Carrot Transformation with Agrobacterium

Carrots Transformed by Agrobacterium infections

Carrots Transformed by Agrobacterium infections

This video­b­log from 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​.you​tube​.com/​w​a​t​c​h​?​v​=​m​S​6​O​j​C​e​k​rNo ; 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/​7​V​5​Z​d​x​s​d​s5s.

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
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.

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.

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.


Editor Pat Heslop-Harrison. ORCID 0000-0002-3105-2167

Pat Heslop-Harrison is Professor of Molecular Cytogenetics and Cell Biology at the University of Leicester. He is also Chief Editor of Annals of Botany.

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