Proceedings of International Workshop on Biotechnology in Agriculture
PRIMARY STUDY OF CELL SUSPENSION CULTURE OF YELLOW PASSION FRUIT (Passiflora edulis F. flavicarpa)
Bui Le Thanh Khiet, Nguyen Ngoc Thi, Phan Xuan Huyen and Duong Tan Nhut Dalat Institute of Biology, 116 Xo Viet Nghe Tinh, Dalat, Lam Dong, Vietnam ABSTRACT Passion fruit is a highly valuable species but it has some problems about pests and diseases. There are some reports about shoot regeneration and plantlet formation of vine crops from various explants and using different techniques, but these are not suitable. Cell suspension culture is a tool to study cell growth, to produce secondary metabolites and a tool for rapid micropropagation of some economically valuable crops. Using this technique, we can understand cell growth parameters and establish an efficient protocol. In this study, we used callus derived from in vitro plantlets of yellow passion fruit to initiate cell suspension culture. Cultures were maintained in MS medium supplemented with various NAA concentrations (0.0-2.0 mgl-1), 30 gl-1 sucrose. Cell growth was evaluated by counting cell numbers on 0, 5, 10, 15, 20 and 25 days of culture. After 20 days of culture, 2 ml of suspension culture was transferred to hormone-free MS medium containing 60 gl-1 sucrose and 8 gl-1 agar to induce callus formation. These calli were used to regenerate shoots. Key words: Callus, cell suspension culture, yellow passion fruit INTRODUCTION The genus Passiflora is regarded as constituted of about 400 known species principally distributed in South America (Aguiar-Menezes et al., 2002). Among these, yellow passion fruit, Passiflora edulis f. flavicarpa, is one of the most economically important fruits. Many reports on regeneration and plantlet formation have been described for the genus Passiflora including regeneration from hypocotyls, leaves and cotyledons (Faria and Segura, 1997; Dornelas and Vieira, 1994), regeneration from leaf disks (Monteiro et al., 2000) and mesophyll and cotyledon-derived protoplasts (Dornelas and Vieira, Liquid cultures were carried out in 250 ml Erlenmeyer flasks containing 100 ml of suspension. The flasks were agitated on an Orbital Shaker SO1 (Stuart Scientific, UK) at 120 rpm at a temperature of 25oC in 16 h photo periods under fluorescent light of 2500 lux. The inoculum’s cells (10 ml) was used to count number of cells under light microscopy (Olympus, Japan) on 0, 5, 10, 15, 20 and 25 days of culture. After 20 days of culture, 2 ml of suspension were transferred to solid hormone-free MS medium containing 60 g/l sucrose for callus formation. 1993). A mature endosperm culture has been reported for P. foetida (Mohamed et al., 1996). Few attempts have been made to initiate cell culture in the genus Passiflora (Dornelas and Vieira, 1993; Dornelas et al., 1995). The aim of this study deals with callus formation followed by cell suspension culture. MATERIALS AND METHODS Plant material All explants of Passiflora plants were obtained from an in vitro collection maintained at Dalat Institute of Biology, Dalat, Lam Dong, Vietnam. The explants were culture in vessels on solid medium. Culture media The solid media (agar, 8 g/l) were based on MS medium (Murashige and Skoog, 1962) with glucose at 30 g/l supplemented with NAA at different concentrations (0.0, 0.5, 1.0, 1.5 and 2.0 mg/l). The liquid medium was the same of solid medium without agar. Culture conditions
Figure 1. Callus formation (a, b) and cell suspension culture taken under light microscopy (c)
Nong Lam University Ho Chi Minh City, October 20-21, 2006
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Proceedings of International Workshop on Biotechnology in Agriculture
13000 12000 Cells density (number cells/ml) 11000 10000 9000 8000 7000 6000 5000 4000 3000 2000 1000 0 0 5 10 15 20 25 Days of culture 0.0 mg/l 0.5 mg/l 1.0 mg/l 1.5 mg/l 2.0 mg/l
Figure 2. Growth curves of Passiflora edulis F. flavicarba suspension culture RESULTS AND DISCUSSION Callus induction. After 1 month on solid medium, callus was produced (Fig. 1a) on media with 0.5 and 2.0 mg/l NAA, but there was different: the former produced callus with root, but the latter just produced callus and disappeared shoot. Other media did not produce callus. The formation of callus may be due to the ratio of cytokinin to auxin as mentioned by Skoog and Miller (1957), Gaspar et al. (2003). This primary callus was friable, globular and yellowish-white (Fig. 1b). These calli subsequently gave rise to different lines when continuously transferred to solid media. Aspects of the cell suspension culture. Under light microscopy, cell aggregates of various sizes and types were present (Fig. 1c). Also, there was brown cell content exhibited by many cells. By counting cell numbers, the growth dynamics was evaluated, this corresponds to a sigmoid curve showing a lag phase, followed by exponential, linear and stationary phases. The curve growth of all treatment was shown in Figure 2. Best result was obtained from MS medium supplemented with 2.0 mg/l NAA. Formation of callus from suspension culture. After 2 months transferred to hormone-free medium, calli appeared as white, slimy type. These calli were used to test regeneration capacity. REFERENCES AGUIAR-MENEZES E.L., MENEZES E.B., CASSINO P.C.R. and SOARCES M.A., 2002. Passion fruit. In: Peòa, J.E., Sharp, J.L., Wysoki, M. (eds.) Tropical fruit pests and pollinators. CAB International, 2002, pp. 361390. DORNELAS M.C. and VIEIRA M.L.C., 1993. Plant regeneration from protoplast cul-cures of Passiflora edulis var. flavicarpa Deg., P. amethystina Mikan, and P. cincinnata Mast. Plant Cell Rep. 13: 103-106. DORNELAS M.C. and VIEIRA M.L.C., 1994. Tissue culture studies on species of Passiflora. Plant Cell Tiss. Org. Cult. 36: 211-217. DORNELAS M.C., TAVARES E.C.A., DE OLIVIERA J.C. and VIEIRA M.C.L., 1995. Plant regeneration from protoplast fusion in Passiflora spp.Plant Cell Reports. 15: 106-110. FARIA J.L.C. and SEGURA J., 1997. Micropropagation of yellow passion fruit by auxillary bud proliferation. HortScience. 32: 1276-1277. MOHAMED M.E., HICKS R.G.T. and BLAKESLEY D., 1996. Shoot regeneration from mature endosperm of Passiflora foetida. Plant Cell Tiss. Org. Cult. 46: 161-164. MONTEIRO A.C.B.A., NAKAZAWA G.T., MENDES B.M.J. and RODRIGUEZ A.P.M., 2000. In vitro regeneration of Passiflora suberosa from leaf discs. Sci. Agri. 57: 571- 573. MURASHIGE T. and SKOOG F., 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant. 15: 473-497. SKOOG F. and MILLER C.O., 1957. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp. Soc. Exp. Biol. 11: 118-131. GASPAR T.H., KEVERS C., FAIVRE-RAMPANT O., CRE’VECOEUR M., PENEL C.L., GREPPIN H. and DOMMES J., 2003. Changing concepts in plant hormone action. In Vitro Cell. Dev. Biol.-Plant. 39: 85-106.
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