ADALYA JOURNAL ISSN NO: 1301-2746 Effect of Fungal Elicitors on Plant Growth in the in vitro Cultures of Oldenlandia umbellata L. Author - S. Saranya Research Scholar, PG and Research Department of Botany Government Arts College (Autonomous), Karur 639 005, Tamil Nadu - INDIA E-mail: [email protected] Co-author - P. Velayutham Research Supervisor, Krishna College of Arts and Science, Kolluthannipatti, Karur Dt., Tamil Nadu - INDIA E-mail : [email protected] Abstract Improvement strategies on plant growth are appreciable for commercially important medicinal plants due to over exploitation. Oldenlandia umbellata L. is one such dye yielding medicinal plant with the wide range of application in pharma and textile industry. An attempt was made in the present study to enhance the growth of O.umbellata in the in vitro by three fungal elicitors, namely, Aspergillus niger, Mucor prayagensis and Trichoderma viride. Among the three fungal elicitors A. niger derived elicitors showed the better callogenic response, shooting and rooting. The optimum concentration for each elicitor was studied. The maximum number of 79 shoots and 47 roots were obtained in 100 µg l-1 A. niger elicited callus. Keywords: Oldenlandia umbellata L., suspension culture, fungal elicitors, callogenesis, regeneration, rooting, hardening. Introduction Plant cell, tissue, and organ culture is one of the promising technologies for commercial production of secondary metabolites when natural resources are limited. The stimulation of defined compounds within carefully regulated in vitro cultures provides better understanding of metabolic pathways under highly controlled growth conditions[1]. The industrial application of plant cell cultures has limited success and only few high-value natural products such as shikonin, paclitaxel, resveratrol, artemisinin, ginsenosides, and ajmalicin have been commercialized [2] so far due to the low content of the desired metabolite, recalcitrant nature and slow growth rate, genotypic variations, chemical instability, and uneconomical downstream processing. Several strategies have been developed to overcome these issues, such as medium optimization, elicitation, precursor feeding, immobilization, in Volome 8, Issue 12, December 2019 731 http://adalyajournal.com/ ADALYA JOURNAL ISSN NO: 1301-2746 situ product removal, genetic transformation and bioreactor engineering[3]. Elicitation is an attractive strategy employed to induce growth of the plant and associated metabolite production due to the addition of trace amounts of elicitors in a plant in vitro culture systems. Plants show a wide range of morphological and physiological responses with respect to the elicitors. It involves numerous molecular changes inside the plants concerning for the particular stress conditions. Fungal elicitors have been widely employed to increase natural product formation in plant cell cultures and this strategy has been effective in stimulating the growth and metabolite production. Oldenlandia umbellata L. is one of the important members of Rubiaceae known for its dyeing and medicinal properties. This plant is used in traditional medicine and Siddha for its styptic property[4]. The leaf and root extracts were considered as good expectorants and used for treatment of asthma, bronchitis, and bronchial catarrh[5]. The decoction prepared from its leaves is used as a rinse to treat poisonous bites[6] and also used as a febrifuge. A novel pH indicator dye was reported from this plant[7]. Extract of the whole plant shows significant antitumor activity[8]. The major dyeing property depends on anthraquinone contents of roots and used to impart red colour to the textile materials[9,10]. The multipurpose usage has made increased usage of this plant and a reliable protocol was developed for enhanced growth of the plant through tissue culture technique. So, the present study aims to improve the plant growth by using fungal elicitors in addition to the plant growth regulators. Materials and Methods Plant Material and Fungal Strains The leaf derived callus of O. umbellata was obtained from previously reported protocol [11]. Three fungal strains such as Aspergillus niger, Mucor prayagensis and Trichoderma viride, were procured from the Institute of Microbial Technology, Chandigarh, and used for elicitation Preparation of Fungal Elicitors The fungal strains were grown separately in 250 ml conical flasks containing 100 ml SD (Sabrouds dextrose) broth. The flasks were incubated at room temperature under static conditions. At stationary phase, after 21 days, the flasks were autoclaved and the fungal mats were separated from the culture medium. Fully grown mycelia with spores were homogenized with mortar and pestle and centrifuged at 4000 rpm and the supernatants were Volome 8, Issue 12, December 2019 732 http://adalyajournal.com/ ADALYA JOURNAL ISSN NO: 1301-2746 collected. The collected supernatant was autoclaved for 20 min at 121°C. The autoclaved fungal extracts were used as elicitors for in vitro culture system. The fungal extracts were stored at 4°C for elicitation purpose. Treatment of Fungal Elicitors All three fungal elicitors of four different concentrations, viz., 25, 50, 100 and 200 µg l-l (Equalent polysaccharide content) were added separately into the standardized MS liquid medium for callus culture (10 µM NAA and 30 g l-l sucrose) and the well-developed callus was aseptically transferred to the respective liquid medium. Cell suspension cultures were established and maintained on the above liquid medium at a subculture interval of 7 days. All the cultures were maintained in an orbital shaker at 120 rpm min-1 for 30 days. The treated calli were then transferred to shooting medium fortified with 6 µM BAP in combination with 2 µM Kin and respective fungal elicitors. The well grown shoots were separated and transferred to rooting medium of 6 µM IBA. Fully matured plantlets with well developed roots were transferred and allowed for the acclimatization process. Determination of growth parameters The different parameters were recorded such as number of shoots, shoot length, number of roots per shoot, and root length were recorded. Statistical analysis All the experiments were repeated three times and each experiment consisted of one explant per tubes and five replicates. Statistical analysis was carried out using Analysis of Variance (ANOVA) comparing the treatments and using Duncan’s Multiple Range Test (DMRT) at the 5% probability and analysed using SPSS for Windows, version 21. Results In the present study, callus induced from the leaf explants of O. umbellata was treated with different fungal elicitors in varying concentrations to screen the suitable elicitor and its concentration for enhanced plant growth. Callus Culture Volome 8, Issue 12, December 2019 733 http://adalyajournal.com/ ADALYA JOURNAL ISSN NO: 1301-2746 Callus was induced from the leaf explants on MS medium fortified with different concentrations of IAA, IBA, NAA and 2,4-D. Maximum rate of 100 per cent callus induction with green compact organogenic callus was observed on MS medium supplemented with 10 µM NAA. Effect of Fungal Elicitors on Callus Growth The calli were grown on suspension media containing 10 µM NAA and four different fungal elicitor concentrations, namely, 25, 50, 100 and 200 µg l-1 of the three elicitors to optimize for higher production of cell biomass (Fig.1.a-d). The treated calli were further subcultured on solid media with respective fungal elicitors and obtained large amount of calli. Of the three fungal elicitors treated, large amount of green compact callus was obtained on medium treated with the extract of A. niger followed by T. viride. However, the callus treated with M. prayagensis showed an equal response for callus growth to that of control at all tested concentration. Regeneration of shoots from fungal elicited calli The well developed calli were grown on regeneration medium containing 6 µM BAP + 2 µM Kin with respective fungal elicitors. The shoot regeneration response was varying among the various elicitor treatments with different concentrations (Table 1; Fig.2.a-d). The maximum shoot regeneration response was obtained from 100 µg l-1 A. niger elicitor treated calli. Maximum number of 78.8 shoots with shoot length of 11.4 cm was achieved on the callus treated with A. niger. The M. prayagensis elicitor treated calli at 50 µg l-1, concentration produced 72.6 shoots with 10.2 cm shoot length, where as T.viride at 25 µg l-1 concentration produced an average number of 68.2 shoots with 9.8 cm shoot length. The shoot regeneration frequency and number of shoots were decreased when the calli were treated with higher dose. Rooting and Hardening The healthy shoots obtained from all the treatments showed better rooting on standardized rooting medium. The rooting response was accompanied with shooting frequency with same concentrations (Table 2; Fig.3.a,b). The maximum root regeneration response was obtained from 100 µg l-1 A. niger elicitor treated shoots followed by 50 µg l-1 M. prayagensis and 25 µg l-1 T. viride with an average number of 46.6, 28.6 and 30.8 respectively from root induction medium containing 6 µM IBA. The root regeneration frequency and number of roots were decreased when Volome 8, Issue 12, December 2019 734 http://adalyajournal.com/ ADALYA JOURNAL ISSN NO: 1301-2746 the shoots were treated with above optimal concentration. Similar to organogensis, during elicitation process also the plantlets developed in vitro flowers in umbels (Fig.3.b). The rooted