roots per plant after 6.8 days of inoculation into rooting medium with an average root length of 2.4 cm. 100% hardening response was obtained in Peat moss after 21 days of transplantation in glass house. The experiments were designed in completely randomized pattern. Key words: Murashige and skoog’s medium, proliferation, banana, micro propagation system.

Sugarcane is an important crop for sugar and bioenergy production and belongs to the family poaceae. Current sugarcane varieties have a highly complex and large genome, with 100-130 chromosomes. The demand for sugar, fiber, bagasse and molasses is increasing in Pakistan due to the rising population. Varieties of sugarcane (Saccharum officinarums L.) that are produced by breeding method take approximately 8-10 years, with the number of pests and diseases causing a decrease in cane yield. Improving the productivity of sugarcane varieties is a major challenge. Tissue culture is a way for quick multiplication of desired varieties and to develop disease free healthy plants. Therefore, using high-yielding sugarcane varieties can help us in increasing production. This research aimed to optimize embryogenic callus for subsequent regeneration in sugarcane varieties, specifically CPF-251, HSF-240, and CP-77-400 to device micropropagation. Callus induction was initiated in four week culture of explant developed from spindle leaves by employing various concentrations of plant growth regulators (PGRs) such as Kinetin (0.2 mgL-1, 0.3 mgL-1, 0.4 mgL-1) and 2, 4-D (1.5 mgL-1, 2.5 mgL-1, 3.5 mgL-1) on Murashige and Skoog (MS) medium. Micro shoots emerged after three weeks of callus culture of media supplemented with differing concentrations of BAP (0.3 mgL-1, 0.4 mgL-1, and 0.5 mgL-1) on MS medium. After three weeks, roots developed, when the shooting plants were placed in the rooting medium containing varying concentrations of PGRs such as IBA (0.3 mgL-1 0.4 mgL-1, 0.5 mgL-1) and NAA (3.5 mgL-1, 4.5 mgL-1, 5.5 mgL-1) on MS medium. The most effective shoot growth of variety HSF-240 was obtained at MS + BAP 0.5 mgL-1 + IBA 0.5 mgL-1 + NAA 5.5 mgL-1 with 4.9 cm shoot length, 2.1 cm length of root and 8 numbers of roots. Similarly, for variety CP-77-400, optimal shoot growth was observed at MS + BAP 0.4 mgL-1 + IBA 0.4 mgL-1 + NAA 4.5 mgL-1, with a shoot length of 3.9 cm, root length of 2 cm, and 6 numbers of roots. For variety CPF-251, the most conducive conditions for shoot growth were at MS + BAP 0.3 mgL-1 + IBA 0.3 mgL-1 + NAA 3.5 mgL-1, resulting in a shoot length of 3.5 cm, root length of 1.7 cm, and 5 numbers of root formation. Plants developed through this study proved useful for producing high-yielding, high-sugar recovery cane of existing commercial varieties.

A regeneration system from protoplast to plantlet for a medicinal plant species, Phellodendron amurense Rupr., has been developed. Leaves of micropropagated shoots or plantlets were selected as plant materials for protoplast isolation. The yield and viability of leaf protoplasts were greatly influenced by enzyme combination, treatment time and osmoticum. The highest viability (86%) with a yield of 7.1×105 protoplasts per gram fresh weight was obtained with a 6-h digestion in 1% Cellulase Onozuka R-10 plus 1% Driselase-20. Sustained cell division and colony formation from the protoplasts were best supported at a plating density of 4×105−6×105 protoplasts per milliliter using a 0.2% gellan gum-solidified or liquid MS (Murashige and Skoog, 1962) medium containing 0.6M mannitol, 2.0μM 6-benzylaminopurine (BA) with 4.0 μM α-naphthaleneacetic acid (NAA), indole-3-butyric acid (IBA), or 2,4-dichlorophenoxyacetic acid (2,4-D). The protoplast-derived colonies formed green compact calluses when transferred to a solidified MS medium containing 2.0 μM BA with 4.0μM NAA of IBA. Shoot regeneration from protoplast-derived calluses was induced on MS medium supplemented with 2.0 μM BA and 1.0μM NAA or 2.5μM IBA. Shoot multiplication and elongation occurred on MS medium containing 1.0μM BA. In vitro-grown shoots were rooted on MS medium with either 0.5–4.0μM IBA or NAA. Regenerants were transferred to the Kanuma soil and successfully established under greenhouse conditions.

Aconitum vilmorinianum, a well-known traditional Chinese herb, is recently being threatened by overexploitation and environment disturbance. This study was conducted to provide propagation methods through in vitro germination and explant cultivation. Germination was stimulated up to 66.00% on Murashige and Skoog (MS) medium containing 2.0 mg L−1 6-benzylaminopurine (BAP), 0.1 mg L−1 1-napthaleneacetic acid (NAA), and 30 g L−1 sucrose. Three bacteria (Pantoea agglomerans, Erwinia persicina, and Pseudomonas tolaasii) would be responsible for consistent contamination during germination. The latter two were effectively eradicated after disinfected. The influence of explant types and hormone combinations on direct and indirect organogenesis was evaluated in the present work. The frequency of shoot induction from axillary bud explants was 100% on the MS fortified with 2.0 mg L−1 BAP and 0.3 mg L−1 NAA. Shoots multiplication was optimized on MS medium supplemented with 0.1 mg L−1 thidiazuron (TDZ) and 0.1 mg L−1 NAA. High callus induction percentage (96.67%) was obtained from stem segments on MS medium with 2.0 mg L−1 2,4-D, then successfully regenerated into shoots on MS medium in the presence of 0.1 mg L−1 TDZ and 0.2 mg L−1 NAA. The present work could be useful for the utilization and conservation of this valuable species.

This research focused on the effects of activated charcoal on the in vitro growing media used in the propagation of Brazilian native orchids. Activated charcoal has been described improving the in vitro plant growing and survival after transplant. Seeds were germinated in vitro and after three months they were transferred to one of the following media: MS (Murashige and Skoog) media; half strength of MS media; MS media supplemented with 1 g.L of activated charcoal; half strength of MS media supplemented with 1 g.L of activated charcoal; MS media supplemented with 2 g.L of activated charcoal; half strength of MS media supplemented with 2 g.L of activated charcoal. After six months the following features were evaluated, plantlet height, number of branches per plant, number of roots per plant, length of the roots, fresh weight and O. trulliferum was also evaluated concerning with the pseudobulb diameter. Just after the in vitro plant features evaluation they were transferred to pots containing the tree fern fiber and sphagnum. Two months after transplant the survival rate was evaluated for each treatment. Activated charcoal improved the in vitro plant quality and increased the survival after transplant of the three species evaluated. To M. flavescens and L. flava best results were obtained with half strength of MS media supplemented with 2 g.L of activated charcoal and O. trulliferum with MS media supplemented with 1 g.L of activated charcoal. INTRODUCTION Orchids are one of the most valuable and appreciated plants in the world. Due to its commercial value and beauty, orchids are very cultivated. Several species are in risk of extinction due to its predatory collection and destruction of its habitat. To preserve orchids using tissue culture technique is an option to produce orchids in large scale (Silva, 1986). An adequate formulation of the nutrition media is essential to tissue culture, because media must supply the essential substances for in vitro growth development (Caldas et al., 1990). Addition of activated charcoal on tissue culture media can be beneficial or adverse to growth and development. Its presence depends on the media, the specie and the tissue used and/or the objectives of the research (Pan and Staden, 1998). Activated charcoal as supplement on the media has been reported through the years by several authors due to its beneficial effects, such as, absorption of phenolic complex (Pan and Staden, 1998); root stimulator (George and Havishankar, 1997; Caldas et al., 1990); rhizome growth (Kim and Lee, 1992); development improvement (Choi and Chung, 1989); and absorption of toxic substances present in the media (Fridborg and Eriksson, 1978). Waes (1987) used activated charcoal for in vitro development of 18 species of European native orchids with concentrations from 0,02 % to 0,03 % (g.L) to improve in vitro development. The MS media contains nitrogen in ammonium and nitrate forms. Vitrification is frequent observed affecting plantlets development. Reduction of nitrogen concentration on ammonium has been used to reduce vitrification (Caldas et al., 1990). Reduction of MS media to half strength for in vitro orchid has been showed positive to reduce Proc. V IS on New Flor. Crops Eds.: A.F.C. Tombolato and G.M. Dias-Tagliacozzo Acta Hort. 683, ISHS 2005