For Southern analysis [22], DNA 10 mg was digested with the appropriate endonuclease, elec-
trophoresed on a 0.8 agarose gel, and blotted on a nylon membrane Hybond, Amersham. Dot
blot was performed with uncut DNA 5 mg. The uid A gene probe was prepared from pUCGUS121
from E. coli strain XL-1 Blue, by releasing a 2.1-kb uid A fragment using Bam H1 and Eco R1.
This fragment was labeled using the random prim- ing kit supplied by Amersham. Hybridization sig-
nals were detected following exposure of X-ray film to the membrane for 16 h at − 70°C.
3. Results
3
.
1
. Optimization of culture conditions and de6elopment of transformation protocol
The feasibility of the transformation strategy adopted in the study was initially evaluated by the
number of peanut embryo axes germinating into normal seedlings following wounding by excision
of one cotyledon and by pricking with a needle, infection with Agrobacterium and decontamination
treatment.
Preliminary experiments to develop transforma- tion protocol involved optimization of infection
time and 6ir gene induction treatments to enhance the transformation efficiency. Fig. 1 shows the
maximum number of embryos expressing GUS when infection was carried out for 16 and 24 h.
Only two to four embryos per ten sampled have expressed GUS when infected for two and four
hours. GUS expression increased with increased time of infection. It attained a plateau when the
infection was for 16 h. Though the blue sectors appeared were more with 24 h infection, a 16 h
period was chosen for further transformation ex- periments because of a reduction in the germina-
tion rate observed on infection for 24 h or longer. As the infection treatment given was for an ex-
tended period, and that any further exposure of explants to bacteria appeared deleterious, a dis-
crete co-cultivation step that generally follows in- fection was not included in the protocol.
The results of different 6ir gene induction treat- ments are shown in Figs. 2 and 3. Infection of
peanut with Agrobacterium previously treated with acetosyringone did not improve transformation
efficiency Fig. 2. Nevertheless, wounded tobacco leaf extract added to the AB induction medium
enhanced the transformation efficiency in terms of the number of embryos expressing GUS. Fig. 3
shows the amount of tobacco leaf extract required to obtain optimal infection conditions.
Fig. 1. Effect of infection time on the transient GUS expres- sion in peanut cultivar TMV-2.
Fig. 2. Effect of varying concentrations of acetosyringone on the transformation efficiency in peanut cultivar TMV-2.
Fig. 3. Effect of varying quantities of tobacco leaf extract on transformation efficiency in peanut cultivar TMV-2.
Fig. 4. GUS expression in the tissues of peanut cultivar TMV-2 explanted at various stages of recovery of transgenic plants. a Embryo axis and the plumule, five days after infection bar = 0.5 mm; b embryo axis of peanut cultivar JL-24 bar = 4.5 cm;
c leaflet from a month old plant bar = 3.5 cm; d embryos with one cotyledon removed T , primary transformant; T
1
, progeny bar = 4.0 cm.
Fig. 5. Greenhouse established peanut transformants in peanut cultivar TMV-2. a A fertile plant of T generation; b plants of
T
1
generation.
Fig. 6. Expression of neomycin phosphotransferase II gene. a Expression in T
plants. Lane 1: total protein extract from uninfected plant negative control. Lane 2: total protein
extract from a transformant. b Expression in T
1
plants. Lane 1: total protein extract from uninfected plant. Lanes 2
and 3: total protein extract from the progeny of transfor- mants.
wounded uninfected as well as infected embryos were germinated in the presence of kanamycin.
Therefore, selection on kanamycin was eliminated. Thirty percent of the seeds survived wounding,
infection and germinated into healthy plants with 16 h of infection. The seedlings transferred to pots
were initially covered with polythene bags while they were grown in the culture incubation room
and before they were shifted to the greenhouse. Six primary transformants resulted from three inde-
pendent transformation events involving a total of 150 embryos. Five plants, which survived harden-
ing and transfer to greenhouse were analyzed for the presence and transmission of the transgenes.
These plants have put forth healthy, green and expanded leaves Fig. 5a. The rate of growth of
these infected seedlings was however slow when compared to uninfected seedlings, which might be
due to wounding and prolonged infection with Agrobacterium.
3
.
2
. Expression of the npt II gene The NPT II assay performed, resulted in a
signal at the expected position of 14 kDa with the total protein extracts of the primary transformant
and the T
1
progeny indicating co-integration, ex- pression and transmission of npt II gene Fig. 6a
and b. There was no signal seen in case of unin- fected peanut plants.
3
.
3
. Expression and inheritance of the uid A gene The susceptibility response of embryo axes of
peanut to Agrobacterium infection that was deter- mined initially by scoring the transient GUS activ-
ity five days after infection, resulted in blue color development throughout the embryonal axes Fig.
4a. The advantage of eliminating the background GUS activity resulting from bacterial presence in
the tissue was conferred by the binary vector pKIWI105. The Agrobacterium LBA 4404 was
found to be the most effective strain on peanut cultivars tested. Deep blue sectors were seen occu-
pying most part of the leaflet area Fig. 4c. GUS activity was expressed in various organs and tis-
sues of the T
, T
1
Fig. 4d, and T
2
plants. None of the control plants expressed GUS.
The expression of uid A gene assayed in the greenhouse-established plants by Western blotting
has shown a protein band at the expected position
Fig. 7. Western blot analysis of b – glucuronidase in T plants.
Lane 1: purified GUS protein 20 mg Clonetech positive control. Lane 2: total protein extract 50 mg from a transfor-
mant. Lane 3: total protein extract 50 mg from an uninfected plant.
Winans’ medium 100 ml added with wounded tobacco leaf extract 2 g in 2 ml sterile water and
a 16 h infection time were used for all the subse- quent transformation experiments. Embryos of
peanut cv. TMV-2 when infected in the absence of acetosyringone or tobacco leaf extract did not
show GUS expression.
In the experiments performed initially to deter- mine the tolerance to kanamycin, it was observed
that uninfected embryos control did not germi- nate beyond 150 mg ml
− 1
of kanamycin. Further, there was a reduction in germination rate when
Fig. 8. PCR of peanut cultivar TMV-2, T plants using a 21
mer primer, which amplifies a 514-bp uid A gene fragment. Lane 1: pKIWI105 DNA positive control. Lane 2: DNA
from the leaves of uninfected plant. Lanes 3 – 7: DNA from the leaves of T
plants.
of 74 kDa in the total protein profile Fig. 7. This band was not detected by the antibody in the total
proteins of uninfected plants.
3
.
4
. Molecular analysis of the T plants
In PCR analysis using primers for the uid A gene, DNA fragments of expected size of 514 bp in length
were amplified from the total DNA of the putative transgenic plants Fig. 8. These DNA fragments
were not detected in the DNA of uninfected plants. Dot blot analysis of DNA samples of the five PCR
positive plants further confirmed the presence of the uid A gene in the primary transformants Fig. 9.
Southern blot of uncut DNA of one of these plants gave a hybridization signal with a 2.1 kb uid
A gene probe. This DNA when digested with Sma I gave a signal at the 10 kb position Fig. 11, lanes
1 and 7. A hybridization signal was not obtained with the DNA from the non-transformed plant
Fig. 11, lanes 6 and 10. The protocol facilitated recovery of transformants in as short a time as 4
weeks.
3
.
5
. Molecular analysis of the T
1
and T
2
plants Seeds of T
plants Table 1 were germinated in the greenhouse Fig. 5b and DNA was prepared
from leaves of all the plants that germinated. PCR analysis of 52 DNA samples showed the presence
of the uid A gene in 36 samples Fig. 10. These 36 DNA samples gave a hybridization signal in the
Fig. 9. DNA 5 mg from the five PCR positive T plants was
loaded on a dot blot apparatus and probed with a 2.1-kb uid A gene fragment. Lanes 1 – 5: DNA from the five PCR
positive T plants. Lane 6: positive control pKIWI105
DNA. Lane 7: uninfected plant DNA negative control.
Fig. 10. PCR to show the inheritance of uid A transgene in the T
1
generation. Lane 1: amplified product size of a 514-bp uid A gene fragment in pKIWI105 DNA. Lanes 2 – 53: DNA from leaves of 52 T
1
generation plants.
Fig. 11. Southern blot of T and T
1
plants. DNA 10 mg from the T
and T
1
plants was digested with Sma I and probed with a 2.1-kb uid A gene fragment. Lanes 1 and 7: uncut and
Sma I digested DNA of a primary transformant T respec-
tively. Lanes 2 – 5: uncut DNA of T
1
plants. Lanes 6 and 10: uncut and digested DNA of uninfected plant negative con-
trol. Lanes 8, 9, 11 and 12: DNA of T
1
plants digested with Sma I.
Fig. 12. Southern blot of T
2
generation plants dot blot. Uncut genomic DNA 5 mg was loaded on a dot blot
apparatus and probed with a 2.1-kb uid A gene fragment. Lanes 1 – 32: DNA of T
2
plants.
Abnormal ratio was observed for the T −
1 plant, which shows its chimeric nature. These data confi-
rmed the inheritance and integration of the uid A gene in both T
1
and T
2
generations. To demonstrate that the method is genotype-in-
dependent, the embryos of a few other Indian peanut cultivars viz., JL-24, ICGS-44, ICGV-
86564 were similarly infected with LBA 4404 pKIWI105 and were allowed to germinate. GUS
histochemical assay performed gave intense blue coloration throughout the embryo axes in all these
cultivars Fig. 4b indicating that the method can be applied to other cultivars which are susceptible
to A. tumefaciens infection.
4. Discussion
