Isolation and Culture of Mesophyll Protoplasts of Phalaenopsis

1990 Fiscal Year Final Research Report Summary

• Project/Area Number: 63560029
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: 園芸・造園学
• Research Institution: Kagawa University
• Principal Investigator: TANAKA Michio Kagawa Univ., Fac. of Agriculture, Associate Professor, 農学部, 助教授 (10115975)
• Project Period (FY): 1988 – 1989
• Keywords: Phalaenopsis / Protoplast isolation / Protoplast culture

Research Abstract

To develop a procedure for isolation and culture of mesophyll protoplasts from the leaves of shoots derived from flower-stalk cutting culture of Phalaenopsis, Phalaenopsis George Moler which shows higher regenerative capability in the in vitro culture of leaf segments was used as a material. About one gram (F. W.) of the youngest leaves (3-4 cm long) was scored with parallel 1 mm cuts using blade and placed the in pretreatment solution (400 ppm PVP in hormone-free B_5 medium (1968) containing 0.4M sucrose) for 30 min. These leaf strips were then treated with 10 ml of a filtered-sterilized enzyme solution (0.1% Pectolyase Y-23, 2% Cellulase YC, and 400 ppm PVP, in hormone-free B_5 medium containing 0.4M sucrose, pH 5.5). After 3 hours of incubation in a 100ml Erlemeyer flask at 25ﾟC in the dark without shaking, protoplasts were filtered through 80 um nylon mesh to remove undsted cells and debris then centrifugated at 90 x g for 5 min. The protoplasts floated were collected and washed 2 times by centrifugation at 90 x g for 10 min with washing medium (the same as the pretreatment solution). Yields of mesophyll protoplasts were appoximately 1.1-1.2 x 10^6 /g F. W. after washing. In approximately 0.01% of protoplasts cultured a cell density of 2.5x 10^4 /ml in 30 x 15 mm plastic petridishes containing 2 ml of Gellan Gum (0.18%)-solidified B_5 medium (pH 5.5) supplemented with 0.1mg/1NAA, 5mg/1BA, 5mg/1 adenine, 10mg/1 L-ornithine HCl, 1g/1 L-glutamine, 400ppm PVP, 0.5M D-mannitol and 2% sucrose at 25ﾟC in the dark, an unequal cell division occurred. The smaller daughter cell continued to divide to give unequal daughter cells. Attempts to isolate protoplasts from PLB (somaticembryo) derived from the leaf segment culture have also made. An unequal cell division and colony formation were observed, when culturing these PLB protoplasts in above described culture medium (2mg/1 NAA, 1mg/1 BA). However, this colony did not develop into plantlet.