International Journal of Applied Agricultural Research ISSN 0973-2683 Volume 4 Number 3 (2009) pp. 261 266 Research India Publications http://www.ripublication.com/ijaar.htm In Vitro Culture of Cassava (Manihot esculenta Crantz): Assessment of Cassava Starch from Different Varieties as Gelling Agent in Culture Medium *C. K Nkere and E.N.A. Mbanaso National Root Crops Research Institute, Umudike, PMB 7006, Umuahia, Abia State, Nigeria *Corresponding author: nkerechukwuemeka@yahoo.com Abstract Shoot tips and nodal segments excised from in vitro Manihot esculenta cultivar. were seeded singly into culture tubes containing 15ml of cassava multiplication medium gelled differently in 0.2% gelrite (Control I), 0.7% agar (control II) and in 7% starch from three different cassava varieties viz: TMS 97/0162, 92/0323, and TME 419 Cultures were maintained at 28 o C ± 2 illumination and 16h photoperiod (30-40µmole m -2 S -1 ) supplied by white fluorescent tubes on shelves for four weeks. Percentage survival ranged from 90-100% for shoot tips while nodal segments ranged from 42.86-100%. Plantlets from gelrite gelled medium performed best in terms the height, number of leaves and nodes, it was not significantly different (p<0.05) from those of TME 419 starch- gelled medium especially with the shoot tip explants. The result highlights the potentials of cassava starch from the genotype TME 419, as an alternative to agar or gelrite as a gelling agent for in vitro multiplication of cassava. Key words: Cassava starch, Manihot esculenta, in vitro multiplivation, shoot tips, nodal segment Introduction Cassava (Manihot esculenta Crantz ) is widely grown in tropical and sub-tropical regions of the world primarily for its starch-rich roots and in some cases its nutritional foliage. It originated in South America (Allen 2002) and was domesticated less than 10,000 years ago. Today, it is the fourth most important basic food after rice, wheat
262 C. K Nkere and E.N.A. Mbanaso and maize and is a fundamental component of dietary energy providers for millions of people. Cassava has indeed risen from obscurity in the past to play a central role in the present in food security, culture and economic prosperity of many African, South American and Asian countries. The primary advantage of micropropagation is the rapid production of high quality, disease-free and uniform planting material. The plants can be multiplied under a controlled environment, anywhere, irrespective of the season and weather, on a year-round basis. Production of high quality and healthy planting material of ornamentals, and forest and fruit trees, propagated from vegetative parts, has created new opportunities in global trading for producers, farmers, and nursery owners, and for rural employment. Micropropagation technology is more expensive than the conventional methods of plant propagation, and requires several types of skills. It is a capital-intensive industry, and in some cases the unit cost per plant becomes unaffordable. The major reasons are cost of production and know-how. During the early years of the technology, there were difficulties in selling tissue culture products because the conventional planting material was much cheaper. Now this problem has been addressed by inventing reliable and cost effective tissue culture methods without compromising on quality. This requires a constant monitoring of the input costs of chemicals, media, energy, labour and capital. For example, the cost of medium preparation (chemicals, energy and labour) can account for 30 35% of the micropropagated plant production. Media chemicals cost less than 15% of micro-plant production (Prakash, 1993). In some cases the cost may be as low as 5%. Of the medium components, the gelling agents such as agar contribute 70% of the costs. Other ingredients in the media - salts, sugar and growth regulators - have minimal influence on production cost and are reasonably cheap. Therefore, low cost alternatives are needed to reduce production cost of tissuecultured plants. Thus, this informed the use of starch as a gelling substitute for the conventional gelling agents. Materials and Methods Source of explants Explants (shoot tips and nodal segments) were from in vitro Manihot esculenta cultivar (OY 001 Egedudu) platelets, obtained from in vitro gene bank housed in the Biotechnology unit (Plant tissue culture lab) of National Root Crops Research Institute (NRCRI) Umudike, Abia State Nigeria. Starch preparation The cassava varieties used (TMS 97/0162, 92/0323 and TME 419) were obtained from NRCRI, Umudike. Freshly harvested roots of cassava varieties were peeled, washed and then crushed separately. The pulp was suspended in excess quantity of water, sieved and the effluent collected in large labeled bowls. The effluent was left overnight for the starch to sediment and the supernatant decanted. The surface of the starch was rinsed with clean water and the starch scooped into trays for sun drying at
In Vitro Culture of Cassava (Manihot esculenta Crantz) 263 ambient temperature to remove excess moisture. Further drying to constant weight was in moisture extraction oven. The dried starch was milled into powder using a household milling machine, packaged in sealed polyethylene bags and stored under room temperature until required (Mbanaso, 2008). Culture medium The basic culture medium (Murashige and Skoog, 1962) with 3% sucrose was used. It was solidified with gelrite, agar and the starches at 0.2, 0.7 and 7% respectively. The ph was adjusted to 5.8. For gelrite and agar, the media were dissolved by heating while for the starches; the dried cassava starch powder was first made into thick slurry with a part of the medium to be gelled. The remaining part was heated to a temperature of 78 ± 1 o C and the corresponding cold slurry stirred vigorously into it. A 15ml aliquot each of the different medium were then dispensed into culture tubes and autoclaved for 15min at 121 o C, 1.05kg/cm 2. Explants culture The shoot tip and nodal segment explants from vigorously growing in vitro shoot culture of cassava (OY 001) were excised and leaves remove under aseptic conditions. Explants were seeded singly into culture tubes containing the prepared medium. Cultures were maintained at 28 o C ± 2, 16h photoperiod supplied by white fluorescent tubes on shelves for four weeks. The plant height, number of leaves and nodes were assessed after two and four weeks. Statistical analysis Tests of significance were calculated using analysis of variance (ANOVA) and multiple comparison least significant difference (LSD) of the GenStat (DE3) ver. 7.2. Results and Discussion The growth and proliferation of explants in the differently gelled medium is shown in Photo.1. The percentage survival ranged from 90-100% for the shoot tips while the nodal segments ranged from 42.86-100% with gelrite recording the best result (Fig.1). The height, number of leaves and nodes of the cassava plantlets from the differently gelled- medium is shown in Tables 1, 2 and 3 respectively. Though plantlets from gelrite gelled medium generally gave the best result, it was not significantly different (p<0.05) from those of TME 419 starch- gelled medium especially with the shoot tip explants. This high survival rate of plantlets (both shoot tip and nodal segment explants) from TME 419 starch gelled medium over that of agar gelled medium is significant because survival is a major key factor in the choice of a gelling agent. Several agar alternatives (wheat flour corn starch, laundry starch, potato powder, rice powder and semolina) have been shown to be good substitutes for the micrpropagation of various plants (Prakash, J. 1993.). It has been shown that addition of 8.0% tapioca starch to
264 C. K Nkere and E.N.A. Mbanaso the MS medium severed as a good substitute for Bacto-agar for potato shoot-culture (Getrudis and Wattimena, 1994). The relatively low performance of explants (Shoot tip and nodal segment) in TMS 92/0323 and 97/0162 starch gelled medium is not unusual as it has been reported that some gelling agents contain inhibitory substances that hinder morphogenesis and reduce the growth rate of cultures (Powell and Uhrig, 1987). However, an earlier study has shown that TMS 92/0323 starch gelled medium gave better result after agar in shoot proliferation of cocoyam with a mean multiplication rate of 7.4 after the second passage (Mbanaso, et al., 2008).This once again brings to the fore that the efficiency of a particular starch as a gelling agent in shoot proliferation of a particular plant, dose not transcend to all the plants. Therefore, the adoption of a starch as a gelling agent would depend on proper screening. Figure 1: Percentage survival of explants in differently gelled medium after 4 weeks in culture. Table 1: Plant height after 2 and 4 weeks in culture. Gelling agent 2weeks in culture 4weeks in culture Shoot tip Nodal segment Shoot tip Nodal segment Gelrite 2.22 1.82 3.75 2.66 Agar 1.93 1.14 3.45 2.22 TMS 97/0162 1.14 0.36 2.48 0.79 TME 419 1.59 0.94 3.19 1.68 TMS 92/0323 1.13 0.26 1.86 0.81 LSD (0.05) 0.38 0.57 0.95 1.04
In Vitro Culture of Cassava (Manihot esculenta Crantz) 265 Table 2: Number of leaves after 2 and 4 weeks in culture. Gelling agent 2weeks in culture 4weeks in culture Shoot tip Nodal segment Shoot tip Nodal segment Gelrite 1.7 1.93 3.5 4.21 Agar 1.6 1.29 3.4 3.29 TMS 97/0162 1.0 0.29 2.4 1.14 TME 419 1.6 1.0 3.6 1.36 TMS 92/0323 1.5 0.29 2.3 1.14 LSD (0.05) 0.59 0.63 0.85 1.03 Table 3: Number of nodes after 2 and 4 weeks in culture. Gelling agent 2weeks in culture 4weeks in culture Shoot tip Nodal segment Shoot tip Nodal segment Gelrite 2.6 2.21 4.5 4.43 Agar 2.2 1.64 3.9 3.5 TMS 1.6 0.57 2.8 1.21 97/0162 TME 419 2.5 1.29 3.9 2.5 TMS 2.0 0.57 2.7 1.5 92/0323 LSD (0.05) 0.48 0.47 0.85 0.75 Photo 1: Cassava proliferation in the differently gelled medium.
266 C. K Nkere and E.N.A. Mbanaso Conclusion The above study has shown that cassava starch from the genotype TME 419, could serve as an alternative to agar or gelrite as a gelling agent for in vitro multiplication of cassava. Further studies are needed in the characterization of the starches and the effect of the different components on shoot multiplication. References [1] Allen, A.C. (2002). The origins and taxonomy of cassava. In: Hillocks, R.J., Tres, J.M. and Bellotti, A.C. (Eds). Cassava: biology, production and utilization. CABI Publishing, pp 1-16. [2] Getrudis, D.J.M. and Wattimena, G.A. (1994). The effect of agar substitution on micro shoot production of two potato cultivars (Solanum tuberosum L.). Acta Hort. 369: 447-450. [3] Powell, W. and Uhrig, H. (1987). Anther culture of Solanum genotypes. Plant Cell Tissue Organ Culture. 11: 13-24. [4] Mbamaso, E. N. A.(2008). Effect of multiple subcultures on Musa shoots derived from cassava starch-gelled multiplication medium during micropropagation. African Journal of Biotechnology 7(24): 4491-4494. [5] Murashige, T. and Skoog, F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473-497. [6] Prakash, S. (1993). Production of ginger and turmeric through tissue culture methods and investigations into making tissue culture propagation less expensive. Ph.D. Thesis. Bangalore Univ. Bangalore