Plant Science 150 2000 41 – 49
Transformation of peanut Arachis hypogaea L.: a non-tissue culture based approach for generating transgenic plants
V.K. Rohini, K. Sankara Rao
Department of Biochemistry, Indian Institute of Science, Bangalore
560012
, India Received 20 April 1999; received in revised form 30 June 1999; accepted 3 August 1999
Abstract
Transgenic peanut Arachis hypogaea L. cv. TMV-2 plants have been produced by a tissue culture-independent Agrobacterium tumefaciens-mediated transformation procedure. Embryo axes of mature seeds with one cotyledon excised were incubated on
Murashige and Skoog MS gelled medium for 2 days, then pricked randomly with a sterile needle and infected by immersion in a suspension of Agrobacterium in Winans’ AB medium that was added with wounded tobacco leaf extract. Agrobacterium strain
LBA 4404 harboring the binary vector pKIWI105 that carries the genes for b-glucuronidase GUS and neomycin phosphotrans- ferase NPT II was used for transformation. Following a 16 h infection, 18 h decontamination with cefotaxime and germination
and growth on soilrite for 16 days under growth room conditions, the seedlings were transferred to greenhouse. About 3.3 of the seedlings were GUS positive as determined by histochemical assay and by PCR analysis. Molecular characterization of
primary transformants as well as the T
1
and T
2
generation plants has shown that the method ensured insertion, expression and inheritance of foreign genes in peanut. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords
:
Arachis hypogaea L.; Genetic transformation; Tissue culture-independent method; Agrobacterium tumefaciens; b-Glucuronidase; Neomycin phosphotransferase
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1. Introduction
The peanut Arachis hypogaea L. improvement programs aimed at the development of varieties
resistant to diseases such as tikka caused by Cer- cospora arachidicola and Cercosporidium person-
atum and rust caused by Puccinia arachidis. Modification of peanut genome using genetic engi-
neering methods would facilitate rapid develop- ment of new varieties with traits that confer
disease resistance. Majority of legume transforma- tion studies has favored the use of Agrobacterium
tumefaciens to generate transgenic plants. How- ever, most strategies utilizing Agrobacterium-medi-
ated gene delivery have been performed under in vitro culture conditions and require identification
of tissues competent for transformation and devel- opment of tissue culture systems that efficiently
convert these tissues into plants. Recently there has been a great deal of interest in the transforma-
tion [1 – 8] and in vitro regeneration [9 – 11,6,12] of peanut because it represents one of world’s most
important legume crops and therefore is a target crop for improvement. Nevertheless, generalities
for peanut regeneration response patterns are difficult to formulate. The different genotypes and
or explant materials may be contributory to the divergent results reported on regeneration re-
sponse. Another problem with current peanut re- generation techniques has been the 4 – 5 month
period required to obtain plants from callus. Transformation procedures that minimize or elim-
inate the tissue culture component would therefore be advantageous under such circumstances. The
goal of the work presented here was to develop a method of transformation for peanut that is quick,
cultivar- and tissue culture-independent.
Corresponding author. Fax: + 91-80-33418143341683. E-mail address
:
baradwajbiochem.iisc.ernet.in K.S. Rao 0168-945200 - see front matter © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
PII: S 0 1 6 8 - 9 4 5 2 9 9 0 0 1 6 0 - 0
2. Materials and methods
2
.
1
. Plant material Seeds of peanut cultivar TMV-2 were soaked
overnight and later surface sterilized with 0.1 mercuric chloride for 5 – 7 min, followed by thor-
ough rinses with sterile water. Embryos with one of the cotyledons cut off at the site of attachment
to the primary axis were incubated on semisolid Murashige and Skoog MS [13] basal medium for
two days prior to infection.
2
.
2
. Bacterial strains and 6ectors The Agrobacterium strain LBA 4404 harboring
the binary vector pKIWI105 [14] was used for transformation. The plasmid carries genes for b-
glucuronidase uid A and neomycin phospho- transferase npt II driven by CaMV 35S and
nopaline synthase promoters, respectively. The uid A gene lacks a functional bacterial ribosome bind-
ing site that prevents its expression in the bacteria. The Escherichia coli strain XL-1 Blue harboring
the plasmid pUCGUS121 was used for probe preparation. This plasmid has the uid A gene
under the control of CaMV 35S promoter and nos terminator.
2
.
3
. Infection with Agrobacterium and reco6ery of transformants
The Agrobacterium strain LBA 4404pKIWI105 was grown overnight at 29 – 30°C in LB medium
pH − 7.0 containing 50 mg ml
− 1
kanamycin. The bacterial cells were later resuspended in
Winans’ AB medium pH − 5.2 [15] and grown for 18 h. Wounded tobacco leaf extract was later
added to this suspension. The embryo axes were pricked randomly with a sterile sewing needle and
dunked in the suspension of Agrobacterium in Winans’ AB medium. The infection was carried
out by gentle agitation at 28 – 30°C. The seedlings were blot-dried, washed thoroughly with 500 mg
ml
− 1
of cefotaxime for 18 h and placed on autoclaved soilrite for germination under aseptic
conditions in capped bottles. After 5 – 6 days, the germlings were transferred to soilrite in pots and
the seedlings were allowed to grow under growth room conditions for at least 10 days before they
were transferred to the greenhouse. The pots were initially covered with polythene bags to maintain
humidity. The growth chamber was maintained at 26 – 28°C under a 14 h photoperiod with fluores-
cent light of intensity 35 mmol m
− 2
s
− 1
. For each experiment 50 embryos were infected and the ex-
periments were repeated thrice. Various transformation conditions viz., infec-
tion time, effect of acetosyringone and addition of wounded tobacco leaf extracts on transformation
efficiency were evaluated.
2
.
4
. Expression of marker genes GUS enzyme activity was assessed in the tissues
sampled from different phases of development of putative transformants following the method of
Jefferson [16]. Expression of GUS was also ascertained by
Western blotting. Total proteins were extracted from about 1g of leaf tissue of two months old
transformants and resolved on a 10 polyacry- lamide gel following the protocol of Laemmli, [17].
Immunostaining of the immobilized proteins was performed
using the
GUS antibody
from Clonetech following the GUS gene fusion system
user manual Clonetech. The expression of the npt II gene in the genome of the transformed peanut
plants was checked by the NPT II nd-PAGE assay performed according to Reiss et al., [18].
2
.
5
. Molecular analysis Plant genomic DNA was isolated following the
method of Dellaporta et al. [19]. Plasmid DNA from A. tumefaciens strain LBA 4404pKIWI105
and E. coli strain XL-1 BluepUCGUS121 was prepared following the method of Sambrook et al.
[20].
For PCR analysis of the uid A gene in the genome of transformants, a 21 mer primer [21] —
5 CTG TAG AAA CCC GTG 3 and 5 CAT TAC GCT GCG ATG GAT CCC 3 that am-
plifies a 514 bp fragment was employed. The PCR was performed in a total volume of 50 ml contain-
ing 200 ng template DNA, 4 ml milliQ water, 5 ml 10X buffer containing 15 mM MgCl
2
. 150 mM dNTPs, 5 ng of the primer set and 1 U of Taq
polymerase Bangalore Genei, Bangalore. The re- action was initiated by a hot start at 94°C for 4
min. The PCR cycles were, 1 min at 94°C, 2 min at 55°C and 2 min at 72°C for 32 cycles. The PCR
products were later analyzed on a 1 agarose gel.
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
