Plant Archives Volume 20 No. 1, 2020 pp. 2030-2036 e-ISSN:2581-6063 (online), ISSN:0972-5210 RAPID CLONAL PROPAGATION THROUGH IN VITRO SHOOT TIP AND NODAL EXPLANTS OF PISONIA GRANDIS R. BR. Thiri Bhuvaneswari Ramachandran1, Karthick Murugan2 and Manikandan Gurusamy1* 1*Department of Botany, Sri Kaliswari College (Autonomous), Sivakasi, Virudhunagar District (Tamil Nadu), India. 2Department of Biotechnology, Sri Kaliswari College (Autonomous), Sivakasi, Virudhunagar District (Tamil Nadu), India. Abstract Pisonia grandis R. Br. is a medium size evergreen tree belonging to the family Nyctaginaceae and locally known as Lechaikattai keerai in Tamil. The leaf extracts were reported with numerous bioactive compounds. The cells under culture accumulate large amounts of secondary compounds under specific culture condition. Therefore, in vitro propagation of medicinal plant can be used as fast and reliable method to synthesis the bioactive compounds. Moreover, P. grandis is reproduced by vegetative means and success rate of production of viable sapling is also very low. Besides, young saplings were also affected with leaf rust diseases. Therefore, effective strategies to be developed for the production of viable saplings of this multipurpose plant species by using plant tissue culture technique. In the present study, maximum mean numbers (13.12 ± 0.42) of shoots per explants were obtained on MS medium supplemented with 1.0 mg/l BAP. IBA at 4.0 mg/l showed the best results, where 8.26 ± 0.17 mean number of roots and 10.82 ± 0.21 mean number of root length were initiated after 15 days of culture. Key words: Mass multiplication, Leaf rust, MS Medium, BAP, IBA. Introduction et al., 2000; Pan et al., 2003). Micropropagation is the practice of rapidly multiplying Pisonia grandis R. Br. is a medium size evergreen stock plant material to produce a large number of progeny tree belonging to the family Nyctaginaceae and locally plants using modern plant tissue culture methods. known as Lechaikattai keerai in Tamil. It is wide spread Micropropagation is used to multiply novel plants such as evergreen glabrous garden tree with young shoots are those have been genetically modified or bread through minutely puberulous. It is a native of Hawai island also conventional plant breeding methods. It is also used to naturalized throughout India. It has been considered as provide a sufficient number of plantlets for planting from an ornamental tree and leaves resembles lettuce in taste. a sufficient number of plantlets for planting from a stock So, it is used as a vegetable and also as salad (Anonymous, plant which does not produce seeds or does not respond 1969) in most part of the India. The plant Pisonia grandis well to vegetative reproduction (Ajithkumar and Seeni, extensively used as leafy vegetable and in Indian 1998). traditional medicine as an anti-diabetic and for other The encourage overharvesting of medicinal plants, medicinal purposes. Leaves, stem and root of this species leading to an extractive exploitation that exposes the are extensively used by the tribals in the preparation of species to the threat of extinction. The interest an in several folk medicines (Shubashini et al., 2013). It has vitro propagation of medicinal plants is clearly on the been extensively used in Indian traditional medicine as rise, because they represent the most important sources an antidiabetic, anti-inflammatory agent, and used in the of medicines and other pharmaceutical products. treatment of algesia, ulcer, dysentery and snake bite Methods of in vitro propagation offer highly effective (Prabu et al., 2008; Radha et al., 2008; Sunil et al.,2009; tools for germplasm conservation and mass multiplication Will McClatchey, 1996). The roots are used as purgative. of many plant species threatened with extinction (Murch The phytochemical study reveals that the presence of *Author for correspondence : E-mail : [email protected] steroids likes octocosanol, betositosterol, alphaspinosterol, Rapid clonal propagation through in vitro shoot tip and nodal explants of pisonia grandis r. br. 2031 dulcitol and flavonoids in the leaves of the plant Materials and Methods (Manogaran, 2002). Collection of Plant materials and Surface Sterilization Pisonia grandis have a numerous bioactive The explants (shoot tip and nodal) of Pisonia grandis compounds includes Pinnatol, Allantoin, - Sitosterol, - were collected from Sri Kaliswari College, Sivakasi, Spinasterol, -Sitostero glucoside, Octocosanal, Dulcitol, Virudhunagar dist, Tamil Nadu, India. The explants were Flavonoids and Quercetin. It has been claimed that the washed under running tap water 30 min. and then they compound possesses insulin like effects and also mediate were washed in an agitated solution of liquid detergent certain actions of insulin (Sivakumar et al., 2010). Among for 5 min. The disinfected materials were removed by the various bioactive compounds, pinitol has been rinsing the material with sterilized cooled distilled water extensively used in Indian traditional medicine as an anti- for 3-5 times. The explants of Pisonia grandis were diabetic and by chewing two leaves of this tree per day surface sterilized with 0.1% Mercuric chloride for 3 min. has been traditionally followed to control diabetes. Pinitol Then, subsequently washed with sterile distilled water. or 3-O-methyl D-Chiro-inositol, has been reported to The explants were again sterilized with 4% Sodium possess antidiabetic and hypoglycaemic activities (Mijares hypochlorite for 3 min. and rinsed with sterile distilled et al., 2013; Sivakumar and Subramanian, 2009). It has water. The material were again surface sterilized with been claimed that the compound possesses insulin like ethanol (70%) for 3 min. finally washed with sterile effects and also mediate certain actions of insulin distilled water and the explants cut the tip end with help (Sivakumar et al., 2010). Clinical trials on the effect of of a sterilized blade. pinitol on glycaemic control and cardiovascular risk factors in patients with type II diabetes mellitus have Culture medium and Condition been successfully carried out (Kim et al., 2005; Min- The sterilized explants were cut into 2-3cm long pieces Jung Kang et al., 2006). Pinitol is reported as safe and and cultured on Murashige and Skoog medium containing nontoxic as an anti-diabetic agent even at high levels 30g/l of sucrose and 8g/l of agar was also added as carbon (Sarah et al., 2000). source and gelling agent respectively with the above Plant products have gained prominence during the chemical composition. Along with boiled medium, growth past few years along with the management of traditional hormones like BAP and Kn were added at different medicinal plant resources and it has become a matter of concentrations (0.1-2.0 mg/l) and dispensed into the culture urgency. An ever increasing demand of medicinal plants vessels. The medium was sterilized in an autoclave at and its drugs warrants their mass propagation. The in 121ºC for 30 min. The sterilized culture vessels with MS vitro propagated medicinal plants are genetically pure elite. medium supplemented with different hormones were In organ culture, plants have high concentrations of transferred to the laminar air flow chamber for the secondary metabolites tend to accumulate in specific cell successful sterilization. types during developmental stages. The tissue cultured Culture Initiation and Shoot Multiplication cells from these plants typically accumulate large amounts The explants were carefully inoculated in the culture of secondary compounds under specific conditions. vessels with MS medium along with various Based on that, efficient in vitro regeneration of the whole concentrations of growth hormones without any microbial plant can be used as a fast and reliable method to contamination. The inoculated glass vessels were finally transform P. grandis for the synthesis of bioactive kept in the culture rack in the room with proper compounds and its active principles. Moreover, P. grandis temperature (25±2oC) and light intensity of 2500 – 3000 non-viable to reproduce through sexually and produced lux. by cool fluorescent lamp. After 30-45 days, the shoot their offsprings through vegetative means only. Besides, development from the culture was observed carefully major percentages of saplings developed through and number of shoots and length of shoots developed vegetative cuttings were affected with leaf rust from the explants were also observed. Callus initiations (Ramasubbu et al., 2015). Due to its specific medicinal and its developments if any were also observed in the cultured tubes. Multiple shoots developed from the compounds and varieties of medicinal practices, the plant explants were transferred from the culture tube to a sterile has been over exploited. Therefore, necessary steps to glass plate using sterilized forceps. The leaves were be taken to save the species for before eradication. In removed from the primary shoots developed in the culture this connection, rapid clonal propagation protocol has been medium and sectioned into one node piece with the leaves. developed the mass multiplication of this medicinally These nodal segments were transferred to the shoot important tree species of P. grandis for sustainable multiplication media with different concentration (0.5- utilization. 2.5mg/l) of 6 Benzyl amino purine (BAP) and kinetin 2032 Thiri Bhuvaneswari Ramachandran et al. (0.5-2.5 mg/l). All the experiments were carried out with explants were transferred to large