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International Institute of Tropical Agriculture(IITA), Nairobi, Kenya
R. Manoharan, E. Nyaboga, J. Tripathi & L. Tripathi
Regeneration and genetic transformation of yam
R4D Meeting
25th November 2015
Venue: IITA Ibadan
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Introduction
Yam (Dioscorea spp.) is an economically important food crop in many tropical
countries especially in West Africa, South Asia, and the Caribbean.
It is the second most important root and tuber crop in the world after cassava in
terms of production
There are 600 Dioscorea species, however, only 10 of about 90 edible species are
regularly cultivated for food.
D.rotundata is the most popular and economically important yam in west and central
africa
The consumer demand for yam is very high in sub-Saharan Africa, but the yam
production is declining in this region due to factors including diseases and pests,
high costs of planting material, and decreasing soil fertility.
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Introduction
Nematodes are of particular concern because, apart from causing
significant reduction in tuber yield and quality, they facilitate fungal
and bacterial attacks.
A major economic pest of yam is Scutellonema bradys, known as
the yam nematode and causal agent of dry rot.
The genetic improvement of yam through conventional breeding is
very difficult due to - long growth cycle (8-10 months), dioecious
and poor flowering nature, polyploidy, vegetative propagation and
heterozygous genetic background.
In view of these problems, development of tissue culture
regeneration systems amenable for transformation is a pre-requisite
for the improvement of yam cultivars.
So far, the application of biotechnological tools particularly genetic
transformation to develop improved varieties has been limited by
the absence of efficient regeneration protocols
Source: IITA images
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Direct Organogenesis
A protocol for direct regeneration was established using nodal explants of
yams (D. rotundata; D. alata)
Nodal explants excised with two nodes placed vertically produced an average of
3 shoots with 5 nodes per explant on MS medium supplemented with BAP (0.05
mg/l), NAA (0.01 mg/l), ascorbic acid (25 mg/l), sucrose (30 g/l) and gelrite (2.4 g/l)
The medium was named as yam basic media(YBM)
The protocol was improved by adding glutamine (10 mg/l) which enhanced
multiple shoot induction (4 shoots per explant) with total of 9 nodes when compared
to YBM which produced about 3 shoots per explant with 5 nodes from single explant
Validation of protocol with other cultivars of D. rotundata and D. alata
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Literature review on somatic embryogenesis
Yam Spp Initial explant Media& harmones Authors Journal
D. rotundata Zygotic embryos MS, NAA Osifo
1988
Journal of plant
physiology
D.Composita, D.
cayenensis
Excised Zygotic
embryos
MS, 2, 4-D, Zeatin, Viana and
Mantell,
1989
Plant Cell Tissue and
Organ Culture
D. opposita Stem segments without
A.buds
MS, B5, 2, 4-D
BAP
Nagasaw
a and
Finer,
1989
Plant science
D. alata
Root segments
MS, 2, 4-D, 2iP,
ABA, GA3
Twyford
and
Mantell,
1996
Plant Cell Tissue and
Organ Culture
D. alata Nodal stem segments MS, 2, 4-D,
Picloram, ABA,
BAP
Belarmino
and
Gonzales,
2008
Annals of Tropical
Research
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Explants (immature leaf lobes, nodal stem, petiole, root tips and axillary bud) of
D. rotundata (TDr 2436) were used for callus induction on MS medium
supplemented with 2,4-D , NAA and Picloram (0.5-10 mg/l).
For the induction of axillary buds, nodal stem segments were excised from 8 week
old In vitro grown plants and cultured in MS medium supplemented with BAP (1 mg/l)
for 3 to 4 days in dark.
Explant for somatic embryo induction
a b c
a. In vitro grown plants b. Excised nodal segments c. Induction of Axillary bud
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Somatic embryogenesis
Immature leaf
Petiole
Nodal stem
Axillary bud
Root tips
MS+ NAA (0.5-9 mg/l)
MS+ 2,4-D (0.5-9 mg/l)
Explants used
Axillary bud
MS+ 2µM Cuso4, 2% sucrose)
+Picloram (0.5-12mg/l)
MS+2µM Cuso4, 2% sucrose
+Picloram(0.5- 12 mg/l)+
CSH (600mg/l)+Proline (1g/l)
Medium and harmones
Callus- NAA
Non Embryogenic, watery,
producing root
2,4-D Lower conc< 1.5mg/l
Compact callus
with globular structures
Result
Morphological appearance
Callus
Embryogenic
with globular structures
Callus
Milky white globular
structures
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Auxins
(mg/l) Explants Immature leaf petiole Node Stem internode Axillary bud Root segments
Percentage
of somatic
embryo
induction
No. of
DSLS/expla
nt
after 4
week of
culture
Callus
inducti
on
after 8
week
of
culture
Percentage
of somatic
embryo
induction
No. of
DSLS/
explant
after 4
week of
culture
Callus
inducti
on
after 8
week
of
cultur
e
Percenta
ge of
somatic
embryo
induction
No. DSLS/
explant
after 4
week of
culture
Callus
inductio
n after 8
week of
culture
Percentage of
somatic embryo
induction
No. DSLS/
explant
after 4
week of
culture
Callus
induction
after 8
week of
culture
Percentag
e of
somatic
embryo
induction
No.
DSLS/
explant
after 4
week of
culture
Callus
induct
ion
after 8
week
of
cultur
e
Percenta
ge of
somatic
embryo
induction
No. DSLS/
explant
after 4
week of
culture
Callu
s
induc
tion
after
8
week
of
cultu
re 2, 4-D
0.5
1.5
3.0
5.0
7.0
9.0
NAA
0.5
1.5
3.0
5.0
7.0
9.0
Picloram
0.5
1.5
3.0
5.0
7.0
9.0
32.3±0.61a
28.3±0.66b
17.6±0.42d
11.6±0.76g
5.6±0.55h
-
-
-
-
-
-
-
-
6.3±0.33h
10.3±0.42g
22.6±0.61c
15.6±0.42e
12.6±0.49f
4.6±0.66a
4.3±0.49ab
2.6±0.42cd
1.3±0.42d
-
-
-
-
-
-
-
-
-
1.3±0.33d
2.0±0.57cd
3.3±0.42abc
3.0±0.36bc
2.3±0.33cd
++
++
+
-
-
-
CWR
CWR
CWR
CWR
CWR
CWR
++
++
++
++
+
+
41.6±0.49a
33.6±0.66b
25.0±0.57c
18.3±0.71d
8.6±0.61f
-
-
-
-
-
-
-
-
7.8±0.40f
13.3±0.80e
24.6±0.66c
17.3±0.49d
14.6±0.49e
5.3±0.49a
4.6±0.55ab
3.6±0.66bc
2.3±0.49cd
1.3±0.42d
-
-
-
-
-
-
-
-
1.3±0.33d
2.3±0.49cd
3.6±0.42bc
3.0±0.57c
2.3±0.49cd
++
++
+
-
-
-
CWR
CWR
CWR
CWR
CWR
CWR
+++
++
++
+
+
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
10.6±0.71d
15.0±0.81c
27.6±0.88b
38.3±0.71a
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2.3±0.42c
3.3±0.49bc
4.6±0.61ab
5.3±0.66a
++
++
++
+
+
-
CWSR
CWSR
CWSR
CWR
CWR
CWR
+++
+++
++
+
+
+
18.3±0.49c
15.3±0.61d
11.3±0.80e
-
-
-
-
-
-
-
-
-
-
-
6.6±0.55f
13.6±0.76d
22.3±0.91b
29.6±0.84a
2.3±0.49bc
2.0±0.57c
1.3±0.42c
-
-
-
-
-
-
-
-
-
-
-
1.3±0.42c
2.3±0.42bc
3.3±0.55ab
4.0±0.57a
+
+
+
-
-
-
CWR
CWR
CWR
CWR
CWR
CWR
+
+
++
+
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
12.6±0.80d
29.3±0.84c
34.3±0.80b
42.6±0.84a
-
-
-
-
-
-
-
-
-
-
-
-
-
-
3.0±0.51b
4.3±0.66b
6.3±0.66a
7.3±0.55a
+
+
+
-
-
-
CWSR
CWSR
CWSR
CWR
CWR
CWR
+++
+++
+++
++
++
++
25.6±0.84a
19.6±0.66b
14.3±0.71c
11.6±0.98d
-
-
-
-
-
-
-
-
-
8.3±0.55e
16.6±0.55c
14.3±0.95c
8.0±0.81e
-
3.6±0.49a
3.3±0.49ab
2.3±0.49abc
1.3±0.49c
-
-
-
-
-
-
-
-
-
2.3±0.49abc
3.3±0.42ab
3.0±0.68ab
1.6±0.55bc
-
+
+
+
-
-
-
CWHR
CWHR
CWHR
CWR
CWR
CWR
++
++
++
+
+
+
Table 1 Influence of auxin concentrations on different
explants of D. rotundata on MS medium after 4 and 8 weeks
of culture
+++ -Proliferative callus; ++ -Moderately proliferative; + -less proliferative; - No response; CWR-Callus with root; CWSR-Callus with shoot and root; CWHR-Callus with hairy roots.
Data represents mean value±SE of three replicates; each experiment was repeated twice. Mean values followed by same letters within a column are not significantly different
according to Duncan’s multiple range test (DMRT) at 5% level.
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Effect of 2, 4-D on callus induction
a
a). Callus after 6 week of culture in 2,4-D
(0.5mg/l) from immature leaf ; b). Axillary bud
explant cultured in 2,4-D (0.5mg/l);c) Callus
from nodal stem explants;d) callus from in vitro
excised root segments
Among the explants tested, the
immature leaf lobes and
petioles produced callus with
globular structures at 0.5 mg/l.
Axillary bud didn’t produce
callus at all concentrations
tested.
The nodal stem segments
produced pale yellow callus
The root tip explants produced
yellow globular structures in
media containing 0.5 mg/l 2,4-D
but the structures could not
multiply in the same medium
b
c d
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a). Immature Leaf
b). Petiole
c). Axillary bud
d).Nodal explants
e,f). Root explants
a b
c d
e
Effect of NAA on callus induction
At lower concentrations
(0.5-1.5 mg/l) the
explants produced a
loose mass of callus
which was whitish and
watery.
All the explants formed
roots at all
concentrations of NAA
tested.
f
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The callus obtained from all the explants did not regenerate on further
transfer to hormone free MS medium and later on MS supplemented with
BAP (0.1 mg/l) and ascorbic acid (25 mg/l) even after 8 weeks of culture.
All the calli produced only roots in MS basal medium and MS
supplemented with BAP medium.
Regeneration
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Picloram
Concentrations(mg/
l)
Percentage of
DSLS after 4
week of culture
No of DSLS
after 4 week of
culture
Percentage of
embryogenic
callus induction
after 8 week of
culture
Fresh weight of
the callus(mg)
0.5 15.6±0.98h 2.3±0.49d 99±0.36a 222.3±0.55a
1.5 17.6±0.76h 2.6±0.55d 98±0.57a 207.3±0.98b
3.0 27.3±0.88g 4.0±0.57d 97±0.57ab 185.3±0.66c
5.0 36.6±0.88f 6.3±0.71c 95±0.96bc 179.3±0.98d
7.0 48.6±0.88e 8.6±0.95b 94±0.89c 171.3±0.88e
9.0 52.6±0.80d 11.0±0.93a 94±0.93c 167.6±0.88f
12.0 64.6±0.80a 12.3±0.66a 94±0.57c 161.3±0.66g
14.0 60.3±0.98b 11.3±0.88a 88±0.85d 153.6±0.91h
16.0 57.3±0.95c 10.6±0.76ab 83±0.81e 145.3±0.55i
Data represents mean value±SE of three replicates; each experiment was repeated twice. Mean values followed by
same letters within a column are not significantly different according to Duncan’s multiple range test (DMRT) at 5%
level.
Table 2 Effect of different picloram concentrations on induction of DSLS
and embryogenic callus from axillary bud of D.rotundata on MS medium
supplemented with 0.318 mg/l copper sulfate and 2% sucrose after 4 and 8
week of culture.
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Effect of Picloram for callus induction
The effect of auxin picloram (0.5-12
mg/l) was tested for callus induction
using axillary buds as explants.
Picloram concentrations (above 7
mg/l) produced compact callus with
globular structures whereas the
axillary buds cultured on lower
concentrations (below 3 mg/l)
produced soft callus
a) Picloram (12 mg/l) b) Picloram (0.5 mg/l)
a b
c). Microscopic view of the callus in
MS+Picloram (12mg/l) after 8 week of culture
c
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Regeneration
a
b
a) Callus placed in 1/2MS BAP (0.05 mg/l)
and IAA (0.1 mg/l)
b) Callus placed in MS BAP (1 mg/l)
The callus derived from Picloram 12
mg/l turned green after transferring to
light on hormone free MS medium and
produced roots.
when transferred to full and half strength
MS supplemented with BAP (1, 3 or 5
mg/l) and combination with BAP (0.05
mg/l) and IAA (0.1 mg/l) produced roots
only.
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c
d
c). Callus placed in 1/2MS GA3 (5 mg/l)
and IAA (0.1 mg/l)
d). Callus placed in 2MS GA3 (5 mg/l)
Regeneration
The callus still produced only roots in full and
half strength MS medium supplemented with
GA3 (1, 3, 5 mg /l) even after 3 subcultures.
The number of roots were even higher on
medium supplemented with GA3 in
comparison to BAP.
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Picloram
concentrations(mg/l)
Percentage of
DSLS after
4 week of culture
No of DSLS
after 4 week of
culture
Percentage of
milkywhite
embryogenic
callus after 8 week
of culture
Fresh weight of
the
callus(mg)/explant
0.5 67.6±0.91c 11.6±0.95c 67.3±0.88a 526.6±0.66a
1.5 69.6±0.88c 14.3±0.95b 61.3±0.71b 488.3±0.66b
7.0 86.3±0.66b 19.0±0.85a 43.6±0.66c 402.3±0.76c
12.0 89.6±0.88a 19.6±0.76a 31.3±0.98d 348.3±0.84d
Table 3 Effect of Casein hydrolysate and Proline concentration on induction of
DSLS and milky white embryogenic callus from axillary bud of D.rotundata on
MS medium supplemented with 0.318 mg/l copper sulfate and 2% sucrose after
4 and 8 week of culture.
Data represents mean value±SE of three replicates; each experiment was repeated twice. Mean values followed by same
letters within a column are not significantly different according to Duncan’s multiple range test (DMRT) at 5% level.
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a). Callus from MS
Picloram in
combination with
casein hydrolysate
(600 mg/l)
and proline (1g/l) after
8 week of culture
a,b) 1.5mg/l;
c, d) 3 mg/l
e, f) 7mg/l
a
e
d c
b
f
c
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Picloram
concentrations(mg/l)
Activated
charcoal(%)
Purple coloration
of the callus
Percentage
of
indirectsom
atic embryo
induction
No of somatic embryos
per callus
0.5 0 + - Roots
0.2 ++ 43.6±0.66c 51.3±0.98c
1 +++ 70.6±0.84a 87.3±0.76a
1.5 0 - - Roots
0.2 ++ 36.6±0.80d 38.6±0.88d
1 +++ 63.3±0.88b 72.3±0.55b
7.0 0 - - Roots
0.2 + 22.6±0.76e 16.3±0.84f
1 ++ 38.0±0.57d 34.6±0.55e
12.0 0 - - Roots
0.2 - 13.6±0.91f -* 1 + 24.6±0.80e -*
Table 3 Influence of Activated charcoal on somatic embryo induction
from the callus derived frompicloram concentrations supplemented with
casein hydrolysate and proline on MS medium after 6 week of culture.
*callus has compact globular structures instead of shiny small globular embryos. Data represents mean value±SE of three
replicates; each experiment was repeated twice. Mean values followed by same letters within a column are not significantly
different according to Duncan’s multiple range test (DMRT) at 5% level.
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The calli turned purple when
transferred to MS medium
supplemented with activated
charcoal (1%) after 6 week of
culture
a, b, callus in activated
charcoal medium
a
b
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Picloram
Concentrations(mg/l)
BAP(mg/l) No of cotyledonary
collar per callus
Germination
response(%) 8
weeks
Germination
response(%)
16 weeks
0.5 0 45.3±0.49d 5.3±0.66f 12.6±0.95gh
0.4 68.6±0.80a 24.6±0.84a 44.3±0.91a
1 51.6±0.49c 16.0±0.89c 28.3±0.88c
1.5 0 32.0±0.93f 4.3±0.49fg 11.0±0.96h
0.4 63.0±0.85b 19.3±0.76b 37.6±0.91b
1 41.3±0.66e 12.6±0.76d 21.6±0.71e
7.0 0 14.6±0.66h 2.6±0.42g 7.3±0.66i
0.4 21.3±0.98g 15.6±0.66c 25.6±0.80d
1 15.3±0.66h 9.3±0.91e 15.6±0.76f
12.0 0 0 0 0
0.4 14.3±0.66h 8.3±0.84e 14.6±0.95fg
1 5.3±0.55i 2.6±0.61g 5.3±0.71i
Table 5 Effect of BAP on germination of somatic embryos in
MS medium after 8 and 16 week of culture.
Data represents mean value±SE of three replicates; each experiment was repeated twice. Mean values followed by
same letters within a column are not significantly different according to Duncan’s multiple range test (DMRT) at 5% level.
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Development of tiny
globular structures which
later formed as a opening
on the surface of the purple
callus in MS medium
supplemented with BAP
(0.4 mg/l)
a, b, c, d. callus in MS medium
supplemented with BAP
(0.4mg/l) at the end of 1st
subculture
a
c
b
d
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Formation of well developed
cotyledonary collar and
subsequent germination from
the collar at end of 2nd
subculture in MS medium
supplemented with BAP (0.4
mg/l).
Hook type of germination with
cotyledonary collars(Twyford
and Mantell 1996)
a, b, c, d, e, f Germination of
callus in MS medium
supplemented with BAP (0.4
mg/l) at the end of 2nd
subculture
a
f
d c
b
e
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a, b, c, d. Germination
of callus in MS medium
supplemented with BAP
(0.4mg/l)+ ascorbic acid
(25mg/l)
3rd subculture
Germination
a b
d c
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The regeneration efficiency of
callus obtained from MS medium
supplemented with 0.5 mg/l
picloram when transferred to
germination medium was 44%.
Total duration of regeneration is
8 to 9 months
a, b, Germinated plants in MS
medium supplemented with BAP
(0.4mg/l)+ ascorbic acid (25mg/l) after
4th subculture
b, c Germinated plants in YBM
medium
Germination
a
d c
b
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In vitro plants
acclimatized in
screen house with
90% efficiency
a, b, c, d). Hardened plants in
screen house
In vitro plants acclimatized in screen house
a b
d c
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Agrobacterium-mediated genetic transformation
of yam (Dioscorea rotundata)
• No report of any existing genetic transformation of yam (Dioscorea
rotundata and alata) with evidence of stable integration of transgenes
• Few reports of transient transformation of D. alata (Tör et al., 1993; 1998)
and D. rotundata (Quain et al., 2011)
• Recently established Agrobacterium-mediated transformation system for
D. rotundata using meristematic tissues
• The results of this study has been published in Frontiers in Plant Science,
5(463), 2014, 1— 14.
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0 mg/l 5 mg/l
7.5 mg/l 10 mg/l 15 mg/l
Concentration (mg/l) Explant development/Regeneration response
0 Green shoots regenerated
5 Green shoots regenerated
7.5 Axillary bud induced but no shoot production
10 No shoots regenerated
15 No shoots regenerated and explants were bleached
Kill curve analysis of antibiotics
Hygromycin (0 – 15 mg/l)
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0 mg/l
150 mg/l
100 mg/l 75 mg/l
175 mg/l 200 mg/l
Kanamycin (0 – 250 mg/l)
Concentration (mg/l) Explant development/Regeneration response
0 Green shoots regenerated
75 Green shoots regenerated with pale patches
100 Shoots regenerated but bleached
125 Albino shoots regenerated, arrested development
150 No shoots regenerated
200 No shoots regenerated
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Left
Border Right
Border
35S
Poly A
Nos
Poly A
gusA 35S P hpt
MCS
pCAMBIA1301
Left
Border Right
Border
35S
Poly A
Nos
Poly A
MCS
pCAMBIA2301
npt II 35S P
35S
P
gusA 35S P
Lac Z
alpha
Catalase
Intron
Catalase
Intron
Lac Z
alpha
Left
Border
35S
Poly A
Nos
Poly A
gfp 35S P nptII
MCS
pCAMBIA2300-gfp
35S
P Lac Z
alpha
Plasmid vectors
(I)
(II)
(III)
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Plasmid
construct
Cultivar Agrobacterium
strain
No. of regenerated plants
on selective media
Transformation
efficiency (%)
pCAMBIA1301 TDr2436 EHA105 9.4 ± 0.50a 18.2 ± 0.37a
LBA4404 8.4 ± 0.24ab 16.2 ± 0.37ab
TDr2579 EHA105 6.2 ± 0.37cde 12.8 ± 0.37cd
LBA4404 7.6 ± 0.50bc 14.4 ± 0.24c
pCAMBIA2301 TDr2436 EHA105 7.4 ± 0.40bc 14.8 ± 0.37c
TDr2579 4.8 ± 0.37e 10.2 ± 0.37e
pCAMBI2300-gfp TDr2436 EHA105 7.2 ± 0.80bcd 14 ± 0.31c
TDr2579 5.8 ± 0.37de 9.4 ± 0.24e
Effect of Agrobacterium strains, cultivars, selectable marker and
reporter genes on transformation efficiency of D. rotundata
Summary: Factors affecting Transformation
Efficiency
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B C
D E F
A
Expression of reporter genes in
transgenic plants
G
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Dot and Southern blot analysis
Optimized DNA extraction protocol (CTAB) of Yam
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Plasmid
construct
Cultivar No. of axillary buds used No. of explants shooting on
selective media ( 3 cycles )
pCAMBIA1301 Amola 50 23
TDr2288 50 18
pCAMBI2300-gfp Amola 50 17
TDr2288 50 14
Transformation of Amola and TDr 2288
Amola TDr2288
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Regeneration of yam
transgenic yam cultivars in
selection medium.
a, TDr Amola with pC2301
b, Tda 00/0060 with pC1301
c) Tda 1304 with pC1301
d) TDr Amola with pC1301
Regeneration of transgenic Yam cultivars
a b
c d
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Embryogenic callus infected
with Agrobacterium strain
EHA 105 harboring plasmid
pCAMBIA 1301(gusA
reporter gene)
Selection of callus in
selection medium (MS+0.4
mg/l BAP) containing 7.5
mg/l hygromycin and
carbenecillin 250 mg/l
(Stage 1 selection)
a, b) Callus in stage 1 selection
Agrobacterium-mediated transformation
in embryogenic callus
a
b
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Embryogenic callus showing
gusA gene expression
a, Control
b,c, d, Callus showing gusA gene
expression
Histochemical staining in callus
a
c d
b
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Selection of embryogenic callus in
Hygromycin (15 mg/l)
Stage 3 selection: 15mg/l Hygromycin
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