Studies on expression of an antifungal gene maize chitinase in tobacco and potato and its effect on growth and the fungal pathogen Rhizoctonia solani

Abstract

To confer resistance against the chitin-containing fungal pathogens, an antifungal maize gene, chitinase (cDNA clone), under the control of a CaMV 35S promoter was successfully introduced into the tobacco (Nicotiana tabacum) and potato (Solanum tuberosum) using Agrobacterium tumefaciens mediated transformation. Transgene integration and expression was confirmed by Southern and northern blot analysis. The transgene was inherited as a dominant trait in a 3:1 Mendelian ratio in most of the transgenic tobacco lines studied. Homozygous lines were identified based on the progeny segregation on kanamycin-containing medium, which was used as selection marker. Some of the homozygous transgenic tobacco lines and potato transformant regenerants showed four to eight-fold increased constitutive enzyme activity. Homozygous transgenic tobacco inoculated with the fungal pathogen Rhizoctonia solani showed a marginally lower susceptibility, as compared to untransformed seedlings, which was statistically not significant. Transgenic and untransformed tobacco seedlings raised in closed plastic containers were less susceptible to R. solani. The fungal susceptibility of the transgenic potato needs to be investigated.Greenhouse grown seedlings of some transgenic tobacco lines were more vigorous than the untransformed seedlings, and a linear correlation (r = 0.74) between seedling mass and endochitinase activity was noted. Among the in vitro grown mixed progeny (progeny of self-pollinated heterozygous parents) a vigor based prediction of the genetic status, homozygous, heterozygous or null for the maize chitinase gene was made. Most predictions were correct, as confirmed by seedling growth on kanamycin-containing medium. Suspension culture studies of transgenic and wild type lines also showed a linear correlation (r = 0.77) between growth and endochitinase activity.Seedlings grown in closed plastic containers were more vigorous than their counterparts in the greenhouse. The endochitinase specific activity in cell wall bound, but not soluble proteins of these seedlings was three-fold higher than the greenhouse grown seedlings, especially in the wild type.Overall results indicate a role for endochitinase in plant growth, but further investigations are needed to confirm this.U of I OnlyETDs are only available to UIUC Users without author permissio