Agrobacterium-Mediated Geen Transfer

2.1. Agrobacterium-Mediated Geen Transfer

It has been duly established that gene transfer through Agrobacterium is invariably accomplished in the following two methods :

2.1.1. Co-culture with Tissue Explants

In actual practice, the ‘suitable gene construct’ is strategically inserted carefully very much within the T-region of a disarmed Ti plasmid ; and for this one may use either a ‘cointegrate’ or a ‘binary vector’. Consequently, the ‘recombinant vector’ is placed carefully in the Agrobacterium that is invari- ably co-cultured with the plant cells or tissues’ to be transformed for about 48 hours.

Importantly, in the instance of several plant species, normally ‘small leaf-discs’ (having a few mm diameter) are cut carefully from the surface of pre-sterilized leaves the subsequently employed for co-cultivation, such as : tobacco, tomato, petunia etc. Consequently, the ‘transgene’** thus accom- plished essentially comprises of a specific selectable reporter gene e.g., the ‘bacterial neo genene’. The

* Such type of transformations failed to pass on to next generation. ** A ‘gene’ into which hereditory material from another organism has been introduced.

GENETIC RECOMBINATION

resulting neo gene is strategically hooked on the appropriate regulatory sequences which are found to

be functional in plant cells. In actual practice, such a ‘gene’ is invariably termed as chimaceric gene because it predominantly consists sequences derived from several different genes.

It has been duly observed that in the course of leaf disc agrobacterium co-culture, a chemical entity known as acetosyringone* duly released by plant cells thereby causing specific induction of the vir genes that particularly affords the plausible transfer of recombinant T-DNA into a plethora of the prevailing plant cells. In doing so the resulting T-DNA would eventually get integrated into the plant genome ; and hence, the transgene would be expressed. Consequently, the transformed plant cells will

become resistant to kanamycin (an antibiotic) on account of the expression of neo gene. After a gap of

48 hours, the treated leaf-discs are meticulously transferred onto a generation medium containing suit- able concentrations of both kanamycin and carbenicillin as shown in Fig. 2.1. The two ‘antibodies’ do play specific roles, namely :

(a) Kanamycin — permits exclusively the ‘transformed plant cells’ to undergo division and regeneration of ‘shoots’ in 3-4 weeks duration ; and (b) Carbenicillin — helps in killing Agrobacterium cells only. At this stage, the shoots are adequately separated, rooted, and ultimately transferred into the prepared soil-bed.

Salient Features : The prevalent salient features of co-culture with tissue explants are as enu- merated under :

(1) The agrobacterium does infect certain specific monocot plant species and ultimately gives rise to the formation of crowngall cells, such as : Asparagus or causes apparent swellings, for instance : Allium cepa, Dioscorea bulbifera, besides Chlorophytum and Narcissus. In

most of the these instances, the critical production of the opines by the corresponding crowngall cells and swelling tissues was noticed evidently.

(2) Besides, the integration of T-DNA into the genomes of at least two plant species, namely : Dioscorea bulbifera and Orzyza sativa has been well established and amply demonstrated. Nevertheless, the efficiency of transformation is observed to be at a low ebb.

(3) Importantly, an appreciably efficient degree of transformation of the monocot cells may be accomplished by the induction of acetosyringone in the course of co-culture of plant cells with the aid of Agrobacterium.

(4) A few plant species are found to secrete such chemical entities that essentially and precisely inhibit the usual induction of the vir operons by acetosyringone. Example : Secretion of 2, 4-dihydroxy-7-methoxy-1, 4-benzoxazin 3-one (DIMBOA) that specifically inhibits vir gene induction ; and this problem may be circumvented by adding an excess of acetosyringone. However, this technique help in the successful transformation in several cereal species e.g., barley, maize, rice and wheat.

* Acetosyringone :

PHARMACEUTICAL BIOTECHNOLOGY

A LEAF

LEAF DISCS

INCUBATED WITH

[SURFACE STERILIZED]

[4-6 mm DIA.]

AGROBACTERIUM

[LIQUID MEDIUM]

INCUBATED OVER NIGHT

LEAF DISCS

[DISCS BLOTTED DRY]

FILTER PAPER FEEDER, CELLS MEDIUM SHOOT REGENERATION MEDIUM CULTURE (2 DAYS)

SHOOT MEDIUM + KANAMYCIN

ROOT MEDIUM

CARBENICILLIN

KANAMYCIN REGENERATION OF SHOOTS

[3/4-WEEKS DURATION]

CARBENICILIN

Fig. 2.1. A Sequential Diagramatic Representation of the Methodology for Agrobacterium-Mediated Gene Transfer.

[Adopted from : ‘Biotechnology’, BD Singh, 1998]

2.1.2. In Planta Transformation

It has been observed that inhibition of Arabidopsis seeds in the freshly prepared cultures of Agrobacterium ultimately gives rise to the stable integration of T-DNA strategically present in the Arabidopsis genome. It seems that the prevailing Agrobacterium cells eventually gain entry into the corresponding seedlings in the course of the germination process, are adequately held up within the plants ; and subsequently, when flowers undergo the process of development (i.e., blooming) they ulti- mately transform either the ensuing ‘zygotes’ or the ‘cells’ which yield zygote.

Another viable and feasible approach is invariably accomplished when the ‘just budding flowers’ of the Arabidopsis plants are duly dipped into a freshly prepared culture of Agrobacterium, and subse- quently a partial vaccum is generated so as to augment and facilitate entry of the corresponding bacte- rial cells right into the body of the exposed plants. Consequently, the plants are allowed to grow, selfed, and the ‘progeny’ thus obtained are subjected to vigorous methodical screening for the identification of the generated transformants.

Advantages : The various glaring advantages of the two aforesaid approaches of Agrobacterium transformation methodologies are as given below :

GENETIC RECOMBINATION

(a) absolute elimination of the need for regeneration from the tissue explants, (b) methodologies involved are relatively not so cumbersome and easy ; and, therefore, may be

adapted with other plant species with an equally high successful rate, (c) possess little risk of ‘somaclonal variation’, although the probability for the mutations

independent of stable T-DNA integrations do prevaily by virtue of the incidence of the abortive T-DNA insertions.