Effects of hornonal balance during leaf-disc infection of Nicotiana plumbaginifolia with Agrobacterium tumefaciens nopaline strain C58

Abstract

The technique of leaf-disc transformation has been used in order to study the influence of different hormonal balances on the process of transformation of Nicotiana plumbaginifolia tissues with Agrobacterium tumefaciens nopaline-strain C58.

C58 Agrobacterium strain incites withish, compact undifferentiated calli on Nicotiana tabacum, whilst allows the recover of shoots in N. plumbaginifolia. Shooting can be modulated through the hormonal balance of the culture medium mostly during the tissue culture phase (phase 2) subsequent to the period of infection (phase 1). Complete absence of hormones in phase 1 and 2 does not result in any callus formation or shooting on the edges of the infected leaf-discs, thus indicating that in this case the transformed cells need hormones at least in initial phase of development. In both the phases, addition of 2,4-D, NAA and kinetin resulted necessary in the stimulation of callus and shooting, namely in phase 2, and kinetin played a determinant role. Direct shooting also leads to an higher frequency of NOS⁺ regenerated shoots.     

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。