Tissue Culture Of Fortunella Margarita (Lour.) Swingle And Its Morphogeny And Physiological-biochemical Changes

Epicotyls of Fortunella margarita (Lour.) Swingle were cultured in vitro in this study. To set up the in vitro regeneration system, the conditions for callus induction and organogeny were studied. The morphologic change during culture was observed through paraffin slice; the content of soluble protein was mensurated through ultraviolet spectrophotography, and changes of isozymes were analyzed by polyacrylamide gel electrophoresis. The results indicated as follows: Appropriate combination of 6-BA and NAA can be effective in callus induction and redifferentiation. The optimal concentration for callus induction is 6-BA 1.5mg/L+NAA 1.0mg/L, with the increment of 20.3 times on 24^th day; as for callus differentiation is 6-BA 1.5mg/L+NAA 0.5mg/L with the differentiation rate of 75%. The in vitro regeneration of Fortunella margarita (Lour.) Swingle epicotyl is organogeny type, most buds are formed inside the callus, some are originated from surface. During root induction, relatively high concentration of NAA (1mg/L) was easy to produce callus, and relatively low concentration of salt is good for rooting, 1/2MS + NAA 0.5mg/L is preferable, with the rate of 95%. Among the peroxidase (POD), POD₁ has relation to the startup of induction and proliferation; POD₂, POD₅ and POD₆ appeared on the 12^th day of induction, they are signs of the fast increase of callus; POD₄ disappeared since the beginning of induction, but reappeared on the 24^th day of differentiation, it has relation to buds forming. Among the superoxide dismutase (SOD), SOD₄ disappeared on the 12^th day of callus induction, it is also a sign of the fast increase of callus; SOD₂ and SOD₇ appeared on the 24^th day of differentiation, they are the signs of bud forming. Catalase (CAT) showed only one band, it's stable expression is important for maintaining the stabilization of cell metabolism circumstance. Malic dehydrogenase (MDH) got intense during callus induction, but showed a fluctuation when buds emerged; MDH₄ appeared since the 2^nd day of callus induction, but disappeared on the 24^th day of callus differentiation, it maybe needed in special physiological process of in vitro culture. During callus induction the concentration of soluble protein presented 'W shape change, there was a little pick on 4^th day, and went up after that, this is consistent to the startup of induction and increase of callus; during callus differentiation, the concentration of soluble protein presented 'U' shape change, from 1^st to 18^th day it fell, but went up later, this is related to the consumption of protein during early stage of differentiation and fast synthesis in bud forming.