Protoplast isolation and genetically true-to-type plant regeneration from leaf- and callus-derived protoplasts of Albizia julibrissin

Protoplast isolation and subsequent plant regeneration of Albizia julibrissin was achieved from leaf and callus explants. Leaf tissue from 4 to 5-week-old in vitro seedlings was the best source for high-yield protoplast isolation. This approach produced 7.77 x 10(5) protoplasts (Pp) per gram fresh weight with 94 % viability; after 60 min pre-plasmolysis with 0.7 M sorbitol followed by digestion in a solution of cell and protoplast wash plus 0.7 M mannitol, 1.5 % cellulase Onozuka R10, and 1 % pectolyase Y-23 for 6 h. Liquid Kao and Michayluk medium containing 2.7 mu M alpha-naphthaleneacetic acid (NAA) and 2.2 mu M 6-benzylaminopurine (BA) was best for sustained cell division and microcolony formation from both leaf- and callus-derived protoplasts at a density of 3-5 x 10(5) Pp ml(-1). Protoplast-derived microcalli became visible after 3-4 weeks on semi-solid medium of the same composition. Microcalli were then cultured on Murashige and Skoog (MS) medium containing Gamborg B5 vitamins or woody plant medium supplemented with different concentrations of NAA plus 4.4 mu M BA for further growth. Proliferated leaf- and callus-protoplast-derived calli differentiated into microshoots on MS medium containing 13.2 mu M BA plus 4.6 mu M zeatin after 2-3 weeks, with an overall shoot organogenesis efficiency of 78-93 %. Rooting of microshoots on half-strength MS medium containing 4.9 A mu M indole-3-butyric acid was successful, and plantlets were acclimatized to the greenhouse with a survival rate of > 62 %. Using ten start codon targeted and ten inter-simple sequence repeat primers, the genetic integrity of nine leaf- and six callus-protoplast-based plants was validated along with the mother seedlings.