Plant Cell Rep (2003) 21:1072–1079
DOI 10.1007/s00299-003-0620-y
GENETIC TRANSFORMATION AND HYBRIDIZATION
S. Dayal · M. Lavanya · P. Devi · K. K. Sharma
An efficient protocol for shoot regeneration and genetic transformation
of pigeonpea [
Cajanus cajan
(L.) Millsp.] using leaf explants
Received: 11 October 2002 / Revised: 18 February 2003 / Accepted: 19 February 2003 / Published online: 7 May 2003
Springer-Verlag 2003
Abstract A protocol for efficient plant regeneration from
leaf explants of pigeonpea [Cajanus cajan (L.) Millsp.]
was developed for the production of transgenic plants.
Leaf explants from 4- to 5-day-old in vitro raised
seedlings were most efficient in producing multiple
adventitious shoots in 90% of the explants on shoot
induction medium [Murashige and Skoog (MS) medium
+5.0 mM benzyladenine +5.0 mM kinetin]. Shoot buds
originated from the petiolar cut end of the explants and
elongated rapidly on medium containing 0.58 mM gib-
berellic acid. Over 80% of the elongated shoots rooted
well on MS medium containing 11.42 mM indole-3-acetic
acid and were transplanted with 100% success. The
procedure reported here is very simple, efficient and
reproducible, and is applicable across diverse genotypes
of pigeonpea. The usefulness of this system for further
studies on the genetic transformation of pigeonpea has
been demonstrated in biolistics-mediated gene transfer by
using nptII and uidA as marker genes, where 50% of the
selected plants showed gene integration and expression.
Keywords Biolistics · Cajanus cajan (L.) ·
Organogenesis · Pigeonpea · Transformation
Abbreviations BA: N
6
-Benzyladenine · GA
3
: Gibberellic
acid · IAA: Indole-3-acetic acid · IBA: Indole-3-butyric
acid · 2-iP: N
6
-[2-Isopentenyl]adenine · MS: Murashige
and Skoog medium · RIM: Root induction medium ·
RT-PCR: Reverse transcriptase polymerase chain
reaction · SEM: Shoot elongation medium · SIM: Shoot
induction medium
Introduction
Pigeonpea [Cajanus cajan (L.) Millsp.], one of the major
grain legumes of the semi-arid tropics (SAT), is cultivated
on over 3.4 million hectares world-wide with an annual
production of 2.7 million tons and an average yield of
790 kg ha
1
(Nene and Sheila 1990). Because of its high
protein content, pigeonpea forms a significant component
of the diet of vegetarians in the SAT. However, its
production and productivity are constrained by several
diseases, including sterility mosaic, Fusarium wilt, Phy-
tophthora blight, Alternaria blight and stem canker
(Reddy et al. 1990), and by Helicoverpa armigera (the
legume pod borer), which is a major insect pest of
pigeonpea.
Biotechnological approaches such as gene transfer for
enhanced disease and pest resistance offer opportunities
for rapid improvement of pigeonpea. However, the
availability of an in vitro regeneration system is a pre-
requisite for effective genetic transformation. The regen-
eration of shoot buds from different seedling explants of
pigeonpea has been reported previously (George and
Eapen 1994; Shiva Prakash et al. 1994; Naidu et al. 1995;
Geetha et al. 1998; Mohan and Krishnamurthy 1998). In
these reported regeneration systems, the time required for
the formation of shoot buds and their complete differen-
tiation into shoots was long, and the recovery of fully
differentiated plants was low thus making such systems
inefficient for genetic transformation work. Hence, in the
present study, the major emphasis was on the establish-
ment of a regeneration protocol that would provide
transgenic plants in large numbers for routine work on the
genetic enhancement of pigeonpea. The usefulness of this
system for further studies on the genetic transformation of
pigeonpea was demonstrated by biolistics-mediated gene
transfer leading to recovery of a large number of
transgenic plants.
Communicated by P.P. Kumar
S. Dayal · M. Lavanya · K. K. Sharma (
)
)
Genetic Transformation Laboratory,
International Crops Research Institute for the Semi-Arid Tropics,
Patancheru, 502 324 Andhra Pradesh, India
e-mail: k.sharma@cgiar.org
Fax: +91-40-23241239
S. Dayal · P. Devi
Department of Botany,
Osmania University,
500 006 Hyderabad, India
Materials and methods
Plant material
Different genotypes of pigeonpea [Cajanus cajan (L.) Millsp.] were
obtained from the gene bank of the International Crops Research
Institute for the Semi-Arid Tropics (ICRISAT). These belong to
different maturity groups, including ICPL 87, ICPL 88039, ICPL
87119, ICPL 85063, ICPL 88009, ICPL 87091, ICPL 2376, ICPL
87051, ICPL 91011, ICPL 332, and ICPL 84031. Unless mentioned
otherwise, all experiments were carried out with var. ICPL 88039.
Explant preparation and shoot regeneration
The seeds of pigeonpea var. ICPL 88039 were surface sterilized
with 70% ethanol for 2 min and further washed with 0.1% (w/v)
mercuric chloride containing 1–2 drops of Tween-20 for 8 min,
followed by rinsing in sterile water 4–5 times prior to soaking for
4 h. The seed coat was removed from pre-soaked seeds and
germinated on semi-solid Murashige and Skoog (MS) basal
medium (Murashige and Skoog 1962). Primary leaves from 4- to
5-day-old aseptically germinated seedlings were used as explants
for initiating tissue cultures. While preparing the leaf explants, care
was taken to excise the petiolar region sufficiently away from the
axillary meristem so as to completely eliminate any preformed
meristematic tissues. In each Petri dish, 10–12 explants were
cultured with the petiolar cut end and the abaxial surface of the
lamina in contact with the medium. To achieve efficient organo-
genesis, the effect of age of the explants was studied by taking
leaves from 2- to 15-day-old aseptically germinated seedlings and
culturing them on shoot induction medium (SIM).
To study the role of the lamina and petiolar region of the leaf
explant, different portions of the lamina were surgically removed so
as to have explants with full, one-half, one-quarter or no lamina
along with the petiolar cut end (Table 1).
The effect of various cytokinins [N
6
-benzyladenine (BA),
kinetin, N
6
-[2-isopentenyl]adenine (2-iP)], used either alone or in
combination, on the regeneration of multiple shoot buds was
studied. Aseptic leaf explants (4–5 days old) were placed on MS
containing four different combinations of BA, kinetin and 2-iP
concentrations (Table 2). In the first combination, the concentration
of BA ranged from 0 to 10 mM, while the kinetin concentration was
kept constant at 5 mM (C1–C6); in the second combination the
concentration of BA and kinetin was vice versa (K1–K6). In the
third combination, the concentration of kinetin ranged from 0 to
10 m M, while the 2-iP concentration was kept constant at 5.0 mM
(P1–P6). In the fourth combination, the concentration of kinetin
was kept constant (5.0 mM), while the concentration of 2-iP varied
between 0 and 10 mM (I1–I6). Furthermore, various concentrations
of BA and kinetin were tested to standardize the best combination
of cytokinin for multiple shoot induction. Based on the mor-
phogenic response, MS in combination with 5.0 mM BA and 5.0 mM
kinetin (SIM) was optimal for shoot bud differentiation. The leaf
explants with multiple shoot buds that were obtained from SIM
were transferred to shoot elongation medium (SEM) consisting of
MS supplemented with gibberellic acid (GA
3
) ranging from 0.58 to
2.89 mM (data not shown).
Table 1 Effect of size of the lamina on shoot bud induction from the petiolar region of leaf explant of in vitro-germinated seedlings of
pigeonpea
Explant No. of explants cultured No. of explants producing shoot buds Frequency of shoot bud induction (%)
Full lamina 30 27 90.0
One-half lamina 30 26 86.6
Three-quarter lamina 30 22 73.3
Minus lamina 30 6 20.0
Table 2 The effect of N
6
-ben-
zyladenine (BA), kinetin and
N
6
-[2-isopentenyl]adenine (2-
iP) on shoot bud induction from
leaf explants of in vitro-germi-
nated seedlings of pigeonpea
Medium Growth regulators (mM) No. of
explants
cultured
Explants
producing shoots
(mean€SE)
Explants forming
shoots (%)
Kinetin BA 2-iP
C1 5.0 10.0 30 15.0€1.4 50.0
C2 5.0 7.5 30 20.5€0.7 68.3
C3 5.0 5.0 30 28.5€0.7 95.0
C4 5.0 2.5 30 26.5€0.7 88.3
C5 5.0 1.0 30 22.0€0.7 73.3
C6 5.0 0.0 30 24.5€0.7 81.7
K1 10.0 5.0 30 15.0€1.4 50.0
K2 7.5 5.0 30 23.5€0.7 78.3
K3 5.0 5.0 30 27.0€1.4 90.0
K4 2.5 5.0 30 20.5€0.7 68.3
K5 1.0 5.0 30 22.0€0.0 73.3
K6 0.0 5.0 30 23.5€0.7 78.3
P1 10.0 5 30 25.5€0.7 85.0
P2 7.5 5 30 21.0€1.4 70.0
P3 5.0 5 30 18.5€0.7 61.7
P4 2.5 5 30 17.5€0.7 58.3
P5 1.0 5 30 21.5€0.7 71.7
P6 0.0 5 30 20.5€0.7 68.3
I1 5.0 10 30 11.0€1.4 36.7
I2 5.0 7.5 30 21.0€0.7 70.0
I3 5.0 5.0 30 23.5€0.7 78.3
I4 5.0 2.5 30 10.5€0.7 35.0
I5 5.0 1.0 30 13.5€0.7 45.0
I6 5.0 0.0 30 21.5€0.7 71.7
1073
Rooting of shoots and transplantation
Shoots over 3 cm in length were transferred to root induction
medium (RIM) consisting of auxins such as indole-3-butyric acid
(IBA) and indole-3-acetic acid (IAA). Various concentrations of
IBA (0.98–9.8 mM) and IAA (1.14–11.42 mM), and MS medium
with reduced sucrose concentration (1% w/v) were tested individ-
ually (data not shown). The elongated shoots were cut and directly
dipped in 11.4 mM IAA solution for pulse treatment and transferred
to culture tubes containing MS containing 1% sucrose (w/v) and
devoid of any growth regulators, where they formed adventitious
roots within 6 days. Regenerated plantlets with well-developed
roots were transferred to small pots, filled with autoclaved sand and
thiram (fungicide), for hardening. The plantlets in the pots were
kept covered with polythene bags for 5 days and later transferred to
the greenhouse. The plants from smaller pots were transferred to
bigger pots (30.5 cm diameter) that contained autoclaved sand and
soil (1:1) supplemented with farm manure and di-ammonium
phosphate.
Culture medium and conditions
MS basal medium containing 3% sucrose was used for all in vitro
cultures. The pH of the medium was adjusted to 5.8 prior to adding
0.8% agar; media were autoclaved at 121C for 15 min. Cultures
were maintained at 26€1C under continuous light provided by
white cool fluorescent tubes of 60 mEm
2
s
1
light intensity. The
growth regulators BA, kinetin, GA
3
and IAA were filter-sterilized
prior to addition to culture media. The explants were cultured on
sterile Petri dishes (9015 mm) containing SIM; explants bearing
adventitious shoot buds were subsequently transferred to culture
tubes (25150 mm) for shoot elongation and rooting of shoots. Data
on the frequency of shoot bud regeneration from each explant was
recorded. All experiments were repeated three times and the data
were analyzed by calculating mean and standard error.
Effect of genotype
To study the effect of genotype of the explant donor seedlings, 11
genotypes of pigeonpea belonging to different maturity groups
were selected (see Table 3). Leaf explants from 4- to 5-day-old in
vitro germinated seedlings were cultured on SIM and their shoot
bud regeneration responses were compared.
Genetic transformation via biolistics
DNA preparation, microprojectile DNA delivery
and analysis of transgenics
Plasmid pRT99GUS (Fig. 1) containing the uidA and nptII genes,
both under the control of the 35S promoter of cauliflower mosaic
virus (Topfer et al. 1988) was used to optimize genetic transfor-
mation of leaf explants. The plasmid was isolated by the alkaline
lysis method (Sambrook et al. 1989) and DNA delivery was carried
out using the particle delivery system PDS-1000He (Bio-Rad,
Hercules, Calif.) following the manufacturer’s recommendations.
Gold particles (1 mm diameter) were coated with 5 mg plasmid
DNA per 50 ml particle preparation by using the CaCl
2
precipitation
method. About 50 leaf explants were placed in each Petri dish
containing SIM and explants were placed in such a way that the
petiolar cut end of all the leaves faced towards the center. In each
experiment, 400 explants were taken and each experiment was
repeated three times. The plates containing the explants were
placed at a distance of 6 cm from the stopping plate under a vacuum
of 22 inches of Hg (74.5kPa) and rupture disk rated for a pressure
of 1,300 psi (8.96 MPa). After each bombardment, the explants
were incubated on the same plate overnight and transferred to fresh
plates containing SIM at a plating density of 10–12 explants per
plate. The regeneration protocol as described above was followed.
Once shoot differentiation was observed from the petiolar ends, the
explants were subjected to 25 mg l
1
kanamycin in a shoot
development medium consisting of half-strength SIM. After
2 weeks of culture, the explants were transferred to elongation
medium consisting of 0.58 mM GA
3
along with 50 mg l
1
kanamycin. In the following 2–3 subcultures of 2 weeks each on
SEM, the concentration of kanamycin was increased to 100 mg l
1
for stringent selection of transformed shoots. This was followed by
rooting of the selected and elongated shoots, and transplantation of
the rooted shoots to the glasshouse.
Analysis of transgenic plants
Genomic DNA from the putative transformants growing in the
glasshouse was analyzed for the presence of the introduced genes
by PCR amplification of uidA and nptII, and Southern hybridiza-
tion for the nptII gene according to Sharma and Anjaiah (2000). For
Southern blot hybridization of the nptII gene, the DNA was
digested with XhoI, which is a unique site within the pRT99GUS
plasmid DNA. The blot was probed with a non-radioactively
labelled (Alkphos Direct Labelling and Detection System; Amers-
ham Biosciences, Uppsala, Sweden) 700 bp PCR-amplified nptII
gene fragment. For the positive control, the plasmid pRT99GUS
was restricted with PstI to release the 700 bp nptII gene fragment.
RT-PCR analysis of the putative transformants growing in the
glasshouse was carried out using the Thermoscript RT-PCR system
(Invitrogen, Carlsbad, Calif.) on total RNA isolated with the TRIzol
Table 3 Effect of explant donor genotype on shoot bud regeneration from leaf explants from in vitro-germinated seedlings of pigeonpea
after culture on shoot induction medium
Genotype Duration type No. of explants
cultured
No of explants producing
shoots
Frequency of shoot bud
induction (%)
ICPL 91011 Extra short 39 24 61.5
ICPL 88009 Short 39 16 41.0
ICPL 84031 Short 42 25 59.2
ICPL 87091 Short 50 31 62.0
ICPL 87 Short 73 52 71.2
ICPL 88039 Short 54 45 83.3
ICPL 2376 Medium 77 34 44.2
ICPL 87051 Medium 57 31 54.4
ICPL 332 Medium 35 19 54.3
ICPL 85063 Medium 69 44 63.8
ICPL 87119 Medium 35 23 65.7
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Fig. 1A–K Regeneration of multiple shoots from leaf explants
derived from in vitro-germinated seedlings of pigeonpea, Cajanus
cajan L. (arrows indicate the petiolar cut end undergoing shoot bud
differentiation). A Leaf explants at day 0 cultured on Murashige and
Skoog (MS) medium supplemented with 5.0 mM N
6
-benzyladenine
(BA) and 5.0 mM kinetin [shoot induction medium (SIM)]. B
Enlargement and swelling of the petiolar cut end within 5 days of
culture on SIM. C Initiation of adventitious shoot buds from leaf
explants observed after 7 days on SIM from the swollen tissue of the
petiolar cut end. D Differentiation of shoot buds from the petiolar cut
end after 8 days on SIM. E Proliferation of multiple shoot buds after
10 days on SIM. At this stage, the explants with reduced lamina are
ready for transfer to reduced SIM for shoot development. F
Development of shoot buds into shoots after 12 days on reduced
SIM. G Formation of multiple shoots after 2 weeks on reduced SIM.
H Explant bearing multiple shoots placed on shoot elongation
medium (SEM) containing MS supplemented with 0.58 mM gib-
berellic acid (GA
3
) for shoot elongation after 7 days. I Arooted
pigeonpea plant on MS containing 1.14 mM indole acetic acid (IAA)
[root induction medium (RIM)] after 3 weeks ready for transplan-
tation. J A well-established and hardened plant successfully
transplanted to the glasshouse at 4 weeks. K In vitro produced plants
after 2 months in the glasshouse showing normal growth, flower
production, and pods that contain viable seeds
1075
reagent (Invitrogen) according to the manufacturer’s procedures.
To study the inheritance of the introduced genes in the T1
generation, five seeds from eight selected primary transformants
were germinated and PCR analysis to detect the uidA gene was
carried out (Table 4).
Results
The genotype ICPL 88039 of pigeonpea exhibited over
95% seed germination on MS basal medium. After the
culture of leaf explants (Fig. 2A) on SIM, their lamina
underwent considerable enlargement and swelling of the
petiolar cut end within 5 days (Fig. 2B). Initiation of
shoot buds from the swollen tissues of the petiolar cut end
had occurred by 7 days (Fig. 2C). Differentiation of shoot
buds from the proximal cut end of the petiole occurred at
8 days (Fig. 2D), and proliferation of these into a large
mass of shoot buds by 10 days (Fig. 2E). Thereafter, the
shoot buds continued to proliferate until transfer to SEM.
Prior to subculture on reduced SIM for shoot develop-
ment, half of the lamina of the explants was removed
(Fig. 2F) and well-developed shoots developed by 2 weeks
(Fig. 2G). After 21 days, explants bearing multiple shoots
were separated and cultured on SEM, on which they
underwent elongation (Fig. 2H). The semi-elongated
shoots rooted easily on RIM within 3–4 weeks (Fig. 2I).
The rooted shoots could be readily transplanted with a
success rate of 100% and showed normal growth in the
glasshouse (Fig. 2J). Upon maturity, the plants produced
fertile flowers and pods that contained viable seeds
(Fig. 2K). Following this protocol, more than 100 plants
with normal morphology and seed fertility have been
produced so far.
The effect of the age of the explant donor seedlings on
shoot regeneration was determined. In general, younger
seedlings (<5 days) provided explants that were highly
regenerative while the regeneration potential declined
with age thereafter (Fig. 3); 4- to 5-day-old leaf explants
exhibited the highest frequency of multiple shoot regen-
eration where 90% of the explants responded. In studies
on the role of the lamina tissue in shoot bud regeneration
from the petiolar cut end, it was found that leaf explants
containing intact lamina (Fig. 2A) were essential for the
regeneration response, with shoot bud induction declining
with reduced lamina tissue (Table 1). A very low
frequency of shoot regeneration occurred if the entire
lamina was removed from the petiolar explants. Hence,
whole leaf explants from 4- to 5-day-old seedlings were
used in the optimized protocol.
In preliminary experiments, it was found that a
combination of two cytokinins was required for the
regeneration of shoot buds from leaf explants. Hence,
various combinations of cytokinins, such as BA with
kinetin and 2-iP with kinetin, were tested to induce shoot
bud differentiation. In general, BA in combination with
kinetin was found to be more efficient in inducing
multiple shoot buds as compared to combinations of
kinetin with 2-iP, where only a single shoot was formed in
a few explants (Table 2). Therefore, equimolar concen-
Table 4 Inheritance of uidA gene in the T1 generation of
transgenic pigeonpea plants
Plant No. of
T1
plants
tested
PCR analysis of uidA gene 3:1 Segregation
a
No. of plants c
2
P
Positive Negative
PP1 5 3 2 0.6 0.4386
PP2 5 3 2 0.6 0.4386
PP3 5 5 0 1.67 0.1963
PP5 5 2 3 1.65 0.1990
PP6 5 4 1 0.03 0.8625
PP7 5 3 2 0.60 0.4386
PP8 5 4 1 0.03 0.8625
PP13 5 1 4 4.03 0.0447
a
df=1; P=0.05; c
2
=3.841
Fig. 2 Restriction map of plasmid pRT99GUS used for biolistic-
mediated transformation of leaf explants from in vitro germinated
seedlings of pigeonpea
Fig. 3 Effect of age of leaf explant donor seedlings on the
regeneration of multiple adventitious shoots in pigeonpea; 60
explants were cultured on SIM
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![Table 2 The effect of N6-benzyladenine (BA), kinetin and N6-[2-isopentenyl]adenine (2- iP) on shoot bud induction from leaf explants of in vitro-germinated seedlings of pigeonpea](/figures/table-2-the-effect-of-n6-benzyladenine-ba-kinetin-and-n6-2-1eomo29g.webp)
