Silicon carbide fibers

transformation

Presented by Pursem Vidula N

Introduction

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.
 Silicon carbide fibers
 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-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.
 Silicon carbide fiber-mediated stable
transformation of plant cells (Kaeppler et al 1991)
 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.
Materials and methods
 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.
 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.
Selection for stable transformants
 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
 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
Other applications
 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
Pollen-mediated method for transformation of maize, tomato
or melon

 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
- 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.)
Use of silicon carbide fiber forAgrobacterium-
mediated transformation in wheat

 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.
Advantages of silicon carbide fibers
transformation

 no use of specialised equipment

 high efficiency
 low cost
 high reproducibility
 genotype independence,
 genetic stability of the transformants
 technical simplicity.
Disadvantages
 Fibres require careful handling

 SIC fibres may be carcinogenic(similar

properties to asbestos)

 Cannot be used with callus that is hard

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