Micropropagation

Plant tissue culture, more technically known as Micropropagation, can be broadly defined as a collection of methods used to grow large numbers of plant cells, in vitro, in an aseptic and closely controlled environment. This technique is effective because almost all plant cells are Totipotent each cell possesses the genetic information and cellular machinery necessary to generate an entire organism. Van over beck and his co-workers in 1941 exhibited for the first time the stimulatory effect of coconut milk on embryo development of callus formation in Datura. Narstog (1965) & others led to the development of synthetic media for the culture of younger embryos. Most of the modern tissue culture media have been derived from the work of Skoog and co-workers during 1950's and 1960's. It was in 1957 Skoog & Miller demonstrated hormonal control of organ formation, in the tobacco plant tissue culture which is not applicable to most plant species.

Tissue culture techniques are becoming increasingly popular as alternative means of plant vegetative propagation. Since the pioneer plant tissue culture studies, one of the main objectives was to design a proper medium that supports sufficient growth of the explants in a totally artificial environment. The first medium formulations used for plant culture work were based on experience of microorganisms. One of the most successful media, is the MS medium, was devised by Murashige and Skoog (1962). SH medium (Schenk and Hildebrandt, 1972) was developed for callus culture of both monocotyledons and dicotyledons. Nitsch's medium (Nitsch and Nitsch, 1969) was developed for another culture. It contains lower salt concentrations than that of MS, but not as low as that of White's medium. Plant tissue and cell culture media are generally made up of some or all of the following components: macronutrients, micronutrients, vitamins, amino acids or other nitrogen supplements, sugar(s), other undefined organic supplements, solidifying agents or support systems, and growth regulators. Several media formulations are commonly used for the majority of all cell and tissue culture work. These media formulations include those described by White, Murashige and Skoog, Gamborg et. al., Schenk & Hildebrandt, Nitsch & Nitsch, Lloyd & McCown. Murashige and Skoog's MS medium, Schenk and Hildebrand's SH medium, and Gamborg's B-5 medium are all high in macronutrients, while the other media formulations contain considerably less of the macronutrients.

Phytochemical

Phytochemical can be defined, as chemicals produced by plants. However, the term is generally used to describe chemicals from plants that may affect health, but are not essential nutrients. Phytochemicals are plant or fruit derived chemical compounds. "Phytonutrients" refer to phytochemicals or compounds that come from edible plants. Phytochemicals exists as long as plants exist but we only know about hundred years about their existence. More than 5000 years ago, the Chinese based their medicine on the influence of yin and yang, and on the five elements. The earliest record about herbal medicine dates back to 2800 BC when the Chinese emperor Shen Nong wrote the text The Great Native Herbal. Hippocrates (460-377 BC) and Aristotle (384-322 BC) introduced the herbal medicine from India and Egypt to Europe. The Greek Physician Dioscorides wrote the book De Materia Medica in the first century AD. Phytochemicals have been used as drugs for millennia. For example, Hippocrates in 400 BC used to prescribe willow tree leaves to abate fever.

Tinospora cordifolia

T. cordifolia is a large, glabrous, succulent and climbing. It is distributed throughout tropical Indian subcontinent, Sri Lanka and China typically growing in deciduous and dry forests, ascending to an altitude of 1,200m. The leaves are heart shaped. The succulent bark is creamy white to grey in color, with deep clefts spotted with lenticels. It thrives easily in the tropical region, often attains a great height, and seems to be particularly fond of climbing up the trunks of large neem trees.

T. cordifolia is a large extensively spreading, perennial woody climber. The stem is rather succulent with long fleshy aerial roots from the branches. The bark is creamy white to grey, deeply left spirally, the space in between being spotted with large rosette like lenticels. The wood is white, soft and porous and the fleshy cut surface quickly assumes a yellow tint on exposure to air. The branches bear smooth heart-shaped leaves, unisexual greenish flowers (summer) and red berries (winter). The leaves are simple, alternate, long petiole and possess a characteristic heart shape, giving the name cordifolia to the plant. The drupes are ovoid, glossy, succulent, red and pea-sized. The seeds are curved. Fruits are fleshy and single seeded. Flowers grow during the summer and fruits during the winter.

Early Micropropagation study of Tinospora cordifolia:

The intent of this research update is to briefly examine "what is being done" and to explore "what can be done" with regard to the tissue culture of ornamental plants. Such a consideration necessarily includes an overview of tissue culture as a propagation tool. Micropropagation allows the production of large numbers of plants from small pieces of the stock plant in relatively short periods of time.

V. Raghu, S. P. Geetha, Gerald Martin, Indira Balachandran and P. N. Ravindran in year 2006 developed a protocol for rapid clonal propagation of Tinospora cordifolia, through in vitro culture of mature nodal explants. Shoots were initiated on both Murashige and Skoog (MS) medium and woody plant medium (WPM) supplemented with 2.32 μM kinetin (KIN). Among the cytokinins tested, N-benzyl adenine (BA) was more effective an average multiplication rate of 6.3 shoots per explant was obtained with WPM supplemented with 8.87 μM BA elongated shoots was rooted in half-strength MS medium supplemented with 2.85 μM indole-3-acetic acid (IAA). Rooted plantlets were successfully transferred to sand and established with 80% survival.

Many works have been done on Tinospora cordifolia by different method of plant tissue culture, with different conditions supplemented to it. Ranjit Avaghamshi, Mundeep jaishwal, P. K. Prajapathi, B. J. Patgiri Gujarat Ayurveda University, Jamnagar. Worked on Phytochemical activity of Tinospora cordifolia in this he classified different types of species of same plant by finding their Phytonutrients and their activity with the standard plant, the estimated the bitterness of different plants. Leaves of this plant are rich in protein (11.2%) and are fairly rich in calcium and phosphorous. Its similarities and differences from amylose were elucidated (Rao et al, 1981). An arabinogalactan has been isolated from the dried stems of T. cordifolia (Chintalwar, et al, 1999). The roots of T. cordifolia contain isocolumbin, palmatine, tetrahydropalmatine, magnoflorine and jatroeehizine (Sarma et al, 1998; Sarma et al, 1995). T. cordifolia is perfectly suited to and grows well in almost any type of soils and under varying climatic conditions especially on some support preferably neem and mango trees. Such plants are supposed to possess better medicinal values (Dina Nath Tewari et al; Gurach 2001). The plant is propagated by stem cutting.

In semi arid areas like North Karnataka the temperature is high and the availability of water is less the standard protocol from micropropagation cannot be used because of the increase in to phenol deposition in the plant. Normally for the survival plant increases the amount of phytochemicals to survive. For standardizing protocol for micropropagation was done to grow it in laboratory. Rapid propagation of Tinospora cordifolia was achieved through plant tissue culture technique. The nodal cuttings of Tinospora cordifolia of size 0.5-1cm were used as experimental plant material. Different conc. of BAP (8, 8.5, 8.7μM) was used in MS media for shoot regeneration from nodal explants.

Conclusion

Micropropagation is widely used technique for the regeneration of whole plant through tissue culture. This has become one of the most beneficial ways to produce species that are difficult to propagate by conventional methods.

For the nodal explant the highest degree of shoot regeneration was found on medium containing 8.5 μM of cytokinins (BAP) with 80% of explants proliferated with average shoot length of 4-5.5 cm within 20 to 25 days, when compared with that of 8-8.7 μM BAP which showed 60% & 50% of explant proliferation with average shoot length of 3-3.6cm within 25 to 30 days respectively. Thus the concentration of BAP (8.5 μM) are found to be responsive and fast growing.

Thus nodal cutting were inoculated on the surface of semi solid MS media supplemented with 8.5 μM BAP, 30gms of sucrose gelled with 7% of agar. The tiny shoot plantlets were regenerated within 25 days of inoculation and incubation.

Percentage of root induction & number of roots per shoot were highly influenced by concentration of auxins. Among different concentration of IAA (0.3,0.4,0.5 μM) the highest degree of root induction was found on the media containing 0.4 μM IAA with 70% of root proliferation and that of the average length of 3-3.5 cm within 20-26 days when compared with others like 0.3 and 0.5 the result was about 40-45% response and length of 2-2.7 cm..

The roots were slowly taken and the roots were cleaned then hardening process was done with composition of sand, red soli, black soil. This was kept in green house then exposed in to the natural environment.

The preliminary phytochemicals test showed positive for Steroids, Terpenoid, Saponin and Alkaloids. The amount of protein in Tinospora cordifolia was found to be 660μg/gm. The amount of phenols was found to be 1.2 mg/gm. The amount of flavonoids was estimated to be 15μg/gm. This proves the amount of Phytochemical produced is high and they are most effective than which are grown in belt of Himalayas.

REFERENCE:-
• A, Malini S, Bairy KL and Rao MS (2002); Effect of Tinospora cordifoliaon learning and memory in normal and memory deficit rats; Indian Journal of Pharmacology 34: 339-349
• Agarwal SS and Singh VK (1999); Immunomodulators: A review of studies on Indian Medicinal Plants and synthetic peptides, Part 1: Medicinal Plants; Proceedings of the Indian National Science Academy (PINSA) B65(3&4): 179-204
• ARTG (2003) Australian Register of Therapeutic Goods. Therapeutic Goods Administration, Commonwealth Department of Health and Aging, Canberra, Australia Atal CK, Sharma ML, Kaul A and Khajuria A (1986); Immunomodulating agents of plant origin I: Preliminary screening; Journal of Ethnopharmacology 18: 133-141
• Badar VA et al (2005); Efficacy of Tinospora cordifoliain allergic rhinitis; Journal of Ethnopharmacology 96: 445-449
• Bairy KL, Rao Y, Kumar B (2004); Efficacy of Tinospora cordifoliaon learning and memory in healthy volunteers: A double-blind, randomised, placebo controlled study; Iranian Journal of Pharmacology & Therapeutics 3: 57-60
• Bishayi B, Roychowdhury S, Ghosh S and Sengupta M (2002);Hepatoprotective and immunomodulatory properties of Tinospora cordifoliain CCl4 intoxicated mature albino rats; The Journal of Toxicological Sciences 27(3): 139-146
• Chintalwar G, Jain A, Sipahimalani A, Banerji A, Sumariwalla P, Ramakrishna R, Sainis K (1999); An immunologically active arabinogalactan from Tinospora cordifolia; Photochemistry 52: 1089-1093
• Dahanukar SA (1986); Study of Influence of Plant Products on Adaptive Processes, Excerpt from Ph.D. Thesis, Department of Pharmacology and Therapeutics, University of Mumbai, Mumbai, p90 & 133


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