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Isolation and Culture of Pineapple (Ananas comnosus) Protoplast

By walaa on June 15,2008

 

 Nabawi A. A. Hagagy, Mohamed G. E. A. Mougheith, Malaka A. E. Saleh,Hassan M. Gendiah, Said K. Mohamed and Sayed A. M. Hassan

Abstract

       In vitro full expended healthy leaves and sterilized in vivo leaves of pineapple (Ananas cv. smoothcayenne) plants were taken and prepared under aseptic conditions as different sources of explants.Also, different enzymes mixtures, incubation periods, osmotic pressure factors, shaking periods andspeeds were concerned in combination with explants sources during protoplast isolation stage. Inaddition, sieve size and centerfugation speed were evaluated in combination with explants sourceduring purification stage. Moreover, medium type protoplast density, auxin/cytokinin concentrationratio, and antibiotic were tested in combination with explants source during protoplast culturing. It isfound that in vitro and sterilized in vivo explants source succeeded in maximizing protoplast yield.Also, using of enzymes mixture consists of 1.0% cellulase + 0.5% macerozyme was superior inincreasing protoplast yield Moreover, using of sucrose at rate of 13.6g /100ml as osmotic pressurefactor and incubation for 20 hours then, shaking for 15 min with speed rate 75 rpm succeeded inenhancing the highest protoplast isolation of pineapple. Meanwhile, using of 25 μM pore size meshsieve and centrifugation at the rate of 1000rpm maximized protoplast purification. Moreover, culturingof protoplast KAO and Michayluk medium supplemented with 3.0 mg/l NAA and 0.2 mg/l BAP aswell as the combination of antibiotic (0.4 mg/l Ampicilin + 0.1 g/l gentamycin + 0.1 g/l tetracycline)and using protoplast density at the rate of 2.5 x 104 induced the best protoplast viability anddevelopment of pineapple explants.

Keywords: Pineapple, tissue culture, cell division, protoplast isolation.



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