Factors affecting micropropagation in Vanilla (Vanilla × Tahitensis J. W. Moore) from node explants

We described culture conditions for direct and indirect regeneration of Iranian Tribulus terrestris L. through epicotyl, hypocotyl and leaf explants. The explants were cultured on MS medium supplemented with different concentrations and combinations of auxin and cytokinin. The results indicated that the mean of callus induction was influenced by explant type and various phytohormones levels. The highest percentage of callus production occurred on MS medium containing 0.1 mg/l naphthalene acetic acid (NAA) and 1 mg/l 6-benzylaminopurine (BAP) from epicotyl explants (91.6%), 0.4 mg/l (NAA) + 2 mg/l (BAP) from hypocotyl explants (94.3%), and 0.4 mg/l (NAA) + 0.5 mg/l (BAP) for leaf explants (100%). Efficient shoot regeneration (22%) was achieved when the epicotyl explants were incubated on MS media amended with 0.1 mg/l (NAA) + 2 mg/l (BAP) and 0.4 mg/l (NAA) + 0.5 mg/l (BAP) within 14 days of culture. Maximum indirect shoot regeneration (28.4%) was achieved from green-yellowish calli derived hypocotyl explants on MS medium with 0.4 mg/l (NAA) + 2 mg/l (BAP) within 21 days of culture. Also, an in vitro regeneration system from nodal segments was developed on MS medium supplemented with different levels of 6-benzyladenine. Maximum number of shoots per nodal explants was developed on a medium containing 3 mg/l (BA) at the rate of 2.5 micro-shoots per nodal explants after 4 weeks of culture. Proliferated shoots were elongated in hormone-free MS medium and also, shoots were rooted on MS medium and MS with 2 mg/l indol-3-butyric acid. Key words: Tribulus terrestris, plant regeneration, callus induction, plant growth regulators, MS (Murashige and Skoog) medium.

Nepenthes plant is a unique plant due to its interesting pitcher at the leaf tip. The objective of the research was to determine the suitable MS (Murashige and Skoog) medium concentration for growth of two species of nepenthes and induction pitcher formation. A factorial experiment arranged in completely randomized design was carried out with the first factor was two species of Nepenthes; N. ampullaria and N. mirabilis. The second factor was several concentrations of MS medium, i.e. 0.500 MS, 0.250 MS, 0.125 MS, 0.0625 MS and 0.000 MS. The results showed that the number of leaves, the number of pitchers were affected by the type of Nepenthes and MS medium while plant height only affected by type of Nephenthes. N. mirabilis had significance higher number of leaf of 13.47 compared to N. mirabilis of 11.10. However, number of pitchers formed was lower in N. mirabilis compared to N.ampullaria. Data obtained also showed that the best MS medium for pitcher formation was 0.0625 MS medium. Keywords : Nepenthes ampullaria, Nepenthes mirabilis, pitcher plant, MS medium

<p>Cephalotus follicularis Labill. is representative of the extraordinary carnivorous group of plants. Carnivorous plants with pitcher traps grow in nutrient poor, sunny and wet habitats, they have adapted themselves to growth in bogs, sandy soils and obtain some nutrients (e.g. nitrogen and phosphate) from insects and other arthropods or protozoa, even from small mammals occasionally (Król et al. 2012). C. follicularis belongs to the monotypic family Cephalotaceae, and it is the endemic plant of south-western Australia. This Australian pitcher plant is heterophyllous with different types of leaves, specialized either for prey capture or photosynthesis. In the spring, non-carnivorous leaves responsible for photosynthesis, while later, in the summer the second type – carnivorous pitcher-shaped leaves occur (Pavlovic 2011). In this paper we present the first observation of tissue culture of C. follicularis for regeneration protocol, taking into account the absence of organic and inorganic nitrogen as well. We kindly achieved the in vitro plants of C. follicularis clones: 00001/01 and 0004/03 from collection of Mr. Kamil Pásek (http://www.bestcarnivorousplants.net/) from Czech Republic. These plants have been maintained on MS (Murashige and Skoog) media, in sterile conditions for several years. Under these conditions the plants are smaller than in the nature, do not develop pitchers properly and do not produce flowers, and intensive multiplication of shoots is observed. Therefore we tried to ensure such conditions, which will enable the proper development of this species in tissue culture. Plants of 00001/01 and 0004/03 clones were transferred to MS solid medium modified by reducing the amount of MS major salts to ½ and ¼ (½ MS and ¼ MS respectively). Plants were placed on these media in two ways: in small groups and individually. These same rules were applied when using subsequent ½ MS based media supplemented with 8,2 μM 2,4-D with 9,3 μM KIN (first fodder) and 4,9 μM IBA with 18,2 μM TDZ (second fodder). In addition, on the same media we put photosynthetic and pitcher leaves separately. All cultures were kept in light conditions. It occurred, that in the long term culture (after 47 days) on the ¼ MS medium the plants growing in small groups, started to develop pitcher and non-carnivorous leaves similar to these in the nature. Plants growing individually on the same medium did not change their appearance. The similar situation was observed for 00001/01 genotype, with one difference, bigger quantity of plants created properly shaped pitcher leaves on ½ MS medium. Decreasing the quantity of MS major salts to ½ gave good results in the case of rooting of plants of the Drosera intermedia Hayne (Kromer et al. 2000) and in Cephalothus regeneration system from root mass (Ko et al. 2010). The first symptoms of morphogenesis were noted on 42 day of culture as the direct organogenesis and somatic embryogenesis. The adventitious shoots and young somatic embryos were observed in 0004/03 on photosynthetic leaves, on the medium supplemented with 2,4‑D and KIN. The pitcher leaves did not induce morphogenesis. Histological analysis confirmed direct morphogenesis. Somatic embryos and adventitious shoots appeared on the petioles without callus formation and originated from epidermal and subepidermal layers of the explants. Taking into account the specific way of nitrogen uptaking by Cephalothus plants, the photosynthetic and pitcher leaves were maintained on the conditions with the lack of organic source of nitrogen and lack of inorganic nitrogen in MS medium. The observations showed that in the absence of inorganic nitrogen in the medium the non-carnivorous leaves become white and slowly degenerate. At the same conditions the pitcher leaves growing in small groups develop well traps, but they are fragile and strong red. In this small group the non-carnivorous leaves are not present. When the medium is free of organic nitrogen the non-carnivorous leaves become white but still alive, while the pitcher leaves are weak and do not develop well shaped traps. Further investigations are necessary for improving the culture protocols for more efficient plant regeneration and improvement of shoots formation, as well as analysis of the nitrogen influence on leaves of this plant.</p>

The present study reports on application of carbendazim for micropropagation and in vitro flowering of Peperomia pellucida. In vitro multiplication of shoot tips and axillary nodal explants were used in 25-100 mgL-1 carbendazim in 70% alcohol treatment for 5 min in MS (Murashige and Skoog) medium and MS hormone medium The explants were cultured on MS medium supplemented with auxins, cytokinins and additives (Casin hydrolyte and glutamine) used to proliferate the shoots. The maximum number of shoots (21.5/shoot tip) were developed on MS medium supplemented with 2.0 mgL-1 KIN (Kinetin) 0.5 mgL-1 NAA (α- Naphthalene acetic acid), 100 mgL-1 casein hydrolysate and 100 mgL-1 glutamine additives. The highest number of roots per shoot (17.6) and highest rooting frequency (94.2%) were obtained when the adventitious shoots were inoculated on MS medium with 1.5 mgL-1 IAA (Indole-3-acetic acid). The highest number of shoot development (75.4 %) was achieved on MS medium supplemented with 2.0 mgL-1 KIN 2.0 + 0.5 mgL-1 ABA (Abscisic acid) with 5% sucrose. It was found that MS medium devoid of 1.5 mgL-1 IAA promote the root formation and maturation of in vitro flower in P. pellucida. Acclimatized plantlets were transferred to botanical garden and the characters compared with mother plant of P. pellucida.

Reliable and reproducible protocols to get healthy and well formed plants from nodal explants of the pomegranate ( Punica granatum L) cv ‘Bhagava’ has been developed. Nodal segments were cultured on two different media at full strength Murashige and Skoog (MS) and Woody Plant Medium (WPM). The media was prepared as a basal medium supplemented with 0.2 to 2 mg/L 6-benzylaminopurine (BAP), 0.1 to 1 mg/L 1-naphthalacetic acid (NAA), 0.5 to 2.5 mg/L silver nitrate (AgNO3) and 10 to 50 mg/L adenine sulphate for establishment stage. For proliferation stage, 0.1 to 0.5 mg/L BAP and NAA was tested. For rooting stage 0.0, 0.25 and 0.5 mg/L 3-Indolebutyric acid (IBA) and NAA on MS and WPM medium were tested. The nodal explants grown on MS medium containing 1.8 mg/L BAP, 0.9 mg/L NAA, 1 mg/L silver nitrate and 30 mg/L adenine sulphate had the highest proliferation rate (10 to 15 shoots/explants) in establishment stage. The same trend was found concerning the maximum leaves numbers (15 to 20 leaves/explants) on proliferation medium containing 0.4 mg/L BAP and 0.3 mg/L NAA. The plantlets grown on MS medium were found to have better survival compared to WPM medium. 0.5 mg/L NAA and 0.5 mg/L IBA showed equal rooting response in both the medium, whereas thick root formation was observed in the medium containing IBA. Key words : Micropropagation, proliferation, rooting, Punica granatum .

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.

The objectives of the studies were to develop a system for callus induction and plant regeneration from immature inflorescences of nilegrass (Acroceras macrum Stapf cv. Taishu No. 1). Explants were cultured on an MS (Murashige and Skoog) medium supplemented with 2.00 mg L^(-1) 2,4-D (2,4-dichlorophenoxy acetic acid) and different concentrations of CPPU (N-(2-chloro-4-pyridyl)-N'-phenyl urea). The effects of CPPU on callus induction frequency were examined. The frequency of callus induction was 72.4% when immature inflorescences were cultured on MS medium with 2.00 mg L^(-1) 2,4-D and 0.10 mg L^(-1) CPPU for 5 weeks. For plant regeneration, the callus was transferred to MS medium with BA (N6-Benzyladenine) or TDZ (N-phenyl-N'-1, 2, 3-thiadiazol-5-lurea). The frequency of shoot regeneration was 16.7% with 0.05 mg L^(-1) TDZ and 13.3% with 1.0 mg L^(-1) BA. To increase the frequency of plant regeneration, callus induced with 2.00 mg L^(-1) 2,4-D and 0.1 mg L^(-1) CPPU was transferred to MS medium with 0.5 mg L^(-1) NAA and 1.0 mg L^(-1) TDZ for 5 weeks. This increased the shoot regeneration significantly by 77.8%. The shoots were transferred to 1/2 MS medium for rooting and then transplanted to the field. The percentage of surviving plantlets was more than 92%. From these results, we established an efficient system for plant regeneration from immature inflorescences of nilegrass. It would be helpful for commercial mass production of nilegrass propagation.

The effect of 2, 4-dichlorophenoxyacetic acid (2, 4-D), 6-benzylaminopurine (BAP), kinetin (KN), IAA (indole-3-acetic acid) and indole-3-butyric acid (IBA) was evaluated for in vitro shoot regeneration of Ocimum canum Sims. This important medicinal plant species belongs to the family Lamiaceae and it is also known as hoary basil. Nodal segment explants were cultured on Murashige and Skoog (MS) medium containing different phytohormones. 2, 4-D resulted in callus induction from nodal segments when supplemented with MS medium. The highest callus induction (90.17%) was recorded on MS medium containing 2, 4-D (2 mg/l). When nodal segments were cultured on MS medium supplemented with BAP (0.5–2.0 mg/l), KN (0.5–2.0 mg/l) as sole and with combination of BAP (0.5 mg/l) and IAA (0.10–1.00 mg/l), multiple shoots proliferation was initiated. The maximum number of shoots (12.75±0.64), with average length 6.04±0.08 cm, was recorded with MS medium containing BAP (1.5 mg/l) as sole. Repeated subculture of in vitro proliferated shoots yielded 35–40 shoots from a single nodal segment explant after 4–5 months. Developed single shoots were transferred for rooting to ½ MS medium containing IBA (1.5 mg/l) for best rooting of each shoot (3.99±0.23). The regenerated plantlets with well-developed roots were transferred to plastic pots containing a mixture of garden soil, farm yard manure and sand (1:1:1) in controlled environment for achieving high survival percentage (80 – 85%). Thus, a comprehensive micropropagation protocol was developed for O. canum Sims first time using nodal segment explants. This protocol can be used for commercial propagation and for future genetic improvement studies.

ABSTRACTOrganogenesis of Carum copticum from hypocotyl explants of in vitro seedlings was examined. The highest callus induction was observed from hypocotyl explants on both Murashige and Skoog (MS) and Nitsch and Nitsch (NN) media. A maximum of 92% of hypocotyl explants produced callus on MS medium supplemented with 1.0 mgL–1 N6-benzyladenine (BA)and 0.5 mgL–1 naphthaleneacetic acid (NAA), whereas, 84% of hypocotyl explants produced callus on NN medium with 1.0 mgL–1 BA and 0.5 mgL–1 NAA. Hypocotyl explants-derived light green and friable callus responded to shoot organogenesis on both MS and NN media with plant growth regulators. A maximum of shoot regeneration frequency (76%) was observed on MS with 1.5 mgL–1 BA, whereas, 55% shoot regeneration frequency was observed on NN medium with 1.5 mgL–1 BA. A maximum number of 8.4 shoots per callus explant having a shoot length of 4.05 cm was observed on MS medium with 1.5 mgL–1 BA, whereas, 5.5 shoots per callus explant with a shoot length of 3.5 cm was observed on NN medium with 1.5 mgL–1 BA. Regenerated shoots were cultured on full strength MS and NN medium for root induction. The highest root regeneration frequency of 96% was obtained on MS medium with 0.25 mgL–1 NAA inducing 7.6 roots per shoot with root length of 4.25 cm, whereas, 83% root regeneration frequency was observed on NN medium with 0.25 mgL–1 NAA inducing 6.4 roots per shoot with a root length of 3.81 cm. In vitro derived plantlets were hardened and acclimatized successfully.

Background:: Explant recalcitrance is one of the major challenges during the tissue culture of a tree species, especially when explants are derived from mature plants. It is mainly as-sociated with the genotypes/species, source and physiological status of explants, seasonal variations, and competency/incompetency of explants towards PGRs for in vitro manipulations. There-fore, to optimize the appropriate conditions and minimize explant recalcitrance, it is necessary to elucidate the different factors influencing the tissue culture of a tree species. Objective:: Several factors influencing in vitro shoot induction from nodal explants derived from mature plants of Sapindus trifoliatus were investigated. Methods:: Nodal segment obtained from mature plants was used as an explant for shoot regeneration. The factors assessed in this study included the types and concentrations of plant growth regulators, nutrient composition of MS (Murashige and Skoog) medium, sucrose concentrations, the position of nodes on their respective branches, seasonal variations, and successive transfers of mother explant. Results:: The performance of nodal explants was better on modified MS medium, i.e., M4 medium (half-strength of MS major salts and full-strengths of MS minor salts, Fe-EDTA and MS organic nutrients each) than the full-strength MS medium. The addition of BAP in the medium resulted in a higher shoot induction rate than other cytokinins i.e., Kin, Zeatin, TDZ. M4 medium supplemented with 3.0 mg l-1 BAP and 2% sucrose was optimum for shoot proliferation. However, elongation of shoots was observed only when induced shoots, along with the mother explant, were subcultured on a medium containing 1.0 mg l-1 BAP. The shoot multiplication was positively affected by the repeated transfer of the mother explant for different passages on the multiplica-tion medium. Mother explant transferred to multiplication medium (0.8% agar-solidified M4 medium + 2% sucrose + 1.0 mg l-1 BAP) proliferated maximum shoots after fourth passages. Regenerated shoots were rooted in vitro and further successfully acclimatized in field conditions. Conclusion:: The in vitro regeneration system developed from an explant obtained from mature trees can be successfully exploited for mass multiplication of Sapindus trifoliatus, a medicinally and commercially important tree.

Paraserianthes falcataria (L.) Nielsen is a fast-growing tree native to Indonesia that has been widely planted throughout the tropics. The growth and wood qualities of P. falcataria should be improved to promote the establishment of plantation forests for this species. Tissue culture technique has a potential to be applied for tree breeding programs for P. falcataria . The objective of this study is to establish the micropropagation and protoplast culture protocol of P. falcataria . In the present study, the respective conditions were investigated for seedling culture, callus induction, protoplast isolation, and protoplast culture. Surface-sterilized seeds were cultured on MS (Murashige and Skoog) medium at 25, 27, and 29°C. After 4 weeks of culture, 27°C gave the best result for average shoot length. Five types of explant (leaflet, petiole, internode, cotyledon, and hypocotyl) obtained from the seedlings were used for callus induction. They were cultured on the MS media containing a combination of 6-benzylaminopurine (BAP) and 2,4-dichlorophenoxyacetic acid (2,4-D) at different concentrations. Green nodular callus was obtained from only leaflet. The most effective medium condition for callus induction from leaflet was the MS medium containing 10.0 µM BAP and 10.0 µM 2,4-D. Leaflet of seedlings was used for protoplast isolation. Based on the results of the yield and viability of protoplasts, the best enzymatic condition was as follows: enzyme solution, 1% Cellulase Onozuka RS, 0.5% Pectolyase Y-23, and 1% Driselase; osmoticum, 0.8 M mannitol; treatment temperature and time, 30°C for 4 hrs. Isolated protoplasts were incubated in liquid AA media with a combination of 1-naphthaleneacetic acid and thidiazuron at different concentrations. Although cell wall formation was observed, cell division did not occur.

Spilanthes calva DC. (Maha Akrnella) is a valuable medicinal plant belongs to Family Asteraceae. Itis widely used in indigenous medicine to treat toothache in most of the Asian countries. Not only it hasanesthetic properties, but also contain secondary metabolites, with the insecticidal properties, whichcould be used as potential bio insecticide. This is an annual plant, which grows to a height about 30ern. After flowering mother plant is dried off. Four to six weeks later seeds are germinated and newseedl ings are produced. Viabil ity of seeds loses with in short period of time. Even though seeds aregerminated percentage of germination is low (about 30%). Rooting of cuttings is also not possible.This is a limitation in using this valuable medicinal plant for commercial production. Therefore it isvery important to develop a protocol for mass propagation through tissue culture and establishing cellcultures will be useful for large-scale chemical extraction in industrial purposes.Leaf discs were used as explant for callus initiation. In order to identify the suitable maturity stage forcallus initiation, leaves were harvested at different maturity stages i.e first, second and third fullyopened leaf.Leaves were washed with Dettol" soap and soaked in a solution of Teepol" for 5 minutes. After thatleaves were washed with running tap water for 45 minutes, In order to surface sterilize. Leaves werewashed with 10% Clorox ™ (5.25% Sodium hypochlorite v/v) for 5 minutes and then with 70% alcoholfor 30 seconds each followed by three successive washings in sterile distilled water. These operationswere carried out inside the laminar airflow cabinet before inoculation. Basal media tested for the study were full strength MS (Murashige and Skoog, 1962) medium and Y2 MS (both macro andmicronutrients) medium. Media were supplemented with different concentrations (1.0 mgl' - 3.0mgl") of BAP and 2,4-0. Cultures were incubated under complete dark at 25± I °C in the growthroom.Study conducted by Haw and Keng (2003) on the same species produced multiple shoots from axillarybud explants without inducing callus in MS medium supplemented with 2.0 mgl.:' BAP. In the presentstudy, callusing was observed within 5 days of incubation in full strength MS medium supplementedwith BAP and 2AO. It took longer period to initiate callus when both macro and micro nutrients in thebasa l rned ium was lowered to ha If and the amount of callus produced was also very low even after 6thweek of incubation. In order to observe the time taken to produce maximum amount callus freshwe iuht was measured after 2".1,4th and 6tltweek of incubation. It was observed that maximum amountofc~llus was produced within 4 weeks in all explant types tested with a maximum of 0.88 g:': 0.23 inleaf discs obtained from first fully opened leaf.In order to determine the best growth regulator combination for callus initiation, calli fresh weightswere measured after fourth week of incubation in different growth regulator combinations tested.Highest amount of calli were in MS medium in the presence of2.25 mgl' BAP and 1.0 mgl' 2.4-0.Fragile calli, which were transulant and mucilaginous in nature were observed within 15 days ofincubation, which could lead to cell suspension cultures.

The nodal explants of C. ovalifolium were cultured on Murashige and Skoog (MS) medium augmented with either [6-benzyladenine (BA) or kinetin (Kn) or thidiazuron (TDZ) or N6-(2-isopentenyl) adenine (2 ip)]. The maximum percentage response (100%) with highest number of shoots (5.0 ± 0.9) with longest shoot (3.7 ± 0.5 cm) per nodal explants were obtained on MS medium containing 5 mg/l BA after 45 days of culture. Shoot regenerated on MS medium supplemented with 5 mg/l BA as well as from other cytokinin shows callus formation at the base of stem; hence these shoots are not suitable for further use. The possible reason of callus formation could be ascribed to the activity of endogenous auxin. To prevent callus formation, nodal explants were cultured on MS medium supplemented with combination of BA and 2,3,5-tri-iodobenzoic acid (TIBA) or BA with TIBA and activated charcoal or BA with TIBA and adenine sulfate (AdS). Nodal explants inoculated on the MS medium augmented with 5 mg/l BA, 3 mg/l TIBA and 30 mg/l AdS have not only prevented callus formation but also produced significantly maximum number of shoots (7.5 ± 0.3) having shoot length of 4.7 ± 0.2. Maximum number of roots (6.3 ± 0.4) and root length (5.3 ± 0.1 cm) per explant was observed on half-strength MS medium containing 2 mg/l indole 3-butyric acid.

Introduction:The present research work was undertaken with a view to developing a suitable protocol forin vitroplant regeneration of economically important plant (Glycine max) (Bangladesh Agricultural Research Institute BARI- 5) variety,viaboth direct and indirect organogenesis fromin vitrogrown seedlings.Methods:For micropropagation explants were cultured on MS and half strength Murashige and Skoog (MS) medium supplemented with various plant growth regulators (cytokinins and auxins). In the present study for inducting of callus, among 3 different hormone combinations, the suitable medium was 3.32 mg/L 2, 4-D containing MS medium and the callus was deep green in color. Different type of media like MS, 1/2 MS and MS with different (6-Benzyl Amino Purine) BAP concentration was used for seed germination ofGlycine max. 100% of seed germination was observed in MS +1 mg/L BAP containing the medium.Results:In the present investigation, different concentration of cytokinins and auxins{BAP, 2, 4-D, and Naphthalene Acetic Acid (NAA)} were used individually or in combinations with MS medium to observe their effect on multiple shoot regeneration from the cotyledonary nodal segment. 100% shoot formation from cotyledonary nodal segment was recorded in 1.5 mg/L BAP and 0.15 mg/L BAP + 0.025 mg/L NAA containing MS medium, the best number of shoot was 10.9±2.0 found in MS + 1.5 mg/L BAP containing medium and highest length of shoot was 2 cm recorded in 1.5 mg/L BAP + 0.3 mg/L (different concentrations of Giberrellic acid) GA3containing MS medium. In addition, for root inductionin vitroraised well developed and elongated shoots were excised and cultured on MS and 1/2 MS medium supplemented with various concentration of Indole-3-Butyric acid (IBA). It was observed that MS medium containing 0.1 mg/L IBA and 1/2 MS medium containing 0.25 mg/L IBA was optimal for root induction. In which 100% shoots rooted well within 13 days of culture. The highest average number of roots per shoot was 6 recorded in MS +0.5 mg/L IBA containing the medium and highest average length of root was 8 cm recorded in 0.1 mg/L IBA containing MS medium.Conclusion:The most effective surface sterilization treatment for explants ofGlycine maxhas been found in 0.1% HgCl2solution for 15 minutes.

This is the first report of in vitro propagation and alkaloid accumulation in callus cultures of Ceropegia juncea Roxb. a source of "Soma" drug in Ayurvedic medicine. Multiple shoots and callus induction was optimized by studying the influence of auxins [IAA (Indole-3-acetic acid), NAA (2-Naphthalene acetic acid) and 2,4-D (2,4-Dichlorophenoxyacetic acid.)] and cytokinins [BA (6-benzyladenine) and Kin (Kinetin)] alone and in combinations. The best response for multiple shoot induction was obtained in nodal explants on MS medium supplemented with 7.5 μM Kin (8.5 ± 3 shoots per explants). The shoots were rooted on half strength MS (Murashige and Skoog's) medium fortified with either IAA or NAA (0.5-2.0 μM). The plantlets were transferred directly to the field with 100 % success rate. Supplementation of MS medium with auxins and cytokinins enhanced the growth of callus but inhibited the shoot regeneration in nodal explants. Best callus induction and proliferation observed on MS + 1 μM 2,4-D+5 μM BA. However the maximum cerpegin content (470 μg/g dry weight) was recorded in dried callus derived on MS+10 μM IAA+5 μM BA. Quantitative TLC (Thin layer chromatography) studies of the callus revealed a phytochemical profile similar to that of naturally grown plants. The calli were maintained by subculturing at 4 weeks interval on fresh parent medium over a period of 34 months. The optimized in vitro propagation and callus culture protocol offers the possibilities of using organ/callus culture technique for vegetative propagation and production of cerpegin alkaloid.