Regeneration and genetic transformation of yam

www.iita.org A member of the CGIAR consortium

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


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.


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


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


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


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


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


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.


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


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


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


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.


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


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.


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.


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.


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


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.


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


Induction of Indirect somatic embryos


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.


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


(0.4mg/l) at the end of 1st

subculture

a

c

b

d


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


a, b, c, d. Germination

of callus in MS medium


(0.4mg/l)+ ascorbic acid

(25mg/l)

3rd subculture

Germination

a b

d c


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


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


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.


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


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)


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


75 Green shoots regenerated with pale patches

100 Shoots regenerated but bleached

125 Albino shoots regenerated, arrested development




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)


Transformation Procedure


A B C

D E F

Transformation of D. rotundata 2346


Transformation of D. rotundata

A B

TDr 2346 TDr 2579


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


B C

D E F

A

Expression of reporter genes in

transgenic plants

G


PCR and RT-PCR analysis

PCR

RT-PCR


Dot and Southern blot analysis

Optimized DNA extraction protocol (CTAB) of Yam


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


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


Transgenic plants in green house


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


Embryogenic callus showing

gusA gene expression

a, Control

b,c, d, Callus showing gusA gene

expression

Histochemical staining in callus

a

c d

b


Selection of embryogenic callus in

Hygromycin (15 mg/l)

Stage 3 selection: 15mg/l Hygromycin


Thank You