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Micropropagation and in Vitro Flowering in Pentanema Indicum Ling

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Micropropagation and in Vitro Flowering in Pentanema Indicum Ling Plant Biotechnology 24, 527–532 (2007) Tissue Culture Note Micropropagation and in vitro flowering in Pentanema indicum Ling Iyyakkannu Sivanesan1, Byoung Ryong Jeong1,2* 1 Department of Horticulture, Division of Applied Life Science (BK21), Graduate School, Gyeongsang National University, Jinju 660-701, Korea; 2 Research Institute of Life Science, Gyeongsang National University, Jinju 660-701, Korea * E-mail: [email protected] Tel: ϩ82-55-751-5489 Fax: ϩ82-55-757-7542 Received January 17, 2007; accepted September 27, 2007 (Edited by S. Ogita) Abstract In this study we have investigated micropropagation and in vitro flowering for a medicinally important plant Pentanema indicum. Maximum callus proliferation was obtained on MS medium supplemented with 2.0 mg lϪ1 BAP and 1.0 mg lϪ1 IBA. The best shoot regeneration (19Ϯ1.0) was achieved in five weeks when callus was cultured on MS medium amended with 4.0 mg lϪ1 BAP and 1.0 mg lϪ1 IAA. Direct multiple shoot initiation was also obtained from shoot tip and nodal explants in the presence of BAP and IAA. Addition of adenine sulfate (1.0 mg lϪ1) to the regeneration medium increased the shoot multiplication. Regenerated shoots rooted best on MS medium containing 2.0 mg lϪ1 IBA. Multiple shoots regenerated from callus, shoot tip and nodal explants flowered (90%) in vitro on a MS medium fortified with 2.0 mg lϪ1 IBA. Plantlets were successfully acclimatized in a soil condition and the survival rate was 96%. Key words: Asteraceae, callus culture, direct regeneration, in vitro flowering. Pentanema indicum Ling. belongs to the family scale for extraction of pure drug. To date only one report Asteraceae (Compositae). It is a female antifertility drug available on regeneration for this important medicinal used by tribes in Bihar state of India. The plant is an plant (Thulaseedharan and Vaidyanathan 1990) reported annual and is distributed throughout India, ascending up callus induction and plant regeneration in Vicoa indica. to an altitude of 1800 m in Himalayas, Pakistan, Burma, However, regeneration using apical bud and nodal China, Sri Lanka, Thailand and Africa. It is an erect, explants derived from mature plants is not available. rigid, leafy herb 1–3 ft in height. The roots are tough, Therefore, the efficiency of apical bud and nodal explants woody and externally pale brown in colour. Stem terete, to regenerate P. indicum plants has been explored in striate, with many pubescent branches. Leaves are the present study. In addition, we have demonstrated a variable in size, sessile, oblong–lanceolate, acute, entire callus mediated plant regeneration system and in vitro or serrulate, rough or scrabid with short appressed hairs flowering. on both sides. Ray florets 12–24, much longer than Actively growing shoots were used as the explants involcure and disc florets scanty. Many compounds have source. The shoots were separated from the mother plants, been isolated from P. indicum namely, sesquiterpene– defoliated and cut into 4–6 cm pieces. The explants (leaf vicolides, monoterpenediol–vicodiol and thymol esters and stem) were surface sterilized with 70% (v/v) ethanol (Saradha Vasanth et al. 1990; Mossa et al. 1997), cis–cis for 60 s and 0.1% (w/v) mercuric chloride for 10 min, germacranolide (Sawaiker et al. 1998), 4,5,6-trihydroxy- washed four times with sterile distilled water. Leaf and 4-7-dimethoxy flavone (Krishnaveni et al. 1997). Vicolide stem segments 0.5–1.0 cm in length, were prepared D showed abortifacient and antifertility activities (Alam et and inoculated on Murashige and Skoog (MS) medium al. 1989). The hexane soluble fraction showed antiviral (Murashige and Skoog 1962) supplemented with plant activity against Ranikhet virus (Chowdhury et al. 1990). growth regulators. The pH of the medium was adjusted In vitro flowering offers a unique system in the study of to 5.7 with 0.1 N NaOH or 0.1 N HCl before autoclaving molecular basis and hormonal regulation of flowering. at 1.06 kg cmϪ2 and 121°C for 15 min. All culture media There is also a possibility of using in vitro flowering contained 3% (w/v) sucrose and solidified with 0.8% in ecological and genetic studies. It is a modern area (w/v) agar. of research and has great potential in plant breeding Basal medium supplemented either with BAP or BAP programmes. In vitro propagation of medicinal plants combined with IBA were used for callus induction could help in raising disease free health clones in a large from leaf and stem explants. The callus induction rate This article can be found at http://www.jspcmb.jp/ Copyright © 2007 The Japanese Society for Plant Cell and Molecular Biology 528 Micropropagation and in vitro flowering in Pentanema indicum Ling (explants with callus formation) was determined after 30 Table1.Effect of plant growth regulators on callus induction from days. After four weeks, the calli proliferated upon the leaf and stem explants of P. indicum. leaf and stem segments. The calli were transferred on Plant growth regulators Callus induction (%) Ϫ MS medium supplemented with different concentrations (mg l 1) MeanϮSD and combinations of BAP and IAA to induce plant BAPIBA Stem Leaf regeneration. Regenerated shoots were dissected out, cut 1.0 — 18.2Ϯ1.7i 12.4Ϯ0.7j into shoot tips and single nodal segments, and subcultured 2.0 — 39.4Ϯ1.9g 20.6Ϯ1.0i on MS medium containing different concentration of an 3.0 — 63.0Ϯ1.0d 36.6Ϯ1.0h Ϯ e Ϯ f auxin and a cytokinin. Multiplication of these shoots was 5.0 — 54.8 1.1 40.8 1.2 1.0 1.0 43.1Ϯ3.3f 34.0Ϯ1.4h tested in the same media or in media supplemented with 2.0 1.0 92.0Ϯ1.0a 64.0Ϯ1.0d Ϫ adenine sulfate (1.0 mg l 1). After four weeks of culture 2.5 1.0 78.0Ϯ1.6c 70.4Ϯ1.2b plantlets were transferred to fresh medium containing The experiments were repeated thrice, each experiment consisting of same composition of ingredients. 25 replicates. Results were recorded after 30 days. Variability around Individual shoots, which were 3–4 cm long were the mean was represented as the standard deviation (SD). MeanϮSD Ͻ excised from the shoot clump and transferred to half followed by different letters are not significantly different (P 0.05) by Duncan’s multiple comparison test. strength MS medium containing IAA and IBA (0.5, 10, 1.5 and 2.0 mg lϪ1). All cultures were incubated at 25Ϯ2°C under cool fluorescent light (3000 Lux; 16-h percentage of explants producing calluses as affected photoperiod). After 35 days the rooted plantlets were by explant type is shown in Table 1. Generally, the removed from the tubes, washed thoroughly with sterile highest frequency of callus formation was obtained in distilled water to remove traces of agar and planted in stem (92%) and leaf (70%) explants. On increasing small plastic pots filled with mixture of sterile soil, sand, the concentration of BAP (1.0–5.0 mg lϪ1), a gradual vermiculite (1 : 1 : 1). The potted plants were irrigated increase in percentage of culture forming callus was with MS basal salts solution (1/4 strength) devoid of noted (12–41%) in leaf explants. Of the various BAP sucrose and myo-inositol every four days for three concentrations, the maximum percentage (63) of light weeks. After three weeks the plants were kept under greenish friable callus was obtained from stem explants shade for two weeks and then transferred to glasshouse. in medium supplemented with 3.0 mg lϪ1 BAP. Higher After callus induction, the primary calli were separated concentration of BAP inhibited callus induction. The from stem explants and they were subcultured on MS combination of BAP with IBA at the range of 2.0 and medium (3% (w/v) sucrose and 8.25 g lϪ1 ammonium 1.0 mg lϪ1 respectively induced better proliferation of nitrate) supplemented with BAP alone or combined with callus. The primary calli were separated from leaf and IAA and adenine sulfate. Regenerated shoots of 3–4 cm stem explants and they were subcultured on MS medium long were excised and cultured on the MS medium (3% containing either BAP or BAPϩIAA. Over a period (w/v) sucrose and 8.25 g lϪ1 ammonium nitrate) fortified of 21 days, green compact callus was beginning to turn with IAA and IBA. The number of flowering shoots was brown, whereas light greenish friable callus produced counted at the end of each subculture period. To study reddish or orange pigment. However, the white friable the effects of various carbon source such as glucose, callus multiplication was good and scoring was possible fructose, lactose, maltose, sucrose and nitrogen source after 60 days of culture. Hence, the white friable callus viz., ammonium nitrate on in vitro flowering. The was used for shoot regeneration and all other callus concentration of each carbon source was 3% (w/v) and types were not useful. Calli were small and friable and ammonium nitrate used was 0, 8.25, 16.5 and 33 g lϪ1. have always a potential for induction of de novo shoot All experiments were repeated thrice. Data were formation. Incorporation of various concentrations of BA analyzed by analysis of variance (ANOVA) to detect in the MS basal medium significantly increased the shoot significant differences between means using SAS proliferation rate and number of shoots (data not shown). computer package (SAS Institute Inc., Cary, NC, USA, The shoot primordial started appearing from the white Release 8.1). Means differing significantly were friable callus obtained from the medium containing BAP compared using the Duncan multiple range test at 5% and IBA on passage to MS medium containing BAP probability level.
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