The Determination And Analysis For Variation Of Content Of Endogenous Hormones In The Tissue Culture Process For Poa Pretensis L.

Poa pratensis L., known as Kentucky bluegrass, is one of the most importantpasture plants and used widely for lawn-making across China. It is an herbaceousperennial, cool-season grass that has smooth, green to dark green leaves. The planttissue culture technology is an efficient tool for breeding improvement of Kentuckybluegrass; however, several factors play different roles in the process of tissue culture.In this study, the mature seeds of three Kentucky bluegrass cultivars, Midnight Ⅱ,Nuglade and Rugby Ⅱ, were used as the explants for callus induction, differentiation andcallus protoplasts separation. By means of seed pretreatment, the treatment of seedsoaking under the low temperature promoted the induction level of callus. In addition,the content of endogenous phytohormones (IAA, ABA and ZT) in the initial material andtheir callus were estimated by high performance liquid chromatography, the detailedinformation and result of the study is following:1. The mature seeds of three Kentucky bluegrass cultivars, Midnight Ⅱ, Nuglade and RugbyⅡ, were used as the explant materials for callus induction with various pretreatments whichincluded different temperature and time periods of seed soaking. In addition, the callus ofdifferent sub cultures was studied for plant regeneration. The result illustrated that:1) With the same time interval of seed soaking, a4℃temperature regime had the highestcallus induction rate for all three Kentucky bluegrass cultivars, this promotion was presentedas the ahead of timing of recovering and higher level of recovering of callus.2) With the same temperature condition, the rate of recovering for mature seeds of all threeKentucky bluegrass cultivars with8hours of seed soaking was higher than the rate ofrecovering with16or24hours of seed soaking. In conclusion, the time periods ranging from16to24hours were considered to be more appropriate for seed soaking.3) With the elongation of time of sub culture, the ability of differentiation presented adeclining trend. The rate of callus differentiation was highest for once only sub inoculationof midnight Ⅱ and rugby Ⅱ, the callus differentiation rate of Nuglade after a double subinoculation was the highest.2. The mature seeds of three Kentucky bluegrass cultivars, Midnight Ⅱ, Nuglade and Rugby Ⅱ, were used as the explant materials for callus induction and differentiation. Thecontent of endogenous phytohormones (IAA, ABA and ZT) in the initial material and theircallus at each growing stage were estimated by high performance liquid chromatography, theresult showed that, the level of content of endogenous phytohormones during the wholeprocess of tissue culture presented a dynamic change, and this fluctuation was considered tobe more related with the growth and development of callus and protoplast during the courseof tissue culture.1) The rate of inductivity of callus had positive correlation with the hormone level of IAAand ABA in mature seeds. The hormone levels of IAA (0.574μg/g,0.393μg/g,0.415μg/g,respectively) among all seeds of the three cultivars were relatively high and fewer amountsof ABA (0.157μg/g,0.088μg/g,0.123μg/g, respectively)was detected, there was nosignificantly difference of the hormone level of ZT among all cultivars and the hormonelevel showed random patterns.2) During the stage of subculture and differentiation of callus of experimental materials, thehormone content of IAA in the cultivar, Midnight Ⅱ was at the relatively high level(0.425μg/g,0.554μg/g, respectively), also it fluctuated within a small range and reached theclimax during the period of growth and bud formation of callus. The hormone content ofboth ABA (0.120μg/g,0.141μg/g) and ZT (0.111μg/g,0.202μg/g) persisted at a relativelylow level during the stage of callus culture and differentiation. However, during the stage offormation of germ callus, these hormone levels rose gradually and, as well, there wasenhancement of differentiation of callus, this variation was thought to confer facilitation tothe germ callus.3) The high ratio values of hormone content ZT/IAA as well as ABA/IAA were beneficial tothe growth of callus and formation of germ callus and the ratio of ABA even have thepositive correction with differentiation of callus.3. The content of endogenous phytohormones (IAA, ABA and ZT) in embryogenic callus ofthree Kentucky bluegrass cultivars, Midnight Ⅱ, Nuglade and Rugby Ⅱ, was estimated andthe protoplast was isolated and cultured. The result illustrated that, the content of IAA amongthe embryogenic callus of all three cultivars were relatively high, with the highest value of0.600μg/g in the callus of Nuglade, the content of IAA in the callus of Rugby Ⅱ was 0.523μg/g and Midnight Ⅱ of0.494μg/g; Endogenous ABA levels ranging from low to highwere Nuglade(0.123μg/g),Midnight Ⅱ(0.167μg/g)and Rugby Ⅱ(0.218μg/g)respectively,the rate of division of protoplast had negative correlation with the hormone level of ABA;The content of Endogenous ZT in callus of three Kentucky bluegrass cultivars presented arelatively lower level, with content value ranging from high to low were Nuglade(0.237μg/g),Midnight Ⅱ(0.200μg/g)and Rugby Ⅱ (0.146μg/g) respectively, the rate offission of protoplast had positive correlation with the hormone level of ZT.