Silicon carbide fibers transformation

Presented by Pursem Vidula N

Genetic engineering of plants entails the isolation

and manipulation of genetic material

Once a gene has been identified, cloned, and engineered, it is still necessary to introduce it into a plant of interest

introduction of that genetic material into a plant or plant cells, offers considerable promise to modern agriculture and plant breeding.

Introduction

This needle-shaped material was selected as a means of perforating cells

Is produced by carbonizing (at high temperature in carbon monoxide) the silicate found within the cells of rice husks.

Most whisker preparations are highly heterogeneous, ranging m length from 5-500 um They can also differ in diameter (usual mean of 1 um) and the degree of hydrophobicity, both factors that are likely to affect their efficiency

Silicon carbide fibers

Silicon carbide-mediated transformation is probably the least complicated transformation method.

Silicon carbide fibers are added to a mixture of plant cells and plasmid DNA and mixed on a vortex.

When cells and fibers collide, small holes result, through which the DNA can enter the cell.

Maize (Zea mays, cv 'Black Mexican Sweet')(BMS) and tobacco (Nicotiana tabacum, cv

'Xanthi') tissue cultures were transformed using silicon carbide fibers to deliver DNA into suspension culture cells. DNA delivery was mediated by vortexing cells in the presence of silicon carbide fibers and plasmid DNA.

Silicon carbide fiber-mediated stable transformation of plant cells (Kaeppler et al 1991)

Cell cultures

'Black Mexican Sweet' (BMS) maize nonregenerable cell suspension cultures and nonregenerable tobacco suspension culture Txd (Nicotiana tabacum, cv 'Xanthi')and were grown in sterile liquid medium with appopriate salts,sugars, amino acid and optimum PH.

Cells to be used as samples were collected following subculture.

Materials and methods

Maize suspension culture cells were vortexed in the presence of silicon carbide fibers and the plasmid pBARGUS.

Tobacco suspension culture cells were treated with silicon carbide fibers and plasmids pNGI and pBI221

pNGI contains a B-glucuronidase (GUS) and a

neomycin phosphotransferase II (NPTI1) gene

Plasmid pBARGUS contains the BAR gene which confers plant cell resistance to phosphinothricin-containing herbicides such as BASTA and a B-glucuronidase (GUS)

Twenty-five microliters of plasmid DNA (1 ug/ul)was combined with 40 ul of a 5% (w/v) suspension of silicon carbide fibers in a 1.5-ml Eppendorf centrifuge tube.

The suspension was mixed by vortexing for 5 s. Suspension culture cells were collected using

vacuum filtration, rinsed with culture medium, and approximately 300 ul packed volume of cells added to each Eppendorf tube containing the plasmid DNA and suspended silicon carbide fibers.

Culture medium(100 ul) was added to the mixture and vortexed.

Samples of maize cells treated with pBARGUS and silicon carbide fibers, and control samples treated with fibers alone, were transferred into 60 x 20 mm disposable petri plates, and 2 ml of MS2D medum was added to each plate.

After some manipulations putatively transformed maize colonies growing in the presence of BASTA-containing medium were observed after 4-5 weeks of selection and were subsequently subcultured

Selection for stable transformants

Tobacco suspension culture cells treated with pNGI+ pBI221 + silicon carbide fibers, and control samples treated with only fibers, were transferred disposable petriplates, and 10 ml medium was added to each plate.

One week later, 10 ml of culture medium containing kanamycin was added.

After some manipulations, putative kanamycin-resistant, transformed tobacco colonies were observed after approximately 6 weeks of selection and these were subsequently subcultured.

Total nuclear DNA was isolated from BASTA-resistant maize colonies, kanamycin-resistant tobacco colonies were identified by southern blot

Making use of restriction enzymes and labelled probes

Frame et al (1994) reported recovery of transgenic maize plants by using regenerable maize suspension cultures

Plants were shown to be transgenic by southern blot

Expression tests and inheritance of transgenes in next generation were observed

Other applications

A method for plant transformation comprising pollination pathway and silicon fiber treatment .

Recipient plants are pollinated by pollen grains carrying the transforming DNA wherein the pollen grains are pre-treated by silicon carbide fibers and the transforming DNA

Pollen-mediated method for transformation of maize, tomato or melon

- preparing silicon carbide fibers solution;

- preparing pollen germination medium;

- mixing the silicon carbide fibers with DNA and with the germination medium;

- putting fresh pollen into the above mixture resulting in a paste;

- vortexing the mixture for 30-60 seconds;

- applying the resulting paste for pollination;

- selecting the transformants.

#

Transformation of Microalgae Using Silicon Carbide Whiskers by: Terri G. Et al (2005)

The ability to genetically engineer these organisms, in order to study and eventually manipulate the metabolic pathways, would greatly enhance the utility of microalgae as scientifically and industrially important species.

Silicon Carbide Whisker-Mediated Embryogenic Callus Transformation of Cotton (Gossypium hirsutum L.) and Regeneration of Salt Tolerant Plants by Shaheen et al, 2008

recovery of fertile and stable transformants was developed for cotton (Gossypium hirsutum L.)

Silicon carbide fibers have been used for causing wounds in the immature wheat embryos for Agrobacterium- mediated genetic transformation.

Agrobacterium-infected explants were stained for GUS activity.

Without wounding the GUS expression was observed in 2.4% of the embryo, while 33.3% of the embryos showed GUS expression after wounding with SCF for 2 min.

Use of silicon carbide fiber forAgrobacterium-

mediated transformation in wheat

no use of specialised equipment high efficiency low cost high reproducibility genotype independence, genetic stability of the transformants technical simplicity.

Advantages of silicon carbide fibers transformation

Fibres require careful handling

SIC fibres may be carcinogenic(similar properties to asbestos)

Cannot be used with callus that is hard

Disadvantages

Effective DNA delivery is affected by the length of time subculturing the cells and the vortex treatment

Other limitations

THANK YOU