6. genetic transformation studies using theobroma cacao...
TRANSCRIPT
66.. GGeenneettiicc TTrraannssffoorrmmaattiioonn SSttuuddiieess uussiinngg TThheeoobbrroommaa ccaaccaaoo CChhiittiinnaassee GGeennee
6.1 INTRODUCTION
Castor is one of the most important non – edible oil seed crop which is
widely used in agriculture, textile chemicals, paper, plastics and rubber,
perfumeries, cosmetics, electronics and telecommunication industry,
pharmaceuticals, paint industry, lubricants, folk medicine, etc. (Atsmon 1989;
Vignolo and Naughton, 1991). Recently, its potential as a biofuel crop has been
realized (www.castoroil.in). The major drawback for the production of the crop
is due to the attack of biotic stresses and the presence of the protein ricin (Auld
et al., 2001). Various biotic stresses are there for the lack of production of castor
such as insect pests, bacterial diseases and fungal diseases. Among the biotic
stresses, fungal deisease - Fusarial wilt caused by Fusarium oxysporum was of a
serious concern. The average productivity of castor is affected due to a number
of biotic stresses (Sailaja et al., 2008). Agricultural practices such as fungicide
application and crop rotation are commonly used to fight this soil borne
diseases, but their effectiveness is limited. A realistic long-term solution to
manage the disease is by breeding fungus resistant cultivars. Nevertheless,
effective resistance genes are not always available in domestic cultivars, and
resistance is rapidly overcome by new races of the pathogen. In addition,
conventional breeding strategies are extremely time-consuming. The
application of biotechnological method for gene transfer provides a powerful
tool to improve the fungal disease resistance in castor.
Plant breeders and genetic engineers share the common goal of plant
improvement. While plant breeders traditionally use selective breeding for
varietals enhancement, genetic engineers continue to develop techniques for
the isolation and insertion of genes for desirable traits. Genes which are
unavailable to a particular plant species due to sexual incompatibility may be
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
85
obtained from other organisms and transferred into plants (Perani et al., 1986).
Through gene transfer programmes herbicide tolerance, insect tolerance and
fungal pathogens tolerance in the higher plant were successfully achieved. By
using modern biotechnology, plants able to survive a continuous onslaught by
pathogens (bacteria, fungi, and viruses) and pests (insects, nematodes, etc.).
Plants deploy an assortment of defensive responses soon after infection or
exposure to abiotic stress. These responses involve activation of host defence
genes that were either inactive or expressed at basal levels previously. The
major defence response involves the biosynthesis and accumulation of
pathogenesis-related proteins (PR proteins). Both plants and pathogens possess
certain sets of resistance and avirulence genes that determine the outcome of
the plant–pathogen interaction in what is known as the gene for gene
hypothesis. The induction of PR proteins has been often interpreted as an
attempt by the plant to prevent or limit the spread of the pathogen (Karabi
et al., 1999).
Chitinases are ubiquitous enzymes of bacteria, fungi, animals, and
plants. They hydrolyze the β-1,4-linkage between N-acetylglucosamine
residues of chitin, a structural polysaccharide of the cell wall of many fungi and
of the exoskeleton of invertebrates and results in the production of fungal
elicitors that induce defence responses in plants (Schlumbaum et al., 1986; Leah
et al., 1991; Collinge et al., 1993; Neuhaus, 1999; Velazhahan et al., 2000; Dennis
et al., 2007). Several of these enzymes inhibit fungal growth in vitro, and
expression of their respective genes in transgenic plant can increase protection
against phytopathogenic fungi (Zhu et al., 1994; Lin et al., 1995; Masoud et al.,
1996; Kim et al., 2003). The studies on genetic engineering of plants with
chitinase gene become essential for fungal disease control mechanism. Several
review and research articles have also stressed the advantages of using
chitinase for plant protection because these enzymes are fungicidal, part of the
plant defence system and are not harmful to host or other plants (Collinge et
al., 1993; Moravcikova et al., 2004). Hence, in the last decade efforts have been
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
86
focused on the transgenic expression of chitinase in plants, and significant
importance in the resistance to fungal diseases have been recorded (Asao et al.,
1997; Lorito et al., 1998; Yamamoto et al., 2000; Kim et al., 2003; Kishimoto et al.,
2004; Dennis et al., 2007). Based on the reports, it is observed that the chitinase
gene plays a vital role to control the plant fungal pathogens. In particular, the
class I chitinases, which accumulate to high levels in vacuoles in response to
wounding and pathogen infection, have been reported to be important in these
regards (Maximova et al., 2003). Using transgenic approaches, chitinase genes
from plants and microorganisms have been introduced into different plant
species in order to enhance resistance against a broad range of fungal
pathogens (Kramer and Muthukrishnan, 1997, 2005; Kramer et al., 1993,
Kramer et al., 1997, Kramer and Koga, 1986).
Agrobacterium - mediated transformation via meristem based culture
has been the most common method for transgenic castor development.
Meristem based transformation was already done by Sujatha and Sailaja
(2005). Meristem explants have been successfully transformed in several crops
(Bilang et al., 1993; Saeed et al., 1997; Sticklen and Oraby, 2005). Hence, in this
present investigation we planned to produce the castor plantlets with Cacao
Class I Chitinase gene (Chi I) through Agrobacterium - mediated
transformation protocol by using cotyledonary node culture technique.
6.2 MATERIALS AND METHODS
6.2.1 Source of Chitinase gene
The Class I Chitinase gene was cloned into Amphicillin resistance vector
pGH00.0126. The lyophilized plasmid was gifted by Dr. Sharon Pishak and
Dr. Mark Guiltinan, Department of Horticulture, College of Agricultural
Sciences, The Pennsylvania State University, USA.
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
87
6.2.2 Confirmation of pGH00.0126 carrying T. cacao class I chitinase gene
For the confirmation of the Chitinase gene, the plasmid was
retransformed into E.coli (DH5α) competent cells and plated into Luria Broth
medium with 100 mg/l Amphicillin. The plate was kept in overnight at 37°C
incubator. The single colony was taken and patched into new plate containing
Amphicillin (100 mg/l). A loop of colony was taken from patched plate and
inoculated into 5 ml of LB medium with antibiotic and kept in 37°C at 220 rpm.
The plasmid was isolated from 10 - 12 hours grown culture using alkaline lysis
method and the plasmid was checked by using 0.8% 1X TAE Agarose gel
analysis. Based on the restriction map (Plate 10) the restriction digestion
analysis was done with SmaI, NotI and XbaI.
Restriction setup
Total reaction volume: 30.0µl
DDH20 - 23.0µl
Plasmid - 3.0µl (1.0ug)
10X Buffer - 3.0µl
(10U/µl ) Enzyme - 1.0µl
The restriction was kept at 37°C for NotI, XbaI and 30°C for SmaI in water
bath for 4 hrs and then, the restricted sample was checked with 0.8% 1X TAE
agarose gel.
6.2.3 Cloning of chitinase gene into Binary vector pBinAR
Restriction digestion for chitinase plasmid and pBinAR binary plasmid
with SmaI
Total reaction volume: 50µl
DDH20 – 31.0µl
Plasmid - 4.0µl (2.0ug)
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
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10X Tango yellow Buffer – 4.0µl
(10U/ µl ) SmaI – 1.0µl
The restriction was kept at 30oC in water bath for 2 hrs. For the
confirmation of restriction, 5µl of restricted sample was loaded in 0.8% gel
which gives the linear fragment of approximately 4.2 kb of pGH00.0126
chitinase plasmid and 12.5 kb pBinAR binary plasmid. The sample was purified
and finally eluted from the column using with Qiaquick PCR purification kit
(Qiagen, catalog no. 28104). The eluted plasmid was checked by 0.8% 1X TAE
agarose gel and kept for further restriction with XbaI. (Plate 11)
Restriction with XbaI
Total reaction volume: 50.0µl
Plasmid – 44.0µl
10X Tango yellow Buffer – 5.0µl
XbaI – 1.0µl
The restriction was kept at 37 oC water bath for 2 hrs. After restriction
sample was loaded into 0.8% 1xTAE agarose gel. The completely runned gel
was stain with Ethidium bromide and visualized with low intensity UV
transilluminator and eluted gel at 1.2 kb of chitinase fragment and 12.5 kb
linear fragment of pBinAR binary plasmid using autoclaved blade and the gel
pieces was transferred into 2.0 ml eppendorf tube for purification using with
Qiaquick gel extraction kit (Qiagen, catalog no: 28704)
Finally the sample was eluted with 20.0µl of DDH2O and the
concentration of eluted fragments were analysed by 0.8% 1X TAE agarose gel,
loaded with 1.0µl of each vector and insert.
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
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Ligation
The ligation for insert and vector was done using following 3:1 molar
ratio.
50ng of vector × size of the insert ×3 = ng of insert?
Size of the vector × 1
Ligation setup
Total reaction volume: 10.0µl
DDH2O – 2.0µl
Vector (pBinAR) – 4.0µl (50ng)
Insert (Chitinase) – 2.0µl (15ng)
10X ligation buffer – 1.0µl
T4 DNA ligase – 1.0µl
The ligation sample was kept in 4oC (fridge) for 16 hrs.
6.2.4 Transformation of ligation product
6.2.4.1 E.coli competent cell preparation: Host-DH5α
DH5 alpha E.coli strain was streaked in LA + Nal 10mg/l plate and the
plate was kept at 37oC incubator for overnight. Single colony was taken and
raised in 5 ml LB culture with antibiotic and kept the culture in 37oC shaker for
overnight with 220 rpm. Next day morning, 1.0 ml of overnight culture was
taken added into 100 ml of LB without any antibiotic and the flask was kept at
37oC with 220 rpm until the O.D was reached at 0.6 - 1.0. The flask was kept in
ice for 30 min, and the culture was poured in pre-cooled 50 ml centrifuge tube
in aseptic condition and centrifuged at 5000 rpm for 5 min at 4oC. The
supernatant was discarded and the pellet was resuspended with pre-cooled
100mM CaCl2, and centrifuge was repeated again as mentioned earlier. The
centrifuge was repeated for two more times and finally the supernatant was
discarded and the pellet was dissolved with 1.2 ml of pre-cooled 100mM CaCl2
and 0.8 ml of 100% glycerol. Aliquot (100µl) the in 1.5 ml eppendorf tube (The
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
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tube was kept in ice condition) and the cell was stored into -70oC freezer for
future use.
6.2.5 Transformation: Heat-shock method
10µl of ligation sample was added into 100µl of comp. Cells (cells
thawed before adding sample). The content was gently mixed and kept in ice
for 45 min. The sample was transferred into 42oC Water bath for 90 - 120
seconds, and immediately kept in ice for 10 min after that 1.0 ml of LB was
added and kept in 370C shaker with 220 rpm for 1hr. The 50 - 200µl of culture
was taken and plated into LA+ Kanamycin 50mg/l medium and the plates
were kept in 37oC incubator for overnight. Single colony was observed and that
colony was patched into new plate. For confirmation of clone, the plasmid was
isolated using alkaline lysis method and the clone was confirmed by restriction
digestion analysis (Plate 13) using different restriction enzymes.
Restriction volume: 30.0µl volume
DDH2O – 23.0µl
Plasmid – 3.0µl
10X buffer - 3.0µl
Enzyme – 1.0µl
The restriction sample was kept at 37oC in the Water bath for 4 hrs and
the restriction sample was checked with 0.8% gel (Plate 11, 12 & 13).
6.2.6 Mobilization of Binary plasmid into Agrobacterium strain LBA4404
using freeze-thaw method
Agro comp. cell preparation and Freeze- -Thaw method for mobilization
A. tumefaciens strain LBA4404 was streaked out on a YEB media
containing 10 mg/l Rifampicin and grown at 28°C incubator for 2 - 3 days.
One loop of inoculums was inoculated into 5 ml LB medium containing
10mg/l Rifampicin and grown overnight at 28°C at 220 rpm. The 0.5 ml of
overnight grown culture was reinoculated into 50 ml of LB medium without
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
91
any antibiotic for grown in an incubator-shaker at 28°C at 220 rpm to obtain
cell densities of 0.5 to 1.0 at OD600. The cultures were chilled on ice for 15 min
and the cells were then harvested by centrifugation at 5000 rpm for 5 min at
4°C. In aseptic condition the supernatant was discarded and the pellet was
resuspended with 2.0 to 5.0 ml of precooled 20mM CaCl2 and centrifuged at
5000 rpm for minutes. The centrifuge was repeated one more time. Finally the
pellet was resuspended with 1.0ml precooled 20mM CaCl2 and 0.5 ml of
glycerol (100%) and aliquots the 50µl cells into eppendorf tube (eppendorf in
ice box) and stored it in -70°C freezer for future use.
Competent cells were kept in ice for thawing and then add 500 to 1000
ng of plasmid into comp. cells in laminar condition, then mixed gently and
kept it in ice for 5.0 min. The eppendorf was put into liquid nitrogen for 2.0 –
4.0 min, then immediately transferred into 37°C water bath. The eppendorf
was kept in ice for 5.0 min and then 1.0 ml of LB was added and kept in 28°C
shaker for 4.0 – 6.0 hrs. The eppendorf containing mobilized plasmid was
centrifuged and the pellet was resuspended in 200 ml LB and plated into LA
agar plate with 50mg/l of kanamycin and 10mg/l of Rifampicin antibiotic. The
plate was kept in 28°C incubators for 2 - 4 days; observed the single colony and
restreaked into new LA agar plate with same antibiotics. Finally the
Agrobacterium harbouring the binary plasmid was confirmed through colony
PCR with nptII and gene specific primer (chitinase) (Plate 14).
(For competent cell preparation and transformation was done in only laminar
condition and all things should properly autoclaved)
6.2.7 Binary plasmid and Agrobacterium strain
The 13.7 kb pBinAR binary plasmid is a pBin19 derivative containing
expression cassette for constitutive expression of chimeric genes in plants
(Hofgen & Willmitzer, 1990) The T-DNA portion of pBinAR having nos-npt II
cassette in RB and 770bp EcoR I/Hind III fragment containing the CaMV 35S
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
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promoter, a partial pUC18 polylinker and the OCS terminator in LB. A 1.2 kb
SmaI - XbaI fragment of T. cacao class I chitinase was insert out from pGH00.0126
vector and cloned into corresponding sites of binary vector pBinAR. The total
size of the pBinAR binary vector carrying class I chitinase is 13.7 kb. For
Agrobacterium mediated transformation in plants the binary plasmid having
class I chitinase were mobilized into A. tumefaciens strain LBA4404. The
mobilized Agrobacterium strain was confirmed and taken for gene
transformation studies in plants. (Plate 11 & 12)
6.2.8 Plant materials, transformation, selection and regeneration
For standardization of gene transfer studies Agrobacterium strain
LBA4404 harbouring the binary plasmid pBAL2 (hptII and nptII as a plant
selection marker and GUS as a reporter gene) were used. Further
transformation was done with Agrobacterium strain LBA4404 harbouring binary
plasmid pBinAR - Chitinase. Agrobacterium mediated gene transfer sequential
process as discussed earlier (see Chapter 5).
Cotyledonary node explants were excised from 10 days old in vivo
seedlings, surface sterilized and planted on the precultue medium for 4 days in
the culture room at 25 ± 2ºC, 16/ 8 h (light / dark) photoperiod. A total of one
fifty explants were inoculated. Agrobacterium strain LBA4404 harbouring
pBinAR - Chitinase culture were initiated by inoculating a loop of colony into 10
ml of YEB liquid medium containing 50mg/l Kanamycin and 10mg/l
Rifampicin at 28C for 24 h. The bacterial cultures (OD600 = 1.0) were pelleted at
5000 rpm, resuspended in 50 ml of mMS basal medium containing the
hormones for multiplication of shoots for co - cultivation. The explants were
co - cultivated on the Agrobacterium suspension along with acetosyringone 100
mg/l and incubated for two days in the culture room at 25 ± 2ºC, 16/ 8 h (light
/ dark) photoperiod. After 2 days of co - cultivation, the explants were
thoroughly washed with 250 mg/l of cefotaxime in sterile water for a period of
five minutes and incubated for ten days on the mMS medium containing
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0.3 mg/l TDZ, 0.6 mg/l of PF – 68, 250 mg/l cefotaxime, and 50 mg/l
Kanamycin with 0.8% agar (selection I medium). After 10 days of culture, the
proliferated regions of the explants were excised and transferred to the MS
medium containing 0.3 mg/l TDZ, 0.6 mg/l PF – 68, 50 mg/l Kanamycin with
0.8% agar (Selection II medium), and were cultured for ten weeks. The
surviving green or creamy white regions were transferred to the regeneration
medium containing mMS + 0.3 mg/l TDZ, 0.6 mg/l PF – 68, 50 mg/l
Kanamycin with 0.8% agar (Selection III medium) for proliferation of shoots.
Regeneration of shoots was observed after 10 days; the regenerated shoots
were transferred to the mMS medium containing 0.6 mg/l PF 68 and 0.3 mg/l
Gibberellic acid (GA3) for shoot elongation. Elongated shoots were rooted on
the half - strength mMS medium containing 1.5 mg/l IBA, 0.6 mg/l AgNO3
and 0.8% agar. The rooted plants were initially transferred to sand, soil and
vermiculite mixture (1:1:1) in green house and were later established in field
(Plate 15).
6.2.9 Molecular confirmation of transformants
6.2.9.1 DNA isolation and quantification
The genomic DNA was isolated from transformed and untransformed
plant sample using CTAB method (Dolye and Dolye, 1990). The detailed
procedure is given in the materials and methods of chapter 5.
6.2.9.2 Polymerase chain reaction
Polymerase chain reaction (PCR) was performed to confirmation of
transgene in putative transgenic plants using npt II and gene specific primer chi
I. The primer sequences for npt II (F) 5’ – GCTTGGGTGGAGAGGGCTATT –
3’, (R) 5’ – AGAACTCGTCAAGAAGGCGA – 3’ to yield 750 bp PCR amplified
fragment, and for Chi I (F) 5’-GGAAAATGGTTGCCAGAGTCAGTGC–3’ (R)
5’– GCTACATTGAGTCCACCGAGGGTC – 3’ to yield a 1.0 kb PCR amplified
fragment. The final concentration of PCR components was prepared as 1X Taq
buffer with 1.5mM MgCl2, 200µM of each nucleotide, 0.4 µM of each primer,
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
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1.0 units of Taq DNA polymerase and 100ng of template in 25.0 µl of total
reaction volume. The PCR sample was kept in Gene Amp thermal cycler
(Eppendorf, Germany). and set the cycling temperature as initial denaturation
at 94ºC for 5.0 min followed by 30 cycles of denaturation at 94ºC for 1.0 min;
annealing at 58ºC for 1.0 min; and extension at 72ºC for 1.0 min. The final
extension for 10 min at 72ºC and the reaction was hold at 4 ºC for short term
storage of the reaction (Plate 16).
6.2.9.3 Southern Hybridization
For southern hybridization 10.0 µg of genomic DNA was extracted from
transformed and untransformed plant sample and digested with HindIII then
electrophoresed on a 0.8 % agarose gel. DNA fragments were transferred to
nylon membrane (Hybond-N+, Boehrigher, Laval, Quebec, Canada) and
hybridized with a digoxigenin-labeled chi I fragment probe described by
Tingay et al. (1997) to detect the integration of chi I gene (Plate 16).
6.2.10 In vivo evaluation of antifungal activity of transgenic castor plants
against Fusarium oxysporum
The pure culture of Fusarium oxysporum was obtained from the National
Chemical Laboratory, Pune. A suspension of spore (2 x 105 spores / ml sterile
water) was prepared from potato Dextrose agar medium. Four weeks old well
acclimatized PCR positive plants maintained in the green house were sprayed
with this suspension. The pots were covered with polyethylene bag for 2 days
to create proper conditions for infection and to prevent the spore dispersal. The
control (non-transgenic) plants were also treated in the same manner (Ganesan
et al., 2006). The resistance of the T0 generation was monitored for several
weeks from the date when the plants were sprayed with the fungal spores.
Plants that appeared resistant as well as those that developed disease
symptoms were grown intermixed after 15 days following infection and
sprayed with spores for a second time. The observation continued for a month.
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6.3 RESULTS AND DISCUSSION
6.3.1 Sensitivity of selection antibiotics
Based on the standardize regeneration protocol, the antibiotic sensitivity
assay was performed in control explants using Kanamycin antibiotic to
investigate the sensitivity of cotyledonary node of R.communis. In addition,
influence of kanamycin concentration (50 mg/l) on shoot induction frequency
was recorded. Several workers used kanamycin as a plant selection agents for
successful selection of putative transgenic plantlets (Plate 15).
6.3.2 Plant materials, transformation, selection and regeneration
Four day pre-cultured mature cotyledon explants were used for
pBinAR - Chitinase gene transfer study. During transformation, pre-culture of
explants, shoot multiplication and regeneration medium prior to co-cultivation
was considered as one of the essential process. Pre - culture of explants was
done for improving the induction of the vir genes activity (Stachel et al., 1985;
Fillatti et al., 1987; Veluthambi et al., 1989). This work was already discussed in
detail in chapter 5. The first round of screening for putative transgenic castor
kept in a short incubation on mMS medium supplemented with TDZ
(0.3 mg/l), PF - 68 (0.6 mg/l), cefotaxime (250 mg/l) and kanamycin (50 mg/l).
In further selection, shoot buds regeneration was observed in the culture
medium from the explants after ten days of culture (Plate 15). Then the shoots
were transferred to elongation and rooting medium having Plant growth
regulators with 50 mg/l kanamycin (Plate 15; Table 6.1). The well rooted plants
were established in pots and carryout molecular confirmation. In each
experiment, nearly 150 explants were used and the experiment was repeated
10 times (Table 6.2) along with controls. Out of 1403 explants, 437 kanamycin
resistance putative transformed plantlets were recovered and the
transformation efficiency of 1.06% was recorded (Table 6.3).
The efficiency and density of Agrobacterium culture were studied using
OD600 = 1.0 when the infection and time period for cocultivation of the explants.
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
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The OD600 = 1.0 was found to be a compromise between cell vitality and
Chitinase expression. The same concentration has been often used in
transformation experiments of castor (Sujatha and Sailaja, 2005) and Sunflower
(Laparra, et al., 1995; Lucas et al., 2000; Mul ler et al., 2001) and other plant
species (Bond and Roaser, 1998).
6.3.3 Molecular confirmation of transformants
6.3.3.1 Polymerase chain reaction analysis
Polymerase chain reaction (PCR) analysis was performed to the presence
of npt II and chi I gene in the kanamycin resistance plants. The expected 750bp
and 1.0 kb amplified fragments corresponding to npt II and chi I gene was
observed (Plate 16), no amplification was detected in the DNA sample from
non-transformed plants. The amplified product of 750bp and 1.0kb was
confirmed the presence of the npt II and chitinase gene in the putative
transgenic plants compare to untransformed plant. No abnormal phenotypes
were observed during the growth of transgenic plants (Plate 16). In each
experiment 2 – 3 plants were obtained when analysing PCR with npt II and
chitinase primers. The transformation efficiency was 1.06% (Table 6.3). The
efficiency was calculated on the basis of PCR positive plants. The observation
indicated the presence of chi I gene in the transgenic plants (Plate 16). The PCR
confirmation was based on previous report on gene transformation in castor
(Sujatha and Sailaja 2005; Malathi et al., 2006) and the Cacao chitinase gene
transformation protocol done by Maximova et al. (2003) and (2006).
6.3.3.2 Southern blot analysis
The integration of transgene copy number and junction fragment
analysis were determined by genomic southern blots. Southern blots analysis
was done using 10µg of transformed and untransformed plant DNA sample
which was digested with HindIII along with E.coli binary plasmid as a positive
control and eletrophoresed on a 0.8% agarose gel then gel was transferred to a
nylon membrane (Hybond-N+, Boehringer, Lavel, Quebec, Canada). The 1.2 kb
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
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chitinase gene was used as a probe labelled with digoxignin and hybridization
was performed as described by Maximova et al. (2006). Out of 15 plants
analysed, 4 plants carried the chi I gene, here chi I probe provide the evidence
for the presence of integrated T-DNA (Plate 16).
6.3.4 Bio-assay for disease resistance in transformed plantlets
Analysis for Fusarium wilt resistance
Plants evoke a series of general defence reaction, including the
production of phytoalexins and anitimicrobial proteins, upon sensing
invading microbes and microbial substances (Lawrence et al., 1996). The wilt
fungus colonizes the root surface and then hyphae penetrate the root cortex,
the endodermis and the xylem. Systemic invasion of the plant results when
successive generations of conidia are produced and then transported by the
xylem’s transpiration stream to the aerial parts of the plant (Garas et al.,
1986; Hill et al., 1999; Daayf, 1997). Binding et al. (1970) was the pioneer to
demonstrate the possibility of in vitro selection of callus cultures for
desirable traits. Many attempts have been made since then to apply such
selection schemes for crop improvement, including resistance to Fusarium
wilt (Arai and Takeuchi, 1993; Scala et al., 1998; Mosquera et al., 1999).
Plant chitinases have been shown to inhibit fungal growth in vitro by
degrading chitin polymers in fungal cell walls (Hüttermann and Cwielong,
1982; Nielsen et al., 1994; Susi, 1997; Susi et al., 1995; Toyoda et al., 1991;
Schlumbaum et al., 1986; Mauch et al., 1988; Sela-Buurlage et al., 1993;
Collinge et al., 1993; Schickler and Chet, 1997; Lorito et al., 1998). Hence, in the
last decade efforts have been focused on the transgenic expression of plant
fungal chitinases in plants, and significant improvements in the resistance to
fungal diseases have been recorded (Asao et al., 1997; Broglie et al., 1991; Lorito
et al., 1998; Tabei et al., 1998).
Genetic Transformation Studies using Theobroma cacao Chitinase Gene
98
In the present investigation, 15 plantlets were selected for Fusarium
wilt testing. During disease resistance analysis, the survival percentage of
non-transformed plantlets was significantly affected by the inoculation of
Fusarium macrospores (2 x 105 spores/ml) during hardening (Table 6.4; Plate
15). Among the 15 transformed plants tested, 10 plants showed healthy
regeneration in fungal spore inoculated soil. These plants were finally
selected as Fusarium wilt tolerant plants (Table 6.4). In support of the above
results the vascular browning was completely absent in the transformed
plants compared to control plant (Plate 15). All the selected disease tolerant
plants showed equal regeneration potential when compared with controls.
In accordance with our results the transgenic Theobroma cacao plants over
expressed with Cacao Chitinase was reproducibly showed enhanced resistance
against the fungus Colletrotrichum gloeosporioides was reported by Maximova
et al. (2006).
6.4 CONCLUSION
This study is the first successful attempt to develop a stable
transformation system for castor via A. tumefaciens LBA4404 strain harbouring
pBinAR Cacao Chitinase mediated transfer using CN from 10th day in vivo
seedlings. Advances in regeneration and transformation protocols have led
to the successful development of transgenic castor with improved
agronomic characters. In our studies, we obtained the significant
improvement in the plant regeneration percentage of transformed plants
and recovery of transgenic plants resistant to Fusarium oxysporum.
Limitations associated with tissue culture protocol make the present protocol
more suitable for rapid development of transgenics in recalcitrant system-like
castor and the method will be useful for genetic engineering of castor for
various agronomical traits including fungal resistance.
Table 6.1
Effect of selection marker sensitivity on growth and multiple shoot induction with growth regulator of cotyledonary node explants
Kanamycin (mg/l)
Percentage of response
Mean number of shoot / explant
00 25 50 75
100 125 150
34.3 21.2 9.4 3.6 - - -
11.3 6.5 4.1 1.2 - - -
Table 6.2
Character gene transfer with Agrobacterium strain pBinAR Chi I and Kanamycin (Selection Marker) on their response on cotyledonary node explants
Experiments No. of explants
Infected (A)
No. of KanR
shoots (B)
Percentage of response (%)
(B/A)
No. of shoots PCR positive
(C)
Transformation efficeiency (%)
(C/A)
1 2 3 4 5 6 7 8 9
10
122 132 150 145 142 150 150 141 138 133
39 42 46 48 47 53 45 49 36 32
31.9 31.8 30.6 33.1 33.0 35.3 30.0 34.7 26.0 24.0
2 0 2 2 1 3 2 2 1 0
1.6 0.0 1.3 1.3 0.7 2.0 1.3 1.4 0.7 0.0
Table 6.3
Summary of character Agrobacterium strain with pBin AR Chi I and Kanamycin (Selection Marker) on their response on cotyledonary node explants
No. of explants Infected
(A)
No. of KanR
shoots (B)
Percentage of response
(B/A)
No. of shoots PCR positive
(C)
Transformationefficeiency
(C/A)
1403 437 31.1 15
1.06
Table 6.4
Survival rate of PCR positive plantlets and control plants on earthen pots inoculated with 2 × 105 spores/ml of Fusarium oxysporum.
Survival rate of regenerants No. of days after inoculation of
spores C C (i) PCR Positive
Plantlets
4 8
12 16 20 24 30
15 15 15 15 14 14 13
11 9 5 2 0 0 0
15 10 10 10 10 10 10
Plate 15 Genetic transformation of Ricinus communis L. using Chi I gene harboured in
pBinAR vector and bioassay with Fusarium oxysporum
a. Preculture of explants (1.0 x) b & c. Shoot bud initiation from cotyledonary node explants in selection media
(1.0 x) d. Shoot bud proliferation (1.0 x) e. Rooting of elongated shoots (1.0 x) f. Hardened plants g & h. Control plants affected by Fusarium (C) i & j. Transverse cut of Fusarium infected control stem and transformed stem k & l. Transformed healthy plants in earthern pots.
Plate 16
a. PCR analysis of putatively transformed plants using npt II
L – Ladder W - Water Control N – Untransformed plant DNA (negative control) L1 to L3 – Transformed plant DNA L4 – Empty well P – Plasmid DNA (positive control)
b. PCR analysis of putatively transformed plants using Chi I L – Ladder W - Water Control N – Untransformed plant DNA (negative control) L1 to L3 – Transformed plant DNA P – Plasmid DNA (positive control)
c. Southern hybridization of putatively transformed plants P – Plasmid DNA (positive control) L1 to L4 – Transformed plant DNA N – Untransformed plant DNA (negative control)