(a) What is the role played by media and growth regulators in micropropagation? How to control exudation and contamination…

(a) Role of media and growth regulators in micropropagation, and control of exudation and contamination (20 marks)

Role of media and growth regulators (10 marks).

Medium. Cultured tissue is heterotrophic and depends on the medium, most commonly Murashige and Skoog (MS) medium, which supplies:

• macronutrients (nitrogen as nitrate and ammonium, phosphorus, potassium, calcium, magnesium, sulphur) and micronutrients (iron as Fe-EDTA, manganese, zinc, boron, copper, molybdenum);
• vitamins (thiamine, pyridoxine, nicotinic acid, myo-inositol);
• a carbon and energy source, sucrose (2 to 3%), which also gives osmotic support;
• a gelling agent, agar (0.6 to 0.8%), a pH of 5.6 to 5.8, and sometimes coconut water, casein hydrolysate or activated charcoal.

Growth regulators. The type and ratio decide the response at each stage of micropropagation:

• Multiplication: a cytokinin (BAP, kinetin, TDZ) at about 1 to 3 mg/l with a little auxin breaks apical dominance and promotes axillary shoots.
• Rooting: an auxin (IBA or NAA, 0.5 to 2 mg/l), often in half-strength medium with low sugar.
• Gibberellin (GA3) elongates shoots; auxin (2,4-D) at high levels gives callus, and a high auxin to cytokinin ratio favours roots and a low one favours shoots.

Control of exudation (5 marks). Cut tissue releases phenolics, which polyphenol oxidase oxidises to toxic quinones that brown the medium and stop growth. It is controlled by:

• using young, actively growing explants from healthy plants, taken in the correct season;
• pre-soaking or adding antioxidants (ascorbic acid, citric acid, cysteine) and adsorbents such as polyvinylpyrrolidone or activated charcoal (0.1 to 0.5%);
• transferring to fresh medium at intervals of one to three days at first, incubating in the dark or in dim light initially, and using a liquid or lower-salt medium.

Control of contamination (5 marks). Bacteria, fungi and yeasts come from the explant, the medium, the instruments, the air or the worker. They are controlled by:

• sterilising the medium in an autoclave (121 °C, 15 psi, 15 to 20 minutes) and heat-labile substances by filtration (0.22 µm);
• surface-sterilising the explant (detergent wash, 70% ethanol, then mercuric chloride 0.1% or sodium hypochlorite, and rinsing in sterile water), and treating the mother plant with fungicide;
• working in a laminar-flow cabinet with sterilised tools, clean gloves and clothing, and a clean culture room;
• using antibiotics or a plant preservative mixture when needed, sealing the cultures, and discarding contaminated ones.

(b) Techniques used in experimental embryology (15 marks)

Experimental embryology studies the development of the embryo and its reproductive processes by growing the parts in vitro under controlled conditions. Its main techniques are:

• Embryo culture: mature or immature embryos are dissected out and grown on defined medium, to study nutrition and development and, in embryo rescue, to save embryos of wide crosses that would abort.
• Ovule and ovary culture: fertilised or unfertilised ovules and ovaries are cultured for seed development, and for haploid production.
• In vitro pollination and fertilisation: pollen is applied to cultured ovules or ovaries, as in test-tube fertilisation in Papaver by Kanta, Rangaswamy and Maheshwari (1962); isolated gametes may be fused, as in maize.
• Pollen culture: pollen is germinated in hanging drops or on agar, in sucrose with boric acid and calcium, to study the growth of the pollen tube and the effect of pistil factors.
• Endosperm culture: gives triploid plants and shows how endosperm supports the embryo.
• Anther, ovary and microspore culture, and protoplast fusion, for haploids and hybrids.
• Staining and microscopy: aniline blue with fluorescence to follow pollen tubes, whole-mount clearing of ovules, histochemistry, autoradiography and confocal microscopy.

The Delhi school of embryology led by Panchanan Maheshwari was a pioneer of these methods in India.

(c) History and progress of ethnobotanical research in India (15 marks)

History (8 marks). Ethnobotany is the study of the relationship between people and plants. The term was proposed by John Harshberger in 1895. In India, knowledge of plants for food, fibre and medicine is old, recorded in the Vedas, the Charaka and Sushruta Samhitas, and in Ayurveda and Sangam literature. Colonial-period works include Hortus Malabaricus (Van Rheede, 1678 to 1693), Roxburgh's Flora Indica, and Watt's Dictionary of the Economic Products of India (1889 to 1896). E. K. Janaki Ammal began ethnobotanical surveys at the Botanical Survey of India in the 1950s, and S. K. Jain built the field in the 1960s and later, so that he is often called the father of Indian ethnobotany.

Progress (7 marks):

• The All India Coordinated Research Project on Ethnobiology (from 1982) surveyed tribes across the country.
• Research at the Botanical Survey of India, the National Botanical Research Institute, CSIR, ICMR and universities, and journals such as Ethnobotany and the Indian Journal of Traditional Knowledge, has documented uses of plants by tribes, such as the Gond, Santhal, Bhil and Toda.
• Leads from folk use gave drugs, such as reserpine from Rauvolfia serpentina, and Jeevani from Trichopus zeylanicus (arogyapacha), developed with the Kani tribe, which became a widely cited early benefit-sharing arrangement.
• Protection of traditional knowledge grew through the Traditional Knowledge Digital Library (2001) and the Biological Diversity Act, 2002 and People's Biodiversity Registers; and the National Medicinal Plants Board and Ministry of AYUSH promote conservation and use.