Multiple copies of virG enhance the transient transformation of celery, carrot and rice tissues by Agrobacterium tumefaciens

Abstract

In an effort to improve the T-DNA-mediated transformation frequency of economically important crops, we investigated the possible enhancement effect of multiple copies of virG genes contained in Agrobacterium tumefaciens strains upon the transient transformation of celery, carrot and rice tissues. Four days after A. tumefaciens infection, we performed histochemical beta-glucuronidase (GUS) assays to determine the frequency of transient transformation of calli from celery and carrot, and explants from rice and celery. Additional copies of octopine- and agropine-type virG genes in A. tumefaciens strains containing an agropine-type Ti-plasmid enhanced the frequency of transient transformation of celery and rice. This enhancement ranged from 25% to five-fold, depending upon the source of the virG gene and the plant tissues inoculated. For both rice and celery, we observed a greater enhancement of transformation using A. tumefaciens strains containing additional copies of an octopine-type virG gene than with strains harboring additional copies of an agropine-type virG gene. Multiple copies of virG genes contained in A. tumefaciens strains harboring a nopaline-type Ti-plasmid had a smaller enhancing effect upon the transformation of celery tissues, and no enhancing effect upon the transformation of rice. In contrast, we obtained a three-fold increase in the transient transformation frequency of carrot calli using an A. tumefaciens strain harboring a nopaline-type Ti-plasmid and additional copies of an octopine-type virG gene. Our results show that multiple copies of virG in A. tumefaciens can greatly enhance the transient transformation frequency of celery, carrot and rice tissues, and that this enhancement is influenced by both the type of Ti-plasmid harbored by A. tumefaciens and by the infected plant species.

EfficientvirGene Induction inAgrobacterium tumefaciensRequiresvirA,virG,andvirBox from the Same Ti Plasmid

“…No increase in transformation efficiency was observed in the nopaline vir helper background. Similarly, Liu et al (46) observed that supplementation of extra copies of octopine (pTiA6) virG or L,L-succinamopine (pTiBo542) virG in nopaline Ti plasmid backgrounds did not bring about a significant increase in transient transformation of celery and rice. This reconfirms our conclusions drawn from Tables 2, 3, and 6 that VirG of Bo542 interacts efficiently with the vir boxes of octopine strains but not with the vir boxes of nopaline strains.…”

Agrobacterium -Mediated Plant Transformation: the Biology behind the “Gene-Jockeying” Tool

“…Normally, vir gene induction is very poor at neutral or alkaline pH or in rich medium; additional copies of virG permitted a substantial degree of induction in rich medium even at pH 8.5. Additional copies of virG also increased the transient transformation frequency of rice, celery, and carrot tissues (199).…”

Uncoupling of the functions of the Arabidopsis VIP1 protein in transient and stable plant genetic transformation by Agrobacterium

“…All experiments were performed in triplicate. For tumor assays (16), 30-50 root segments per tested plant were submerged in an OD 600 ϭ 0.1 culture of A. tumefaciens A208, incubated for 10 min at 25°C, cultivated for 48 h at 25°C in HFMS, washed, and cultured for 4 weeks in HFMS with 300 g͞ml carbenicillin. Tumors were counted, and their phenotype was determined.…”

“…3C), indicating equal efficiency of their transient transformation. Because the uidA reporter gene contained an intron which prevents its expression in bacteria (30), our measurements represented the GUS activity directed by the T-DNA after its transfer to plant cells. Thus, the N-terminal portion of VIP1 in the vip1-1 mutant most likely contained protein activities sufficient to promote transient expression of the Agrobacterium T-DNA.…”