Agrobacterium-mediated Transformation of Pearl Millet (Pennisetum typhoides (L.) R.Br.) for Fungal Resistance
A new Agrobacterium-mediated transformation system was developed for pearl millet using shoot apex explants, conferring resistance to leaf blast disease by inserting a rice chitinase (chi11) gene. Transgenic pearl millet lines (Pennisetum typhoides (L.) R.Br.) expressing rice chitinase gene with high levels of resistance to rust pathogen, Puccinia penniseti, were developed using Agrobacterium-mediated gene transfer method. The emryogenic calli derived from shoot apex of CO9 cultivar were transformed with LBA4404 (pSB1/pKAN-Rchit1.1) that harboured rice chitinase gene (chit11) under the control of maize ubiquitin (Ubi 1) promoter intron. Transgene (chit 11) in the middle of the T-DNA as used as probe in southern analysis. Out of six independent T0 plants tested for southern, three had single copy T-DNA insertions and three had two copies T-DNA insertions. All the six T0 plants carried complete T-DNA with the chitinase transgene. A segregation ratio of 3:1, reflecting T-DNA insertion at a single locus, was observed in the progeny of all the T0 plants which showed normal Mendelian pattern of transgene segregation. Western blot analysis of T1 plants revealed constitutive expression of chitinase at high levels. Bioassays of T1 plants indicated enhanced resistance to the rust pathogen, P. penniseti, in comparison to control plants. This is the first report on Agrobacterium-mediated transformation of pearl millet and first transgenic pearl millet with fungal resistance. This study underpins the introduction of numerous agronomically important genes into the genome of pearl millet in the future.
Received: July 26, 2012;
Accepted: August 07, 2013;
Published: September 19, 2013
Pearl millet (Pennisetum typhoides (L.) R.Br.) is one of the major cereal
crops worldwide (Girgi et al., 2006). It is a
high yielding summer crop tolerant to drought and acidity. It can be grown in
low rainfall areas where maize and sorghum do not thrive (FAO,
2004). It occupies 40 million ha in the drought-prone semi-arid and arid
tropics of Asia and Africa. It meets 80-90% of the calorie necessities of millions
of people inhabiting these regions (Lambe et al.,
2000; OKennedy et al., 2004a). In the
past one decade the net productivity of pearl millet has been limited because
of various reasons such as fungal diseases, insect pests, drought stress, high
soil temperature and inferior agronomic characteristics (Rai
et al., 1997; Gueye and Delobel, 1999; Grover
and Pental, 2003; Ceasar and Ignacimuthu, 2009).
The most important yield constraints of pearl millets are fungal diseases contributing
to yield losses (Grover and Pental, 2003; Latha
et al., 2006; Girgi et al., 2006).
Pearl millet is susceptible to several fungal diseases such as rust disease
caused by basidomycete Puccinia penniseti, Downey mildew caused by the
Oomycete sclerospora graminicola and smut caused by Ustilago sp.
(Wilson, 2000; Grover and Pental,
2003). Among the different fungal pathogens P. penniseti is one of
the most damaging fungi in many countries (Singh et al.,
1993; Girgi et al., 2006). Breeding for resistance
to above mentioned diseases is a high priority for pearl millet breeders (Anonymous,
Traditional breeding has been the main opportunity for crop development in
pearl millet. A number of approaches were taken to improve fungal resistance
in plants such as expression of Pathogenesis Related (PR) proteins (Zhu
et al., 1994; Van Loon and van Strien, 1999;
Kishimoto et al., 2002) and phytoalexins (Hain
et al., 1993; Dixon, 2001). Gene transfer
has become an established and routine technique in many laboratories. Genetic
transformation of pearl millet with antifungal genes would help in the management
of various fungal pathogens (Sharma and Ortiz, 2000;
Thakur and Mathur, 2002; Devi et
al., 2000; Girgi et al., 2006; Ceasar
and Ignacimuthu, 2011).
Taylor and Vasil (1991) and Taylor
et al. (1993), using microprojectile bombardment, incorporated gus
A gene into the scutellum of immature embryos of pearl millet and reported GUS
expression. Lambe et al. (1995) transferred gus
A, hpt, nptII and bar genes into pearl millet through microprojectile method.
Girgi et al. (2002) transferred gus A and bar
genes into the scutellar tissue of immature embryos. Devi
and Sticklen (2002) described a rapid, reliable method for the microprojectile
bombardment and transient expression of GUS in the multiple-shoot tip clumps
of pearl millet. Transgenic pearl millet plants harbouring bar and gfp genes
were also developed using Particle Inflow Gun (PIG) (Goldman
et al., 2003). A transformation protocol was established with the
herbicide resistance selectable marker gene, bar, using PIG (Girgi
et al., 2002). The manA selection system was used as selectable markers
in pearl millet transformation (OKennedy et al.,
2004b). Recently, using precultured immature zygotic embryos and embryogenic
tissue of maize and pearl millet, transformation was developed by biolistic
method (OKennedy et al., 2011).
Latha et al. (2006) reported that a reproducible
method for genetic transformation employing gus A gene with PIG method in pearl
millet. A chemically synthesized antifungal pin gene was used for producing
transgenic pearl millet (ICMP451) with resistance to downey mildew S. graminicola.
Transgenic pearl millet was developed against rust (P. substriata) and
downey mildew (S. graminicola) by introducing a cDNA encoding the antifungal
protein AFP from the mould Aspergillus giganteus (Girgi
et al., 2006). To the best of knowledge, there are no reports on
Agrobacterium-mediated gene transformation in pearl millet so far. Further
transgenic pearl millet with rice chitinase gene resistant to rust disease has
not yet been developed. Hence, the present study was aimed at using Agrobacterium-mediated
transformation system to stably introduce chitinase gene into pearl millet for
developing rust disease resistance.
MATERIALS AND METHODS
Plant materials and Agrobacterium strain: Pearl millet (Pennisetum
typhoides (L.) R.Br.) cultivar CO9 was obtained from Tamil Nadu Agricultural
University, Coimbatore, India. Mature, healthy seeds were surface sterilized
with 70% alcohol for 30 sec and in 0.1% HgCl2 (w/v) for 5 min, followed
by five rinses with sterile double distilled water. Sterilized seeds were cultured
on MS basal medium (Murashige and Skoog, 1962) with 3%
sucrose for germination. Three-day-old shoot apices (2-3 mm) removed from the
seedlings were used for transformation experiments. Agrobacterium tumefaciens
strain EHA 105 harboring LBA4404 (pSB1/pKAN-Rchit1.1) was used for transformation.
A 3.1kb Hind III fragment obtained from pCAMBAR CH11 (kindly provided by Dr.
Muhtukrishnan, Kansas State University, USA) containing rice chitinase gene,
ubiqutin promoter and Ubi1 was subcloned into a binary vector LBA 4404 (pSB1),
Kanr (Fig. 1).
||Plasmid construct LBA 4404 (pSB1/pKAN-Rchit 1.1), showing
restriction sites. LB, RB Left and right borders, respectively
T- DNA of the vector carried highly active maize ubiqutin promoter with its
intron 1 fused with rice chitinase gene driven by CaMV 35S promoter and nos
polyA terminator. The plasmid contained GUS as reporter gene and kanamycin neophosphotransferase
(npt II) as selection marker.
Agrobacterium infection and cocultivation: A. tumefaciens
strain was grown for three days on AB minimal, medium (Chilton
et al., 1974) with 50 mg L-1 kanamycin 10 mg L-1
rifamycin and 10 mg L-1 tetracycline. A single colony was transferred
to AB minimal liquid medium containing the above mentioned antibiotics and the
culture was allowed to shake overnight at 28°C at 200 rpm. The overnight
culture (0.5 mL) was transferred to 50 mL of fresh AB medium containing the
selective antibiotics. The cultures were grown over night under the same conditions.
When the Optical Density (OD) of Agrobacterium reached 0.6 (600 nm),
the bacterial culture was centrifuged at 5000 rpm for 10 min at 4°C and
pelleted. It was resuspended in equal volume of AA medium (Toriyama
and Hinata, 1985) to obtain a density of 108 cells mL-1
which contained 30 g L-1 sucrose and acetosyringone (AS) 100 μM,
pH 5.6 (AA-AS). The culture was allowed to shake again for 3 h under the same
conditions as described above and this Agrobacterium culture was used
for infection of the shoot apex calli explants.
The shoot apex calli (0.5-1.0 cm) were immersed in the bacterial suspension
for 30 min with occasional shaking in the shaker at 150 rpm. Excess bacteria
were removed from the surface of explants by placing them on sterile Whatman
No. 1 filter paper before transfer to semisolid (0.6% agar) cocultivation medium
consisting of 2, 4-D 2.0 mg L-1 and BAP 0.5 mg L-1 with
100 μM AS (PM-CCM). The effect of cocultivation period was evaluated and
the shoot apex calli were assayed for transient GUS activity. After cocultivation
period, the explants were removed from the cocultivation media and rinsed 4-5
times with sterile distilled water containing 250 mg L-1 cefotaxime
and 300 mg L-1 carbenicillin to eliminate Agrobacterium.
Selection and regeneration of transgenic pearl millet: The inoculated
calli were transferred to MS medium supplemented with 2,4-D 2.0 mg L-1+BAP
0.5 mg L-1+ 120 mg L-1 kanamycin+300 mg L-1
carbenicillin (PM-SL1) for inhibition of bacterial growth and selection of transformed
tissues. The kanamycin resistant shoot apex calli were transferred to fresh
selection medium consisting of 2, 4-D 2.0 mg L-1+BAP 0.5 mg L-1+120
mg L-1 kanamycin+300 mg L-1 carbenicillin (PM-SL2). After
three rounds of selection and total of 6 weeks on selection medium the calli
were transferred to regeneration medium fortified with BAP 2.0 mg L-1+NAA
0.5 mg L-1+ 120 mg L-1 kanamycin+300 mg L-1
carbenicillin (PM-RE). Small pieces of the kanamycin resistant calli as well
as their respective untransformed control calli were assayed for GUS activity
every three weeks during subculture. The kanamycin resistant regenerated shoots
(about 3-5 cm length) were transferred to MS medium containing BAP 2.0 mg L-1+NAA
0.5 mg L-1+120 mg L-1 kanamycin+ 300 mg L-1
carbenicillin for shoot development. The regenerated shoots were further transferred
to MS medium containing 0.5 mg L-1 IBA+120 mg L-1 kanamycin+300
mg L-1 carbenicillin for rooting (PM-RIM). Two weeks old rooted plantlets
(8-10 cm length) were individually transferred to plastic cups (10x50 cm) containing
sterile garden soil and vermiculite at 1:1 ratio and watered with sterile half
strength Hoaglands solution (Hoagland and Arnon, 1950).
After covering the cups with polythene bag, the set up was maintained for 7-10
days in the culture room at 27±2°C under a photoperiod regime having
16 h light. Hardened plants were transferred to earthen pots containing garden
soil and maintained in the green house.
Histochemical assay for the GUS gene: The expression of β-D-Glucuronidase
(GUS) gene in shoot apex calli, leaves and roots were assayed with 5-bromo-4-chloro-3-indolyl
glucuronide (X-Glu) substrate essentially following the method of Jefferson
(1987). The pearl millet tissues were incubated in sodium phosphate buffer
(50 mM NaPO4, pH 6.8) that contained 1% Triton X- 100 at 37°C
for 1 h. Fresh phosphate buffer containing 1.0 mM X-Gluc and 20% methanol replaced
the buffer. The reaction mixture was incubated overnight at 37°C and then
tissues were examined visually for dark blue sectors.
PCR analysis of putative transgenic plants: Total genomic DNA was extracted
from young leaf tissues of T0 GUS positive and untransformed control plants
(Roger and Bendich 1994). Genomic DNA of putative transformants
was subjected to PCR analysis with chit11 and npt-II gene primers. Polymerase
Chain Reaction (PCR) was carried out in a 25 μL reaction mixture containing
template DNA (50 ng), 120 μM of each dNTPs, 2 pmol of specific primers
for npt-II gene (Forward (5 GCCATTTGAAGCCGATGTCAC 3) and reverse
primer (5- TCTGCCCCAACTGCCTCTGCT-3), chit11 (Forward (5-CCCCGCGGCCGTAGTTGTAGT-3)
and reverse primer (5 AGAGAGGTTAAAGGCCGACAGC 3), 1 unit Taq DNA
polymerase, 3 mM MgCl2 and 1x Taq DNA polymerase buffer. The reaction
was carried out at 94°C for 1 min, followed by 30 cycles of 94°C for
1 min, 55°C for 1 min and 72°C for 1 min. This was followed by one cycle
of 10 min at 72°C. The reactions were carried out in an eppendorf thermocycler.
The amplified products were assayed by electrophoresis on 0.8% agarose gels,
stained with ethidium bromide (EtBr; 0.5 g mL-1), visualized and
photographed under ultraviolet light.
Southern hybridization: Southern analysis was performed as described
by Southern (1975). The genomic DNA of transformed and
control plants were digested with Hind III restriction enzyme to express the
3.1 kb chitinase gene and electrophoresed in 1% agarose gel to get the band.
The DNA bands were denatured and transferred to a nylon membrane by standard
method. Southern hybridization was carried out using radioactive labeling method.
The probe DNA of GUS gene was labeled [α-32P]dCTP (BRIT, Mumbai,
India) using a random primer labeling kit (Amersham International, Plc. Ltd.,
London, UK). To confirm the presence of chitinase gene in transgenic plants,
1.1 kb chit11 coding sequence was used as probe. Hybridization was carried out
at 65°C. Post-hybridization washes were done sequentially with 3xSSC (1xSSC
is 0.15 M NaCl plus 0.015 M trisodium citrate), 0.5xSSC and 0.1xSSC, along with
0.1% Sodium Dodecyl Sulfate (SDS); each wash was carried out for 30 min at 65°C.
After hybridization washes, the membrane was exposed to X-ray film and incubated
for 48 h at -80°C.
Western blot analysis: Western blot analysis was performed to detect
the expression of chitinase enzyme in T0, T1 progenies
and untransformed plants. Protein extraction and western analysis were performed
as described by Chen et al. (1998). Total protein
was isolated from 1 g young leaves of 35-day-old of T0 progeny, T1
progeny and untransformed plants were ground to a fine powder using liquid nitrogen
and homogenized with extraction buffer (Bradford, 1976)
supplemented with 10 mM β-Mercabtoethanol. The extract was centrifuged
at 18,000 x g for 20 min at 4°C and the supernatant was used for western
analysis. The protein concentration was estimated using the method of Bradford
(1976). Twenty microgram aliquots of total protein were separated by SDS-PAGE
in a 10% gel and transformed to a nitrocellulose membrane using a semi-dry transfer
apparatus. Molecular weight markers (Rainbow marker) were purchased from Amersham
Pharmacia Biotech, Little Chalfot, England. The membrane was blocked using 3%
gelatin and Tween-Tris Buffered Saline (TTBS) and probed with the chitinase
antibody (a polyclonal rabbit antibody raised against barley chitinase kindly
provided by Dr. S. Muthukrishanan, Kansas State University) diluted to 1:1000
(v/v). The second antibody, goat anti-rabbit IgG (H+L) alkaline phosphates conjugate
from Bangalore Genei Pvt. Ltd., Bangalore, India was used at a dilution of 1:2,000.
The membrane was treated with 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and
nitroblue tetrazolium (NBT) colour reagent until bands appeared.
Chitinase enzyme assay: Chitinase enzyme assay was performed to confirm
the level of chitinase enzyme produced in kanamycin resistant plants and compared
with control plants. The colorimetric assay of chitinase was carried out following
the method of (Mauch et al., 1988; Ceasar
and Ignacimuthu, 2012) using colloidal chitin as the substrate.
Leaf blast assay: Fungal bioassay was carried out on T1 plants (2T1,
5T1 and 9T1) and on untransformed seedlings under controlled conditions in the
green house. Puccinia spores were obtained from Division of Plant Pathology,
Tamilnadu Agricultural University, Madurai, India. Spores of Puccinia
were suspended in sterile distilled water and the spore suspension (106
spores mL-1) was sprayed on 10-days old seedlings maintained at 20-25°C
and 85-90% relative humidity. Symptoms of the disease were recorded 10-days
after spore inoculation and disease intensity was graded on 0-9 scale, where
0 indicated maximum resistance and 9 indicated maximum susceptibility resulting
in seedling death. The size of the leasions was measured using the scale provided
in the microscope. Plants falling in the range of 0-4 scale were classified
as resistant and seedlings in 5-9 scale were scored as susceptible. The experiments
were carried out in triplicate and repeated twice. The Chi-square test for goodness
of fit was also applied to confirm the inheritance of rice chitinase gene in
plants grown to maturity under controlled conditions in the greenhouse.
Segregation analysis of transgene in T1 progenies: A segregation
analysis was performed to check the pattern of inheritance. Seeds collected
from selfed T0 plants were screened for kanamycin resistance; the
seeds were germinated initially on MS basal medium containing 3% sucrose (solidified
using 0.8% agar) and placed in dark. The germinated seedlings were then transferred
to the same medium supplemented with 120 mg L-1 kanamycin and placed
in light. After 8 days, seedlings were scored for kanamycin resistance (kanS
and kanR) and the data were validated using χ2 test. Root and
leaf segments were used for GUS assay. From this, segregation was analyzed.
Data analysis: The entire experiments were carried out in an absolutely
randomized design. All experiments were replicated three times, each replicate
consisting of more than ten explants based on the research. The mean frequency
(%) of transient GUS appearance (number of explants producing blue spots/total
number of explantsx100) was calculated 3 weeks after infection and mean frequency
(%) of constant transformation (number of transgenic plants regenerated/total
number of explantsx100) was calculated after 7 weeks under regeneration on kanamycin.
Data were analysed statistically (ANOVA or χ2 test) and the
mean and standard deviation were calculated for each experiment. The Fishers
Least Significant Difference (LSD) (Fisher, 1935) was
calculated at 5% level with the statistical package for social science (Version
12.0 for Windows, SPSS Inc.).
Pearl millet transformation and regeneration: The gene for npt-II encoding
kanamycin phosphotransferase confers resistance to kanamycin. In order to determine
the usefulness of kanamycin resistant gene (npt) for the selection of transformed
pearl millet tissues, killing curve was established for pearl millet shoot apex
calli with different concentrations of kanamycin. The selection media contained
MS basal medium supplemented with 2 mg L-1 of 2, 4-D and 0.5 mg L-1
BAP (PM-SL1). The cultures were incubated for one month. It was seen that no
shoot apices survived on 120 mg L-1 kanamycin. Therefore, 120 mg
L-1 was chosen for selection of transformants throughout the transformation
experiments. Higher concentration of kanamycin induced necrosis of the explants
very rapidly and reduced the survival rate of the explants.
The chitinase gene was introduced into shoot apex derived calli of the pearl
millet cultivar CO9 by Agrobacterium mediated gene transfer method. A
total of three experiments were carried out to determine the transformation
efficiency. Shoot tip calli cocultivated with LBA 4404 (pSB1/pKAN-Rchit 1.1)
for 3 days produced kanamycin resistant calli after 35 days. The selection medium
(PM-SL2) containing 120 mg L-1 kanamycin and 300 mg L-1
carbenicillin (Fig. 2a) was used. Once in 21 days the calli
were subcultured for vigorous selection. Growth of the calli which were not
infected with Agrobacterium LBA 4404 (pSB1/pKAN-Rchit 1.1) was efficiently
inhibited in a medium containing 120 mg L-1 kanamycin (negative control).
The positive control calli (not infected with Agrobacterium) efficiently
proliferated in the callus induction medium in the absence of kanamycin. A high
frequency of kanamycin resistant calli was observed in three different experiments.
Out of 961 calli cocultivated in three experiments 437 kanamycin resistant calli
were obtained; most of the kanamycin resistant calli exhibited blue staining
for GUS activity in the histochemical GUS assay (Table 1).
After 63 days of selection the kanamycin resistant calli were transferred to regeneration
medium containing 120 mg L-1
kanamycin and 300 mg L-1
(PM-RE). Small shoot clusters (Fig. 2b
) were observed on the
transformed calli after three weeks of growth and the regenerating calli were
subcultured in fresh regeneration medium and maintained for another three weeks.
In three experiments, a total of 27 kanamycin resistant plants were regenerated
from 60 calli. After four weeks in regeneration medium the plantlets rooted (Fig.
) on the rooting medium (PM-RIM). Small leaf pieces
of various parts of the putatively transformed shoots were used for GUS histochemical
assay. In these assays, 25 of the 71 regenerated shoots showed blue precipitate
). These plantlets were hardened (Fig.
) and transferred to field conditions to set seeds. This cultivar CO9 showed
a transformation frequency ranging from 6.0- 8.7% (Table 1
PCR and southern analysis of transgenic plants: PCR was carried out
on the genomic DNA isolated from the T0 tranformants and the untransformed
control using primers of npt-II, GUS and chitinase coding sequences. All the
transformants were found to be positive for the amplification of 0.7 kb (npt
II) and 1.1 kb (chitinase) genes by PCR. In all transformed plants the npt II
fragment (0.7 kb) was amplified; only a faint band was observed in plants (lanes)
4, 11 and 12 while no such band was amplified in the untransformed control (Fig.
3a). Of the six plants used to check the chitinase (chit11) transgene fragment
(1.1 kb), all the plants showed the expected fragment size (Fig.
3b). A similar band was also noticed in the positive plasmid control while
no such band was observed in the untransformed control. This indicated that
the tissues were completely free of Agrobacterium.
||Transformation efficiency (TF) by Agrobacterium LBA4404
(pSB1/pKAN- Rchit1.1) with rice chitinase in shoot apex calli of pearl millet
||Agrobacterium-mediated transformation of pearl millet (Pennisetum typhoides (L.) R.Br.cv. CO9) (a)
Transient expression of GUS gene in shoot apex derived calli of pearl millet after cocultivation with
A. tumefaciens for 3 days (scale bar = 3 mm), (b) Pearl millet plant regeneration from shoot apex calli
explants after 21 days, (c) Root induction and whole plant formation in selection (regeneration) medium,
black arrow showing root formation, (d) A transformed pearl millet plants recovered from selection
medium, (e) GUS expression in explants of pearl millet transformed with rice chitinase gene (chi11) using
A. tumefaciens LBA4404 (pSB1/pKAN-Rchit 1.1), black arrow showing blue colour, (f) Transformed
plants in green house condition, (g) Leaf adaxial surface of the transgenic plants (right) not showing fungal
infection, susceptible untransformed control (left)
Southern blot analysis was performed in order to prove stable integration of
chitinase gene within plant genome. DNA from six of the GUS positive transgenic
shoots was used for molecular analysis. Total DNA (20 μg) of putative transformants
was digested with Hind III restriction enzyme which released an internal fragment
of expected size of 3.1kb rice chitinase gene along with npt-II and GUS reporter.
The probe for chitinase gene was prepared from pCAMBAR CHI 1.1 plasmid by digesting
with sac I restriction enzyme and it was radioactive labeled. Chitinase gene
probe hybridized only to DNA from transgenic plants (Fig. 4a)
but not to the DNA from the untransformed control plants. The result indicated
that chitinase gene was integrated in the pearl millet genome.
Segregation analysis of transgene: Selfed progeny of four independent
transformants of CO9 cultivar transformed with LBA4404 (pSB1/pKAN-Rchit 1.1),
were examined for npt-II gene resistance.
|| (a) PCR analysis of transgenic plants with 0.7 kb fragment
of npt-II gene. Lane 1: 1 kb Marker ladder, Lane 2: Untransformed plant
as negative control, Lane 3: pCAMBIA2301 plasmid as positive control and
Lanes 4-14: Putative transgenic plants of CO9, (b) PCR analysis of DNA of
transgenic plants with 1.1 kb fragment within the chit 11 gene, Lane 1:
One killobyte ladder, Lanes 2-7: Putative transgenic plants of CO9, Lane
8: Untransformed plant as negative control and Lane 9: pCAMBIA 2301 with
chit 11 plasmid as a positive control
||(a) Southern blot analysis of T0 transgenic plants.
A chit 11 gene fragment was used to probe genomic DNA isolated from leaves
of transgenic and untransformed lines. The blot was digested with Hind III
and loaded in each lane. They were hybridized with radiolabeled dCTP- chit
11 (3.1 kb) coding sequences, Lane 1: λ Hind III digested marker, Lane
2: Undigested DNA from T0 plants, Lane 3: Untransformed control,
Lanes 4-9: Transformed plants and Lane 10: One killobyte Marker, (b) Western
blot analysis of T1 plants of three T0 lines for chitinase
expression. Twenty microgram aliquot of total protein was loaded in each
lane and separated by SDS-PAGE in 10% polyacerylamide gels, Lane 1: Rainbow
marker, Lanes 2-6 and 8: Transgenic plants of CO9 and Lane 7: Untransformed
|| Segregation of kanamycin resistance gene in T1
generation of pearl millet plants transformed with Agrobacterium
Resistant and sensitive seedlings were distinguishable on MS basal medium
containing 120 mg L-1 kanamycin (PM-GM). These sensitive plants died
within two weeks after the treatment while the resistant plants were as healthy
as untreated plants. Further GUS expression was also observed in the corresponding
plants. The sensitive plants died after the treatment while the resistant plants
were alive. All the four transgenic lines exhibited a segregation ratio of 3:1
Western blot analysis of putative transgenic pearl millets: Western
blot analysis was performed with six T1 transgenic plants. Total
soluble protein was extracted from the leaf tissues of control and transgenic
plants. In each lane 20 μg of total protein was loaded for SDS-PAGE analysis.
Barley chitinase antibody served as the primary antibody. The results showed
high accumulation of 35 kD chitinase in transgenic plants. No signal was seen
in control plants. The size of the protein matched the expected relative mass
of 35,000 for chitinase (Fig. 4b) in addition to the 35 kD
protein; a band at 28 kD was also detected in all six T1 plants.
The 28 kD protein may have been released by proteolysis. Comparable levels of
chitinase were accumulated in all the six transgenic lines. There was slight
difference in the levels of chitinase in transformed plants presumably because
of the degree of the expression of chitinase gene.
Bioassay of transgenic pearl millet plants with rust pathogen P. penniseti:
Fungal bioassay on T1 progenies of three lines of T0 transformants
was done to assess the antifungal activity of chitinase gene against the fungal
pathogen P. penniseti. The symptom was observed for 10 days after fungal
infection. The size of Puccinia pustules on control untransformed plants
was bigger at 15 days post inoculation on the leaves. Rust infection of the
leaf segments from the transformed pearl millet plants was significantly reduced.
The trangenics showed significant disease resistance based on the degree of
symptoms. The untransformed control seedlings were very much susceptible to
fungal spores showing severe disease symptoms (Fig. 2g). Bioassay
on T1 progenies of T0 transformants showed monogenic segregation
of 3 resistant: 1 susceptible plants.
One of the major challenges in agriculture worldwide is to control the great
yield loss caused by pests and fungal pathogens. Realization of this objective,
however, in an environmental-friendly way necessitates integrated efforts from
plant breeders, pathologists and genetic engineers. A major application of gene
transfer technology is the introduction of agronomically useful traits into
crop plants. Genes of agronomic importance such as those that confer resistance
to disease and insects have been isolated from plants and other microorganisms.
Establishment of a high-frequency regeneration system is an essential prerequisite
for generation of transgenic plants. In this investigation, an efficient highly
improved protocol for plant regeneration from shoot-tip-derived embryogenic
calli of pearl millet, var. CO9, has been established. Starting from a single
shoot-tip explant, >500 plantlets could be regenerated within 55 days which
offers ample scope for adopting this technique for successful genetic transformation
of pearl millet.
Recently established efficient transformation systems for improvement of cereals
have made it possible to test the general usefulness of these genes in protecting
food crops. Pearl millet is one of the important coarse cereal crops for which
efficient transformation system has not been available (Vasil,
2008). We had previously studied various factors influencing the Agrobacterium-
mediated transformation with improved transformation efficiency of two cultivars
of pearl millet (unpublished data). Though, cultures of pearl millet are known
to be recalcitrant to in vitro manipulation, we have been able to achieve
high transformation efficiency with highly optimized conditions necessary for
successful transformation of pearl millet. Transgenic pearl millet lines derived
from CO9 genotypes were generated in this study. The significant number of GUS
positive independent transgenic lines showed improved transformation frequencies
of 4.5-6.5. This indicated that the Agrobacterium-mediated transformation
enhanced transformation efficiency compared to other direct methods (Arockiasamy
and Ignacimuthu, 2007; Gasparis et al., 2008;
Ceasar and Ignacimuthu, 2011).
Fungal diseases constitute a major challenge to millions of pearl millet farmers
throughout the tropical regions where pearl millet is grown. Rust is one of
the major biotic constraints in pearl millet production (Rachie
and Majmudar, 1980; Girgi et al., 2006).
Only recently transgenic pearl millet plants with improved agronomic traits
have been produced by introducing useful genes such as pin and afp for downey
mildew and rust resistance, respectively (Latha et al.,
2006; Girgi et al., 2006). The transformation
rates achieved using these methods are low compared to other cereals such as
rice, maize and wheat (Christou et al., 1992;
Becker et al., 1994; Brettschneider
et al., 1997).
This study also proved the efficiency and effectiveness of the super virulent
Agrobacterium tumefaciens LBA 4404 (pSB1/pKAN-Rchit 1.1) in transforming
pearl millet. The plasmid pSB1 contained extra copies of vir genes, thus broadening
the choice of Agrobacterium for monocot transformation. It has been suggested
that the presence of additional vir gene sequence may be important to transform
pearl millet cultivars as well as increase the transformation efficiency (Gelvin,
2000). In this study we report for the first time a significant enhancement
of fungal resistance in pearl millet. Similar superbinary vectors have been
used in other crop plants. Previous reports have also shown elevated chitinase
activity in transgenic canola (Brogue et al., 1991),
strawberry (Asao et al., 1997), rice (Nishizawa
et al., 1999), tobacco (Broglie et al.,
1989; Carstens et al., 2003), cotton (Emani
et al., 2003) and Italian ryegrass (Takahashi
et al., 2005) enhancing resistance to fungal diseases, although the
level of chitinase activity does not always correlate with the degree of disease
resistance (Nishizawa et al., 1999; Emani
et al., 2003).
The transgenic plants exhibited normal growth in terms of phenotype and yield
of seeds. PCR and Southern hybritization analysis proved the integration of
the transgenes into the pearl millet genome. The copy number of transgene varied
from 1-2. Multiple gene copies might cause unstable inheritance and transgene
silencing; therefore transgenic plants with single copy insertion are more important
(Ye et al., 2009; Ignacimuthu
and Raveendar, 2010). PCR analysis proved the integration of the transgene
and none of the amplification of the sequences beyond the T-DNA border was seen
when total DNA was subjected to PCR analysis with nptII and chitinase primers.
Southern blot analysis with Hind III digested DNA suggested that all the six
transgenic lines showed expected 3.1 kb size, indicating the integration of
the rice chitinase gene into the genome of pearl millet and proved that they
were derived from independent transformants. Segregation of the chitinase gene
in the next generation was examined by kanamycin resistance and GUS assay experiments.
Segregation analysis of these transgenics (independent T0 lines)
demonstrated that the transgenes were stably inherited to T1 progeny.
Fungal bioassays on T1 progenies of three primary transformants,
untransformed control (CO9) and susceptible check were done using a highly virulent
rust fungal pathotype. Initially the disease symptoms appeared as chlorosis
at the base of leaf lamina on the second leaf of susceptible T1 seedlings,
untransformed control and susceptible check. The disease symptoms developed
in all the subsequent leaves showed the progression of severe damage caused
with the pathogen giving a rusty appearance. All susceptible seedlings were
invariably stunted and eventually died. Conversely the resistant T1
seedlings which grew rapidly, did not show any of the disease symptoms; they
were healthy and attained maturity with normal seed fertility. The results of
bioassay study revealed a significant reduction of the spore germination sprayed
on transgenic pearl millets than the untransformed control plants. The results
of fungal bioassays on T1 progenies of three transformants amply
testified that the expression of chit11gene in transgenic pearl millet imparted
high-level of resistance against P. penniseti. The transgene used in
other crops viz., rice (Sridevi et al., 2005)
and italian ryegrass (Takahashi et al., 2005)
showed enhanced resistance to fungal pathogen.
In conclusion, transgenic pearl millet plants were regenerated utilizing optimized
Agrobacterium cocultivation and selection conditions. GUS staining and
chit11 gene southern blot analyses confirmed the successful introduction, integration,
inheritance and Mendelian R1 segregation of transgenes in pearl millet. The
introduced chit11 gene was found to be stably integrated into the genome of
pearl millet. The transgenic pearl millet plants showed improved fungal resistance
to rust pathogen, P. penniseti. Many protocols were developed and further
refined during the earlier period for the Agrobacterium-mediated transformation
of monocot cereals. Many of these transgenic cereals have previously reached
the field for large-scale cultivation. However, a genetic improvement program
for millets has been initiated only in recent years and has received less attention
despite their nutritional importance. No report is available till date for Agrobacterium-mediated
transformation of pearl millet (Ceasar and Ignacimuthu,
2009). This study proved the amenability of pearl millet to Agrobacterium-mediated
gene transfer and development of transgenic plants. The system developed here
may be utilized in future for inserting many agronomically important genes into
We thank the University Grants Commission, (New Delhi) for financial support.
We gratefully acknowledge Dr. S. Muthukrishnan, Kansas State University, USA
for providing the plasmid harbouring rice chitinase gene and Dr. R. Terada,
National Institute of Basic Biology, Japan for providing the binary vector for
cloning. We thank Tamil Nadu Agricultural University, Madurai, for assistance
in fungal bioassay. We thank Tamil Nadu Agriculture University, Coimbatore and
ICRISAT, AP, for proving seed materials. We are greatful to Dr. K. Veluthambi,
School of Biotechnology, Madurai Kamaraj University, Madurai for technical training.
We thank Dr. M. Gabriel Paulraj, Scientist, Entomology Research Institute, Loyola
College for guidance. We thank Mr. M. Ramakrishnan, ERI, Loyola, College, for
his Scientific support.
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