Agrobacterial Transformation of Uninjured Plants

The capacity for agrobacterial transformation (using Agrobacterium tumefaciens strain GV3101 with the activated vir genes) was investigated in uninjured seedlings of tobacco (Nicotiana tabacum L.), rice (Oryza sativa L.), and wheat (Triticum aestivum L.) along with the effect of various factors on the frequency of tobacco seedling transformation. Transformed cells were found at the base of the rice leaf sheath and in the upper part of tobacco leaves, whereas they were absent from wheat leaves. Illumination and temperature, which were below and over the optimum, were the factors limiting agrobacterial transformation of uninjured plants. Preincubation of tobacco seedlings in 100 M ABA, 10 mM EGTA, and 0.1 mM colchicine decreased the frequency of agrobacterial T-DNA penetration into plant cells. The authors conclude that transformation occurs predominantly in young growing cells, and its frequency is related to the state of stomata and plasmodesmata of the target plant cells. The elements of the cytoskeleton probably participate in T-complex transport.     

Colonization of Wheat Root Hairs and Roots by Agrobacteria

Formation of extracellular structures in pure culture and in interaction with wheat root surface was studied by scanning and transmission electron microscopy. The effects of various factors (growth temperature as well as pretreatment of agrobacteria with kalanchoe extract, acetosyringone, and centrifugation) on formation of extracellular structures was tested. The data on Agrobacterium tumefaciens (wild-type strain C58 and mutants LBA2525 (virB2::lacZ) and LBA288 (without the Ti plasmid)) adhesion to wheat root surface and root hairs after pretreatment of agrobacteria with inducer of virulence genes (vir) acetosyringone were obtained. Formation of agrobacterial cell aggregates on wheat root hair tips was demonstrated. The proportion of root hairs with agrobacterial aggregates on the root hair tip insignificantly changed after pretreatment with acetosyringone but considerably increased after treatment of A. tumefaciens C58 and LBA2525 with kalanchoe leaf extract. The most active colonization of root hairs and formation of agrobacterial aggregates on hair root tips was observed at 22°C. The capacity of agrobacteria for adhesion on monocotyledon surface could be changed by pretreatment of bacteria with various surface-active substances. Bacterial cells subjected to centrifugation had a decreased capacity for attachment to both wheat root surface and root hairs. The relationship between the capacity for adhesion and pilus production in agrobacteria was considered.