为从Nicotiana tabacum-N.plumbaginifolia杂种(2n=58)回交N.tabacum的后代中筛选对黑胫病具有较强抗性的植株,利用离体叶片侵染法对存活的120株后代植株进行了初步筛选。筛选分为两次,第1次为初选,对第1次接种后不发病和发病较轻的植株第2次接种进行复选,对经第2次接种后叶片不发病和发病较轻的植株进行基因组原位杂交(GISH)分析。结果显示:第1次接种后5株植株的叶片不发病和8株植株的叶片发病较轻;第2次接种后4株植株的叶片不发病,1株植株的叶片发病较轻。GISH鉴定结果显示:第2次接种后4株叶片不发病的植株携带1-4条N.plumbaginifolia染色体,叶片发病较轻植株则未见N.plumbaginifolia染色体,携带1条外源染色体植株的染色体数目为48。对这5株材料的离体叶片接种病原菌以对其离体叶片的黑胫病抗性进行再次鉴定,结果与复选时所得结果相近。综上所述,自Nicotiana tabacum-N.plumbaginifolia杂种回交一代(BC1)中筛选获得了4株叶片对黑胫病具有较强抗性的异源染色体植株,其中一株可能为异源单体代换植株。
To obtain strong black shank resistant plants with alien chromosomes in Nicotiana tabacum-N. plumbaginifolia backcross generations, 120 plants were primarily screened by in vitro leaf infection method, and plants without scabs and with smaller scabs on leaf were selected in the first screening, plants without scabs and with smaller scabs on the leaves were investigated by genomic in situ hybridization(GISH) in the second screening. 5 plants without scabs on leaf and 8 plants with smaller scabs on leaf were found during the first screening, and 4 plants without scabs on leaf and 1 plant with smaller scabs on leaf were found in the second screening. GISH results showed that there were 1-4 chromosomes from N. plumbaginifolia genome in 4 plants without scabs on leaf, and no N. plumbaginifolia chromosomes in plant with smaller scabs on leaf. Leaves resistance to black shank of these 5 plants were selected to be investigated by GISH and were tested in vitro again, presenting similar results to that of second screening. Thus, 4 tobacco plants with leaf that highly resistant to black shank, were obtained in Nicotiana tabacum-N. plumbaginifolia hybrid BC1 plants, and they had 1-4 alien chromosomes, and one of them could be monosomic alien substitution plant.