Isolation of cells and protoplasts from leaves of in vitro propagated peach (Prunus persica) plants
✍ Scribed by David Mills; Freddi A. Hammerschlag
- Publisher
- Springer
- Year
- 1994
- Tongue
- English
- Weight
- 841 KB
- Volume
- 36
- Category
- Article
- ISSN
- 0167-6857
No coin nor oath required. For personal study only.
✦ Synopsis
Yields of 106-108 peach mesophyll cells and protoplasts-gfw -1 were obtained depending on factors such as digesting enzymes, and leaf size. Onozuka R-10 (2%) in combination with Macerase (0.5%) was found best for protoplast isolation and mediocre for cell isolation among several enzyme combinations tested. Viability was 90% for protoplasts and 60% for cells. Pectolyase Y23 was found to be ineffective in our investigation. Small leaves, 4-10 mm in length, were a superior source for protoplast isolation than medium or big expanded leaves, 22-30 mm in length. The high yields of protoplasts could be obtained only when keeping the ratio of leaf biomass to volume of digesting enzyme solution under 20 mg ml -I. Purification of protoplasts on a sucrose gradient yielded about 10' protoplasts, gfw -1, however, the preparation was still contaminated by intact cells. Protoplasts were cultured under different growth regulators and physical conditions. Limited growth and division of protoplasts embedded in agarose drops were observed.