Carrot Transformation with Agrobacterium

This videoblog from http://www.AoBBlog.com is about one of the Cell and Developmental Biology practicals that I run at the University of Leicester for course #BS1003. It involves infection of carrot root slices with three strains of Agrobacterium, two of which cause the plant cells to divide. An earlier video showed how we set up the cultures www.youtube.com/watch?v=mS6OjCekrNo ; here we review the results and look at the Agrobacterium plasmid structure.

The videoblog is best viewed in HD 1080 if you have a fast internet connection; shortlink is http://youtu.be/7V5Zdxsds5s.

Plant development can be disrupted dramatically by certain pathogens. Here, we saw how the bacterial pathogen, Agrobacterium tumefaciens, causes tumours on differentiated plant tissues by activating cell division. The process involves the transfer of bacterial genes into the plant chromosomes at wound sites, resulting in the genetic transformation of the plant cells which then divide in an uncontrolled fashion because they have incorporated the genes for hormone production into the carrot cells. These genes are located in a circular DNA molecule present in the wild type Agrobacterium tumefaciens known as T37. This is the Ti plasmid, some 206,000bp long. The part that is transferred into the nuclear DNA of the carrot is known as the T-DNA and it contains the genes which make the hormones that induce the plant cells to divide. A different group of genes – both for hormones and controlling plant developement – are present in Agrobacterium rhizogenes and these lead to production of differentiated roots from the carrot cells at the site of wounding – in our case cutting – the root tissue. The third strain of Agrobacterium tumefaciens that we used was similar to the wild type T37 but it had the genes for hormone production removed.

Practical Booklet Introduction:
Cell proliferation and organogenesis mediated by Agrobacterium
Introduction
Plant and animal development can be perturbed by disease. Both plants and animals can suffer oncogenic diseases, in which the normal control over cell division is lost and tumours form. In plants, species of the soil bacterium Agrobacterium cause disease symptoms in infected plants that are characterised by tumours, the so-called Crown Gall disease (Agrobacterium tumefaciens) or by the aberrant production of ‘hairy roots’ (Agrobacterium rhizogenes). The disease symptoms are caused by the transfer from the bacteria, and expression in the plant nucleus, of genes carried on a circular DNA molecule (a plasmid, the ‘tumour-inducing’ Ti-plasmid; or the ‘root-inducing’ Ri-plasmid), following excision and transfer of part of that plasmid. This transfer of genes from bacterium to plant represents a natural form of genetic engineering, and has been exploited experimentally as a means of modifying plant growth, development and metabolism, for both basic and applied research purposes.
Plant tumour tissues are characterised by an ability to grow on media lacking hormones, since the genes transferred from the bacteria to the plant cells encode genes that promote the synthesis of auxins and cytokinins.

Experimental
In this experiment you will inoculate plant tissues (carrot tap roots) with three different strains of Agrobacterium, and study the effects on the infected tissue. The explants will be maintained on a minimal culture medium that contains no added hormones. Therefore, any callus or other outgrowth of the explants will be determined by the transforming effect of the bacteria.

LBA4404 [control] = a ‘disarmed’ A. tumefaciens strain (lacking hormone biosynthesis genes).
T37 = a wild type A. tumefaciens strain.
LBA9402 = a wild type A. rhizogenes strain.

SUMMARY OF RESULTS
In the carrots which were used as control, with no infection with the Agrobacterium, there has been a minimal amount of cell division on the surface cells, and the tissue looks slightly dry. In the carrots with the so-called disarmed strain, LBA4404, there as also been minimal growth and division of the cells. The other two have extensive cell division and proliferation, in the case of Agrobacterium rhizogenes, differentiating to form roots.

In one of the biotechnology lectures, I discussed the T-DNA structure a little more and another clip I will upload soon will show parts recorded live from this lecture, summarizing some of the material discussed above and in this video.