| In this paper, to study the effect of subculture times on endogenous hormone and DNAvariation in Kentucky bluegrass tissue subculture and as well as the relationship between callusdifferentiation capacity and endogenous hormone, the rhizome buds of Kentucky bluegrass(Poa pratensis L.) 'nassu' were cultivated on N6 medium containing different levels of 2,4-D,6-BA and NAA. And the regeneration system from rhizome buds of Kentucky bluegrass wasdeveloped. Additionally, based on the above mentioned research, we also studied the changesof endogenous hormones via ELISA and genome DNA variation via AFLP of Kentuckybluegrass callus after different times of subculture. The main results are as follows:Based on N6 medium, the optimum concentration of plant growth regulator combinationsfor callus induction is 2, 4-D2.0mg·L-1 and 6-BA0.2mg·L-1; the optimum concentration forcallus subculture is 2,4-D1.0mg·L-1 +6-BA0.5mg·L-1; and the optimum concentration forcallus differentiation 6-BA1.0mg·L-1+KT0.5mg·L-1; When the adventitious buds grew to1-2cm, we cut it and then inoculated to MS medium without any plant growth regulator, afterabout 30 days it developed into whole plants and the survival rate is 100%. The callusdifferentiation frequency was influenced by subculture time, adventitious buds began to growafter subcultured 2 times, and differentiation frequency is increased gradually as subculturetime increasing, the highest callus differentiation frequency is 40% when subculture 5 to 6times, then it is decreased gradually as subculture time increasing.When subcultured 1 to 4 times, the contents of IAA and ABA in Kentucky bluegrasscallus increased gradually as subculture time increasing, when subcultured 5 times it reach thepeak, and then decreased gradually, accordingly, the differentiation rate of callus changed withthe same trend. It showed that the raising and high levels IAA and ABA can promote the callusdifferentiation capacity. GA content is high as subcultures 1 time, then its content decreasedgradually, and it is low when subcultured 4 to 6 times, then its content increased gradually assubculture time increased, which indicate that endogenous hormone GA can inhibit thedifferentiation of callus to some extent. The content of endogenous hormone ZR increasedaccordingly as the increasing subculture time increased, and it had no apparent relevancy withthe callus differentiation capacity. The callus subcultured 3 times lost a lot of bands in AFLP electrophoresis, comparing withthe callus subcultured 1 time, it lost 550 bands and add 73 bands. In the callus of subculture 5,7,9,11 times, the percentage of amplified polymorphic fragments was 40.57%,49.73%,50.88%,53.74% respectively, their polymorphic fragments were 501,500,521,699respectively when comparing with the callus subcultured 1 time, It indicated that DNAvariation in callus increased gradually as subculture time increased, and DNA variationfrequency is high; By comparing the DNA variation of callus among different subculture time,we discovered that polymorphic fragments between subculture 3 times and subculture 1 time,subculture 5 times and subculture 3 times, between subculture 7 times and subculture 5 timeswas, subculture 9 times and subculture 7 times, subculture 11 times and subculture 9 timeswere 623,617,597,523 and 494 respectively. The research showed that the variation of callusbecome smooth with the increasing of subculture time. Which can presume that the subculturetimes can be controlled 5 to 9 times in order to decrease the variation if conserve the resourceof seed or make transformation; The subculture times can be controlled 3 to 5 times or morethan 9 times in order to increase the variation if gain more variation. Considering the balancebetween variation rates and differentiation rates of callus if induce breeding, the subculturetimes can be controlled 5 to 9 times. AFLP for its characters such as abundant, clearpolymorphism bands, is an effective technique for the analysis of somaclonal variation ofkentucky bluegrass callus.
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