Virus elimination in potato through meristem culture followed by thermotherapy

SAARC J. Agri., 11(1): 71-80 (2013) VIRUS ELIMINATION IN POTATO THROUGH MERISTEM CULTURE FOLLOWED BY THERMOTHERAPY M. A. Ali, K. M. Nasiruddin, M. S. Haque and S. M. Faisal*1 Department of Biotechnology, Faculty of Agriculture, Bangladesh Agricultural University, Mymensingh-2202 ABSTRACT Virus elimination in potato through meristem culture followed by thermotherapy and virus indexing was studied. Three levels of thermotherapy, viz. 27±1oC (control), 30±1oC and 35±1OC, sixteen combinations of BAP (Benzyl Amino Purine) plus GA3 (gibberellic Acid) concentrations viz. 0.0+0.0 (control), 0.0+0.2, 0.0+0.4, 0.0+0.6, 1.5+0.0, 1.5+0.2, 1.5+0.4, 1.5+0.6, 3.0+0.0, 3.0+0.2, 3.0+0.4, 3.0+0.6 , 4.5+0.0, 4.5+ 0.2, 4.5+0.4 and 4.5+0.6 were used in this study in three potato varieties viz. Diamant, Heera and Lalpakri. Among the thermo therapies, 27±1OC showed the highest (24.55) survival response followed by 30±1OC, 35±1OC, respectively. The poorest (20.47) survival response of meristem derived plantlets was noticed in 35±1OC which gave the highest percentage (43.79) of virus free plantlets followed by 30±1OC. The best (25.85%) survival response was found in Lalpakri and the lowest (19.08%) survivality was recorded in Diamant. The highest (33.27) percentage of PVY (Potato Virus Y) free plantlets was observed in Heera. The combined treatment 3.0 mg L-1 BAP and 0.2 mg L-1 GA3 showed the highest (63.39) percentage of virus free plantlet production followed by 4.5 mg L-1 BAP and 0.2 mg L-1 GA3. Key words: Potato, meristem, ELISA (Enzyme Linked Immuno-Sorbent Assay), BAP, GA3. INTRODUCTION Potato (Solanum tuberosum L.) is the 4th most cultivated food crop after wheat, rice and maize and therefore, the most important dicotyledonous and tuber crop in the world (Jones et al., 1994). Potato is a good and cheap source of carbohydrates, vitamins, minerals and proteins and provides most of the trace * 1 Corresponding author email: Senior Scientific Officer, Horticulture Division, RARS, BARI, Hathazari, Chittagong, Bangladesh Received: 13.11.2012 72 M. A. Ali et al. elements which can meet the energy requirements of the people living in the developing countries. It is cultivated over an area of 345 thousand hectares with an annual production of 5167 thousand tones with an average yield of 14.83 t ha-1 (BBS, 2007). This yield is very low compared to other countries like the Netherlands (45 t ha-1), Germany (46 t ha-1), Scandinavian countries (48-52 t ha-1) and neighbouring India (21 t ha-1) (Beukema and Vander Zaag, 1990; Rashid et al., 1993). Lack of quality seed potato is one of the most important factors for this low yield. Most of the farmers in Bangladesh use degenerated seeds obtained from cold storage owners, farmers and traders. At present Bangladesh imports only a few hundred tones of high quality seed potatoes of some modern varieties, mostly from the Netherlands, for further multiplication by BADC (Bangladesh Agricultural Development Corporation) and private seed potato producers. On the other hand, the local varieties of potato which are tolerant to diseases and environmental stresses cover nearly 36% of the total potatoes cultivated in the country (Siddique and Hussain, 1988). In tropical and subtropical areas like Bangladesh, it is difficult to produce seed tubers of potato due to lack of appropriate storage facilities and transport, as well as the presence of active virus diseases vectors (Omidi et al., 2003). Plant tissue culture offers an efficient method for production and rapid propagation of pathogen-free material and germplasm preservation of plants to overcome this unwanted situation. Diseased free good quality seeds and pathogen free planting materials are produced through tissue culture. Thermotherapy is an important pre-requisite for meristem culture. Thermotherapy reduced the incidence of PLRV (Potato Leaf Roll Virus) and PVS (Potato Virus S) by 45% and 50%, respectively (El-Amin et at., 1994). More virusfree plants were obtained when heat treatments were included (Zapata et al., 1995). Seed health is the prime consideration for potato production, which can be retained by lowering the number of field generations. The conventional method of clonal selection is time consuming and requires intensive control over large areas of land with low rates of multiplication (Van der Zaag, 1987). Rapid multiplication with a reduction to three generations of seed multiplication from 7 in conventional clonal selection gave a 25% increased yield and better seed quality in Ecuador (Bryan, 1989). Based on the above facts, the present work was undertaken to fulfill the following objectives- 1) to see the effect of thermotherapy on virus elimination in meristem cultured seed potato and 2) to establish a suitable and reproducible in vitro regeneration protocol of potato varieties for quality seed potato production. MATERIALS AND METHODS The investigation was conducted at the USDA sponsored (United States Department of Agriculture) Biotechnology Laboatory at Bangladesh Agricultural University, Mymensingh during January 2005 to December 2008, as a part of Ph.D. research works. There were three factors in this experiment. Factor A consisted of VIRUS ELIMINATION IN POTATO 73 three levels of thermotherapy, viz. 27±1OC (control), 30±1OC and 35±1OC, Factor B consisted of three potato varieties viz. Diamant, Heera and Lalpakri and Factor C consisted of sixteen combinations of cytokinin (BAP) plus gibberellin (GA3) concentrations viz. 0.0+0.0 (control), 0.0+0.2, 0.0+0.4, 0.0+0.6, 1.5+0.0, 1.5+0.2, 1.5+0.4, 1.5+0.6, 3.0+0.0, 3.0+0.2, 3.0+0.4, 3.0+0.6 , 4.5+0.0, 4.5+ 0.2, 4.5+0.4 and 4.5+0.6. So, total number of treatments were 144 (3 × 3 × 16). Four explants were placed in a test tube and replicated 3 times. The experiment was done in Completely Randomized Design (CRD). In this study MS (Murashige and Skoog, 1962) medium was used as basal medium. Two sets, each containing plant materials of three potato varieties infected by PVY were selected. Each set consisted of 18 plants- affected by PVY, field symptoms and serological confirmation followed by ELISA test. After maturity the PVY infected potato tubers of three varieties were collected and stored carefully. PVY infected potato tubers of three varieties were kept under dark with 1 minute deeping in 1 ppm concentration of GA3 for sprouting. For meristem collection 0.2 0.5mm long size meritems were selected. After dissection the excised meristems were cultured in MS medium supplemented with the sixteen hormonal combinations. Calli derived from excised meristems were treated with three temperature regimes. All inoculations and manipulations were carried out aseptically unde laminar airflow cabinet. The physical conditions for growth and development of cultures were maintained at the temperature of 25 ± 10C and a light intensity of 2000-3000 lux provided by fluorescent tube. The photoperiod was maintained at 16 hours light and 8 hours dark (1 (...truncated)