Isolation and Culture of Pineapple (Ananas comnosus) Protoplast

Hagagy, Nabawi; Mougheith, Mohamed; Saleh, Malaka; Gendiah, Hassan; Mohamed, Said; Hassan, Sayed

Isolation and Culture of Pineapple (Ananas comnosus) Protoplast

Document Type : Original Article

Authors

¹ Faculty of Agriculture, Moshtohor, Benha University, Benha, Egypt

² National Research Center, Dokki, Giza, Egypt

Abstract

In vitro full expended healthy leaves and sterilized in vivo leaves of pineapple (Ananas cv. smooth
cayenne) plants were taken and prepared under aseptic conditions as different sources of explants.
Also, different enzymes mixtures, incubation periods, osmotic pressure factors, shaking periods and
speeds were concerned in combination with explants sources during protoplast isolation stage. In
addition, sieve size and centerfugation speed were evaluated in combination with explants source
during purification stage. Moreover, medium type protoplast density, auxin/cytokinin concentration
ratio, and antibiotic were tested in combination with explants source during protoplast culturing. It is
found 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 in
increasing protoplast yield Moreover, using of sucrose at rate of 13.6g /100ml as osmotic pressure
factor and incubation for 20 hours then, shaking for 15 min with speed rate 75 rpm succeeded in
enhancing the highest protoplast isolation of pineapple. Meanwhile, using of 25 μM pore size mesh
sieve and centrifugation at the rate of 1000rpm maximized protoplast purification. Moreover, culturing
of protoplast KAO and Michayluk medium supplemented with 3.0 mg/l NAA and 0.2 mg/l BAP as
well 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 and
development of pineapple explants.

Keywords