以甜叶菊(Stevia rebaudiana Bertoni)茎段为外植体,采用L9(34)正交表对腋芽诱导培养基中的激素种类和浓度(0.0、0.1和0.2 mg.L^-1NAA;0.0、0.5和1.0 mg.L^-16-BA;0.000、0.005和0.010 mg.L^-1TDZ)进行筛选;在此基础上对诱变过程中NaN₃溶液的浓度(3、6和9 mmol.L^-1)和处理时间(1、2、4和8 h)进行比较,并初步筛选出最佳的NaN₃诱变条件;采用聚丙烯酰胺凝胶电泳法(PAGE)对经上述NaN₃诱变处理后培养5、10和15 d的试管苗POD同工酶酶谱的变化进行了分析。结果表明:在添加了不同激素组合的培养基上均能诱导出腋芽,但腋芽数和苗高有差异;经综合比较后可确定适宜于甜叶菊腋芽诱导的培养基为添加1.0 mg.L^-16-BA和0.1 mg.L^-1NAA的MS培养基(含5.0 g.L^-1琼脂粉和30 g.L^-1蔗糖,pH 6.0)。经NaN3诱变处理后,茎段在腋芽诱导培养过程中的死亡率随NaN3浓度提高和处理时间延长逐渐增加,平均腋芽数和苗高则下降,且多数处理组试管苗矮化;根据半致死剂量确定的最佳NaN3诱变处理方法为将甜叶菊茎段置于9 mmol.L^-1 NaN₃溶液中浸泡4 h。PAGE分析结果表明:甜叶菊诱变试管苗的POD同工酶谱均可分为a、b和c区,但在不同培养时间各处理组POD同工酶的条带数和活性有所变化。在培养5和10 d后各处理组诱变试管苗POD同工酶的活性和条带数量有明显差异,而在培养15 d后各处理组诱变试管苗POD同工酶活性有差异,但条带数量没有明显变化,表明用适宜浓度的NaN₃进行诱变处理可导致甜叶菊试管苗短期的应激效应。

Taking stem segments of Stevia rebaudiana Bertoni as explants， the type and concentration of hormones ( 0． 0， 0． 1 and 0． 2 mg·L^-1 NAA; 0． 0， 0． 5 and 1． 0 mg·L^-1 6-BA; 0． 000， 0． 005 and 0． 010 mg·L^-1 TDZ) in induction medium of axillary bud were selected by L9( 34 ) orthogonal table．On the basis， NaN₃ concentration ( 3， 6 and 9 mmol·L^-1 ) and treatment time ( 1， 2， 4 and 8 h) in mutation process of stem segments were compared and the optimal mutation condition of NaN₃ was screened preliminarily． And change of POD isoenzyme zymograms of plantlets cultured for 5， 10 and 15 d after above-mentioned NaN ₃ mutation treatment was analyzed by polyacrylamide gel electro-phoresis ( PAGE) ． The results show that axillary bud can be induced on all media containing different hormone combinations， but their axillary bud number and plantlet height are different． By comprehensive comparison， it is determined that the appropriate medium for axillary bud induction of S． rebaudiana is MS medium ( containing 5. 0 g·L^-1 agar powder and 30 g·L^-1 sucrose， pH 6.0) added 1.0 mg·L^-1 6-BA and 0. 1 mg·L^-1NAA． With rising of NaN₃ concentration and prolonging of treatment time， the death rate of stem segments treated by NaN₃ increases gradually during induction culture process of axillary bud， and average number of axillary bud and plantlet height decrease， moreover， mutation plantlets in most treatment groups dwarf． According to the median lethal dose， it is confirmed that the optimal NaN₃ mutation treatment method is putting stem segments of S． rebaudiana in 9 mmol·L^-1 NaN₃ solution soaking for 4 h． The PAGE analysis result shows that POD isoenzyme zymogram of all S．rebaudiana mutation plantlets can be divided to a， b and c regions， but band number and activity of POD isoenzyme in different treatment groups change during different culture times． After cultured for 5 and 10 d， activity and band number of POD isoenzyme of mutation plantlets in different treatment groups have obvious differences， while there is difference in activity of POD isoenzyme but without obvious change in band number of POD isoenzyme after cultured for 15 d． It is suggested that NaN₃ mutation treatment with suitable concentration can cause stress effect of S． rebaudiana plantlets in short term．