National Refresher Course in Plant Biotechnology

Sponsored by the Andhra Pradesh Netherlands Biotechnology Programme

Practical Manual

Introduction

Genetic transformation systems have been developed for a variety of crop plants. The development of protocols for producing transformed plants by Agrobacterium-mediated gene transfer depend on 1) Agrobacterium tumefaciens containing modified Tumor inducing (Ti) plasmid vectors and 2) the development of cell culture techniques that permit efficient DNA delivery, selection of transformants and regeneration of intact plants.

The "wild type" Ti plasmid of the soil micro-organism A. tumefaciens is the causative agent of crown gall disease in dicot plants. The plasmid is large, approximately 200 kb, and contains two regions essential for the production of transformed plant cells (i) the Transfer DNA region (T-DNA) which is excised from the Ti plasmid and transferred to the plant cell nucleus and (ii) the virulence region which encodes proteins essential for T-DNA excission, transport and nuclear integration.

Agrobacterium causes plant cell tumors, because hormone biosynthesis genes, located on the T DNA, are transcriptionally activated when transferred to the plant nuclear environment. The tumorous tissue is permanently transformed and no longer requires the presence of the causative organism. It is however disorganised and undifferentiated.

Clearly with modifications, such a system ought to be capable of being used to introduce foreign DNA sequences into plants. Recently many modified versions of the Ti plasmids have been produced.

Two types of Ti –plasmid-derived vectors can be distinguished:

Cis systems (Co-integrate vectors) in which new genes are introduced via recombination into a non-tumorigenic Ti plasmid.

Trans or binary systems in which new genes are cloned in a plasmid containing a T-DNA, which is subsequently introduced into an Agrobacterium strain harboring a Ti plasmid with an intact vir region, but lacking the T-DNA region. The vir genes or its source will be in another plasmid acting in trans to the T-DNA. This vector is called binary because of the separate location of the virulence region on a second plasmid located in the host A. tumefaciens (Hoekema et al., 1983).

Perhaps the most widely used versions are the so called Binary vectors e.g. pBIN19 (Bevan, 1984), pBI121 (Jefferson, 1987). This modified Ti based vector consists of a small (10kb) broad host range replicon capable of replication and selection both in E. coli and A. tumefaciens. It contains a T DNA region from which the hormone biosynthesis genes have been removed and into which has been incorporated a polylinker-cloning site and kanamycin resistance gene, which facilitates selection of transformed plant cells. This vector is called Binary because of the location of the virulence region on a separate plasmid located in the host A. tumefaciens.

An improved binary vector called the super binary vector was developed by incorporating certain virulence genes from strain A281, in the plasmid that carries T-DNA (Komari, 1990). Using in this class of vector system high efficiency of transformation was recorded. These super binary vectors play an important role in the development of transformation technologies for monocotyledons, especially in cereals like rice (Nagadhara et al., 2003).

The transformed plant cells can be regenerated into whole plants simply by manipulating the hormonal concentration in the culture medium. Regenerated shoots will be selected in culture initially on the basis of their resistance to kanamycin. If the plasmid contains any one of the reporter genes such as: gusA, gfp, anthocyanin biosynthesis genes (C and R genes) one can screen for the expression of GUS, GFP and pigment synthesis.

MEDIA & SOLUTIONS
Stock solutions for Agrobacterium mediated transformation

Antibiotic stocks

Hygromycin (50 mg/ml). Dissolve 500 mg in 10 ml sterile distilled water and filter sterilise with 0.40mM millipore filter into a sterile tubes, which are then stored at –20°C.
Carbenicillin (100 mg/ml). Dissolve 1g in 10ml sterile distilled water, filter sterilise as above and store at –20°C.
Carbenicillin (250mg/ml). Dissolve 2.5g in 10ml sterile distilled water, filter sterilise as above and store at –20°C.
Acetosyringone stock (10mg/ml): Dissolve 100mg of Acetosyringone in 1ml of 70% Ethanol and make up the volume to 10ml with sterile distilled water, filter sterilised and stored and –20°C.
2,4-Dichloro Phenoxy acetic acid (2,4-D): Dissolve 40mg of 2,4-D in about 1ml of 90% Ethanol and make up the volume to 100ml sterile distilled water. This stock is then autoclaved and stored at 4°C.


 

Murashige and Skoog (MS) medium (1962)

pH of the media is adjusted to 5.6 - 5.8 and 1.5% Agar is added and autoclaved

 Table 1: Different media used for rice transformation

Tissue culture and genetic transformation protocol for rice

1)  Dehusk the mature seeds of rice and surface sterilize with 0.1% HgCl2 for 10-12 min followed by thorough washing with sterile water.

2)  Leave the sterilized seeds overnight in sterile distilled water at room temperature.

3)  Excise the swollen embryos next day under aseptic conditions and place on MS medium (Murashige and Skoog, 1962), supplemented with 2mg L-1 2,4-D for callus induction (Table 1) and incubate for three weeks at 26 ± 1° C in dark.

4)  Sub-culture the calli onto 3MN62 (Table 1) for 7 days and observe for embryogenic calli. Separate the embryogenic calli (2-3mm) by visually observing for the characteristic compact nature, pale yellowish colour and nodular appearance.

5)  Incubate the embryogenic calli on the proliferation medium for 4 days before transfer to co-cultivation medium. Transfer the actively growing embryogenic calli onto the co-cultivation medium (CCM) (Table 1).

6) Inoculate a single colony, from a plate not more than one week old in YEP + 50ppm Hygromycin and left overnight on a shaker at 29°C for preparation of Agrobacterium suspension.

7) Pellet the cells and resuspend in PIM2 media (Table 1) supplemented with 100µM AS and keep on the shaker for a further 16 hours at 29°C.

8)  Add 100mM AS to the culture before infection and on the each explant, (placed on CCM) poure 15 µl of the culture using a micropipette. Keep the plates in the dark at 290C for three days (72 hours).

9)  Wash the co-cultivated explants after three days thrice with MS basal medium containing 100mg/l Cefotaxime + 250mg/l Carbenicillin for ten minutes each. Blot dry on a sterile tissue paper and place on Cefotaxime carbenicillin medium (Table 1). Now place the calli in the dark for one week.

Assay for reporter gene gusA activity.

1.  The superbinary vector pTOK233 (Fig. 1), carries the reporter gene gusA coding for b-glucuronidase. The activity of the enzyme is very easy to detect using either colorimetric or fluorimetric methods.

2.  Transient GUS expression in calli infected with pTOK233 is determined as follows (Jefferson 1987) :- At the end of 7 days, place the calli in GUS staining solution (50mM sodium phosphate buffer, pH 7 + 0.1% Triton X 100 + 10mM EDTA + 1mM X-Gluc) at 37°C overnight in dark. The next day the gusA gene expression is visualised by the presence of characteristic blue colour.

References

Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA, 1983. A binary plant vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens,

Ti-plasmid. Nature. 303:179-180.

Bevan M, 1984. Binary agrobacterium vectors for plant transformation. Necleic Acids Res. 12: 8711-8721.

Jefferson RA, 1987. Assaying chimeric genes in plants: the gus gene fusion system. Plant Mol. Biol. Rep. 5: 387-405.

Komari T, (1990). Transformation of cultured cells of Chenopodium quinoa by binary vectors that carry a fragment of DNA from virulence region of pTiBo542. Plant Cell Rep. 9: 303-306.

Nagadhara D, Ramesh S, Pasalu IC, Rao KY, Krishnaiah NV, Sarma NP, Bown DP, Gatehouse JA, Reddy VD, Rao KV, (2003). Transgenic indica rice plants resistant to sap sucking insects. Plant Biotech. J. 1 (3): 231-240.