Isolasi Protoplas secara Enzimatis pada Tanaman Kecipir

<p>The<br />kind and concentration of enzyme that appropriate affected<br />isolation process and result of plant protoplast. The research<br />was conducted to enhance the protoplast rendements of winged<br />bean (Psophocarpus tetragonolobus L.) that was isolated by<br />Cellulase RS and Macerozyme R-10 enzyme as single and its<br />combination in a solution. Concentrations of enzyme used<br />were as much as 2.0-3.0% w/v for Cellulase RS and 0.4-0.6%<br />w/v for Macerozyme R-10. The solution containt mannitol 25<br />mM as an osmotycum. Isolation process was done on shaker<br />with 50 rpm (rotation per minute) speed in dark room for 5<br />hours. Results showed that C3 treatment (concentration of<br />Cellulase RS enzyme as much as 3.0% w/v) resulted<br />protoplasts density 3.49 x 105 protoplasts/g fresh weight of<br />hypocotyl and M2 treatment (concentration of Macerozyme R-<br />10 enzyme as much as 0.5% w/v) resulted 3.19 x 105<br />protoplasts/g. Whereas the best combined enzymes treatment<br />was achieved by C3M2 (combination between Cellulase RS as<br />much as 3.0% and Macerozyme R-10 enzyme as much as 0.5%<br />w/v) which resulted protoplasts density 4.94 x 105<br />protoplasts/g fresh weight of hypocotyl. The protoplast was<br />intact and viable.</p>

Changes of DNA methylation and hydroxymethylation in plant protoplast cultures.

Cytosine methylation patterns in higher eukaryotes are important in gene regulation. Along with 5-methylcytosine (5-mC), a newly discovered constituent of mammalian DNA, 5-hydroxymethylcytosine (5-hmC), is the other modified base in higher organisms. In this study we detected 5-hmC in plant protoplast DNA and demonstrated its increasing content during the first 72 hrs. of protoplast cultivation. In contrast to 5-hmC, the amount of 5-mC decreased during protoplast cultivation. It was also found that 5-hmC did not primarily arise as a product of oxidative DNA damage following protoplast culture.