湿加松无性系组培快繁技术

Tissue Culture and Rapid Propagation of Pinus elliottii×Pinus caribaea

以澳大利亚种源湿加松为试材,对其茎尖离体组织培养技术进行了系统研究。取2年生实生苗茎尖,通过腋芽诱导构建完整植株再生体系,研究了湿加松外植体消毒方法,基本培养基筛选,细胞分裂素6-BA、生长素NAA、蔗糖、光照强度、光照时间等外界因子对芽的诱导、增殖、伸长以及生根的影响。从2年生实生苗采集嫩枝,选择无木质化带芽茎尖,以GD＋1.0 mg/L6-BA＋0.1 mg/L NAA＋2%蔗糖为基本培养基,在光照时间12h/d、光强40μmol/（m2.s）条件下培养,诱导率较高,芽的生长速度也较快。继代培养以GD为基本培养基,最初继代6-BA质量浓度为1.0、0.5 mg/L,然后6-BA质量浓度直接降至0.1 mg/L,继代4~5次,芽的增殖系数可以达到3~4,培养期间无玻璃化和褐变现象。以GD＋2%蔗糖＋0.1~0.3 mg/L IBA为生根培养基,生根率可达45%。将诱导生根的组培苗,在自然光照下炼苗1周,然后移栽于草炭土和蛭石混合基质里（V（草炭土）∶V（蛭石）=2∶1）,成活率可达80%,10 d后苗高可增加2 cm。

A plant regeneration system for Pinus elliottii×P.caribaea was established by axillary bud induction using shoot tips of two-year-old seedlings as experimental materials,in order to study the in vitro culture of shoot tip.Effects of explant sterilization,basic culture medium,contents of cytokinin 6-BA,growth hormone NAA and sucrose,light intensity,and light duration on the induction,proliferation,elongation,and rooting of the adventitious buds were analyzed.Tender branches were collected from two-year-old seedlings of P.elliottii×P.caribaea,and the selected non-lignified stem tips with bud were cultivated on the GD culture medium supplemented with 1.0 mg/L 6-BA,0.1 mg/L NAA and 2% sucrose.The induction rate and growth rate of adventitious buds were higher under light duration of 12 hours per day and low light intensity（40 μmol·m-2·s-1）.GD medium was the best basic culture medium for successive subculture.The 6-BA contents were 1.0 mg/L for the first subculture,0.5 mg/L for the second and 0.1 mg/L for the third.The proliferation coefficient could reach 3-4 after 4 or 5 times of subculture.Vitrification and browning did not occur during the successive subculture.Rooting rate was 45% for the culture medium GD supplemented with 0.1-0.3 mg/L IBA and 2% sucrose.The test-tube plantlets were transplanted to the mixed medium of vermiculite and turfy soil（volume ratio is 1∶2） after hardening under natural light for a week.The survival rate of the test-tube plantlets reached 80% and 2 cm of elongation of the plantlets could be found after 10 days.