广藿香叶盘法高效再生体系主要影响因素的研究

广藿香是重要的芳香药用植物,利用基因工程技术对广藿香进行品种改良,需要建立一个高效的广藿香植株再生体系。该研究以广藿香无菌苗叶片为材料,将叶盘外植体分别置于不同条件下培养,观察、统计其再生植株的数量及生长状况。通过研究15~50 d的苗龄、第2~4节上的叶及培养基中2,4-D、NAA、BA和KT的浓度和配比等因素对广藿香叶盘再生植株的影响,在此基础上优化培养条件,建立广藿香高效再生体系。结果表明:广藿香无菌苗的苗龄、叶片在茎上的着生位置以及培养基中的植物生长调节物质浓度和配比都对广藿香的植株再生有显著影响;优化培养条件为以培养30 d的广藿香无菌苗顶芽下第2对展开叶片切割的叶盘为外植体,在含0.1 mg·L-1NAA和0.5 mg·L-1BA的MS培养基中培养28 d,叶盘的不定芽发生频率达到100%,单个叶盘的平均再生芽数为96.5个,经生根培养及温室炼苗,再生植株的移栽成活率达到96%。广藿香叶盘植株再生体系的建立为其基因转化研究及优良品种的快速繁育奠定了基础。

Main influential factors for efficient plant regeneration system established from leaf-disc of Pogostemon cablin

Pogostemon cablin syn. P. patchouli,is a perennial bushy herb introduced from Southeast Asia and has been extensively cultivated in South China,for both utilization in pharmaceutical and perfume industry. Patchouli cultivated in different regions has diverse morphological characteristics and essential oil constituents,which infect its therapeutic properties directly. It is necessary to established a cultivar with high quality of essential oil. This study aims to establish an efficient system of plant regeneration from the leaf-disc of Pogostemon cablin. The leaves of in vitro patchouli seedlings were used as materials. The effects of donor plant''s age,leaf position and the concentration of plant growth substances used single or combined on plant regeneration of P. cablin were studied. The results showed that low concentration''s benzylaminopurine(BA)combined with naphthaleneacetic acid(NAA)induced the high frequency of prolific adventitious shoots regeneration directly from leaf with less or without callus,the age of donor plant and position of leaf on stem both effected the frequency of shoot regeneration and the mean number of shoots. The high efficient plant regeneration system was established from leaf disc explants prepared from the leaves of the second nodes of thirty-day old-in vitro plant. The culture medium for the prolific shoot inducing was MS culture medium supplemented with 0.1 mg·L^-1 NAA and 0.5 mg·L^-1 BA. The regeneration frequency was 100% and the maximum mean number of shoot per leaf disc was 96.5. Regenerated shoots were elongated and rooted on the half strength MS medium with 0.5 mg·L^-1GA₃ and 0.1 mg·L^-1 NAA. The rooting frequency was 100%. The plantlets with well-developed roots were successfully acclimatized in the greenhouse,and planted in the field with a survival rate of 96%. The established plant regeration system from leaf-disc of P. cablin would lay foundation for the gene transformation of P. cablin and its fast breeding for good cultivars.