somatic embryogenesis, organogenesis and plant regeneration in taro (colocasia esculenta var....

ORIGINAL PAPER

Somatic embryogenesis, organogenesis and plant regenerationin taro (Colocasia esculenta var. esculenta)

Pradeep C. Deo Æ Robert M. Harding ÆMary Taylor Æ Anand P. Tyagi Æ Douglas K. Becker

Received: 27 May 2009 / Accepted: 21 July 2009 / Published online: 9 August 2009

� Springer Science+Business Media B.V. 2009

Abstract Callus was initiated in three different ‘‘escu-

lenta’’ taro cultivars by culturing corm slices in the dark on

half-strength MS medium supplemented with 2.0 mg/l 2,4-

dichlorophenoxyacetic acid (2,4-D) for 20 days followed

by subculture of all corm slices to half-strength MS med-

ium containing 1.0 mg/l thidiazuron (TDZ). Depending on

the cultivar, 20–30% of corm slices produced compact,

yellow, nodular callus on media containing TDZ. Histo-

logical studies revealed the presence of typical embryo-

genic cells which were small, isodiametric with dense

cytoplasms. Somatic embryos formed when callus was

transferred to hormone-free medium and *72% of the

embryos germinated into plantlets on this medium.

Simultaneous formation of roots and shoots during ger-

mination, and the presence of shoot and root poles revealed

by histology, confirmed that these structures were true

somatic embryos. Plants derived from somatic embryos

appeared phenotypically normal following 2 months

growth in a glasshouse. This method is a significant

advance on those previously reported for the esculenta

cultivars of taro due to its efficiency and reproducibility.

Keywords Somatic embryogenesis � Callus � Taro �TDZ � Colocasia esculenta var. esculenta

Introduction

Taro, Colocasia esculenta var. esculenta is an important

food crop in the South Pacific Island countries, Africa, Asia

and in the Caribbean (Opara 2001). About 400 million

people include taro in their diets with the highest per-

centage contributing to the diet in the Pacific Islands

(Ivancic 1992) where Colocasia esculenta var. esculenta is

highly preferred over the antiquorum variety. In addition to

its role in the diet, taro also has significant cultural and

economical importance.

Taro is mainly propagated by asexual multiplication

(Strauss et al. 1979; Ivancic 1992) which has resulted in

limited genetic variation. Consequently, the crop is par-

ticularly susceptible to newly emerging diseases or existing

diseases inadvertently introduced into regions where sus-

ceptible taro cultivars are grown. Conventional breeding is

one of the available approaches for genetic improvement of

taro, with techniques for pollination and seed recovery well

documented (Wilson 1990; Tyagi et al. 2004). However,

this strategy is limited by the availability of germplasm

containing the required trait and the low fertility in widely

accepted cultivars.

Molecular breeding by genetic transformation represents

an attractive alternative strategy to conventional breeding

as a single trait can be added to an existing elite cultivar.

However, prerequisite to the success of this approach is the

availability of suitable target tissue for transformation and

the development of an efficient regeneration system

whereby plants can be regenerated from single transformed

cells. Embryogenic callus is generally considered to be a

P. C. Deo � A. P. Tyagi

School of Biological and Chemical Sciences, Faculty of Science,

Technology and Environment, University of the South Pacific,

Suva, Fiji

P. C. Deo (&) � R. M. Harding � D. K. Becker

Centre for Tropical Crops and Biocommodities, Faculty of

Science, Queensland University of Technology, Brisbane,

QLD, Australia

e-mail: [email protected]

M. Taylor

Centre for Pacific Crops and Trees, Secretariat of the Pacific

Community, Suva, Fiji

123

Plant Cell Tiss Organ Cult (2009) 99:61–71

DOI 10.1007/s11240-009-9576-0

highly desirable target tissue because of the high popula-

tion of totipotent cells and embryos are unicellular in origin

thus reducing the likelihood of chimerism. Further, the

potentially large numbers of plants which can be produced

using embryogenesis provides an attractive system for

mass propagation of plantlets.

Although various types of explants have been used to

initiate organogenic callus in taro, (Abo El-Nil and Zettler

1976; Jackson et al. 1977; Yam et al. 1990), the use of

corm explants for the initiation of embryogenic or orga-

nogenic callus has not been reported to date in either of the

taro varieties, esculenta and antiquorum. In this paper, we

examined the effects of culture medium and genotype on

indirect somatic embryogenesis from corm explants of

Colocasia esculenta var. esculenta and report on the

development of a protocol for the initiation of embryogenic

callus from this explant.

Materials and methods

Plant material

Three cultivars of the esculenta taro variety were used in

this study: CPUK and CK-07 were both originally derived

from the Cook Islands while THA-07 was originally

derived from Thailand. Plants were provided by the Centre

for Pacific Crops and Trees (CePaCT)—Secretariat of the

Pacific Community (SPC) as virus-free accessions and

were multiplied using standard procedures (Tuia 1997) as

follows. Briefly, basal growth medium consisted of

Murashige and Skoog (1962) and contained 30 g/l sucrose,

7.75 g/l agar and the pH was adjusted to 5.8. Multiplication

was a three-stage process involving successively, plantlet

growth on basal medium supplemented with: (1) 0.5 mg/l

thidiazuron (TDZ) for 4 weeks, (2) 0.8 mg/l benzylami-

nopurine (BAP) for 3 weeks, and (3) 0.005 mg/l TDZ for

3 weeks. Following multiplication, small suckers were

removed and subcultured as individual plantlets in hor-

mone-free liquid MS medium for 2–4 weeks without

shaking followed by culturing in hormone-free agar-

solidified MS medium for 12–16 weeks.

Explant preparation

Leaves, leaf bases and roots were removed from the

plantlets to leave only the exposed corm. Transverse sec-

tions (1–2 mm thick) of the corms were cut by hand with a

scalpel and placed on callus initiation medium. Unless

otherwise stated, conditions for all cultures consisted of

25�C and 16 h photoperiod using cool white fluorescent

lamps and a photon flux density of 24 lmoles photons

m-2 s-1.

Culture medium for callus initiation

The basic culture medium used for callus initiation con-

sisted of half-strength MS medium, 30 g/l sucrose, 7 g/l

agar and various concentrations of growth regulators (2,4-

dichlorophenoxyacetic acid [2,4-D] and TDZ) with a pH of

5.8. Explants were cultured in 90 9 15 mm Petri dishes

containing 25 ml of medium. Corm slices were first placed

on callus initiation medium-stage I (CIM-I), which con-

sisted of the basic culture medium supplemented with

different concentrations of 2,4-D (0, 1.0, 2.0, 4.0 mg/l).

The corm slices were placed horizontally with the lower

cut surface (in relation to their original orientation within

the corm) in contact with the medium and cultured in the

dark for 20 days. After CIM-I treatment, explants were

transferred to callus initiation medium-stage II (CIM-II),

which consisted of the basic culture medium with various

concentrations of TDZ (0, 0.25, 0.5, 1.0, 2.0 mg/l). The

explants were placed on the culture medium in the same

manner as in CIM-I and continued to be cultured in the

dark. The specific combinations of growth regulator treat-

ments used in CIM-I and CIM-II are shown in Tables 1 and

2. In a separate experiment, the incubation time on CIM-I

was examined by culturing the explants for 10, 20, and

40 days using 2.0 mg/l 2,4-D only followed by transfer on

1.0 mg/l TDZ. The cultures were treated in the same

manner and same culture conditions as above.

Effect of genotype on callus initiation

The medium used to determine genotypic effects on

explant response was the basic culture medium with CIM-I

and CIM-II containing 2 mg/l 2,4-D and 1 mg/l TDZ or

1 mg/l 2,4-D and 0.5 mg/l TDZ, respectively. The explants

from the three cultivars, CPUK, CK-07 and THA-07 were

treated in the same manner as described above and under

the same culture conditions.

Regeneration, plant development and recovery

Callus was transferred to regeneration medium (RM) and

maintained in the dark for 2 weeks then cultured at low

light intensity (5 lmoles photons m-2 s-1). Three media

were examined as candidates for regeneration; the basic

culture medium contained either (1) no hormones, (2)

1 mg/l abscisic acid (ABA) or (3) 0.2 mg/l naphthalene

acetic acid (NAA) plus 0.1 mg/l kinetin. The cultures were

routinely checked for the presence of somatic embryos.

Any embryos formed were allowed to mature and germi-

nate on the same medium. After 2 months without sub-

culture, data was recorded and the percentage of callus

pieces producing somatic embryos and total number of

62 Plant Cell Tiss Organ Cult (2009) 99:61–71

123

embryos were recorded. Fully germinated embryos were

transferred to basic culture medium without hormones in

individual 28 ml McCartney bottles and cultured at higher

light intensity (24 lmoles photons m-2 s-1) for further

development. After the plantlets reached a height of

4–5 cm, they were acclimatized in a shade house. Plants

were potted in a 1:1 mix of soil and perlite. Humidity was

maintained by covering the pots with clear polyethylene

bags for 1 week and watering on alternate days. Percentage

survival was recorded 1 month after acclimatization while

the phenotype of surviving plants was assessed visually

after 2 months.

Histology

Embryogenic callus and putative somatic embryos were

fixed in formaldehyde: alcohol: acetic acid (FAA) (1:1:8 v/v)

for 4 days, dehydrated in a xylene and ethanol series, and

then infiltrated and embedded with paraplast and wax,

respectively. Thin sections (6 lm) were cut using a rotary

microtome. The sections were heat fixed to 3-aminopro-

pyltriethoxysilane (APES)-coated glass slides, dewaxed

and stained with either Ehrlich’s HX and Eosin or Safranin

O-Fast Green and observed under a compound microscope

(Olympus BX41).

Table 1 Effect of 2,4-D and TDZ on callus initiation and regeneration in taro (Colocasia esculenta var. esculenta) cv. CPUK

Plant growth regulator (mg/l) Number of explants

inoculated

% Explants producing

callus


embryogenic callus


organogenic callus2,4-D TDZ

0 0 100 0 0 0

1 100 85.8 0 0

2 100 97 0 0

1 0 83 78.9 0 0

1 91 94.6 4 ± 3.1 b 45.1 ± 3.9 b

2 93 99 8.8 ± 2.8 b 67.8 ± 5.1 a

2 0 100 96.9 0 0

1 100 98 34.8 ± 2.2 a 63.3 ± 6.0 a

2 110 98.2 7.3 ± 2.6 b 62.8 ± 3.5 a

4 0 96 65.5 0 0

1 90 82 2.4 ± 1.6 b 28.4 ± 5.5 c

2 92 95 3.5 ± 1.8 b 34.0 ± 5.2 c

Corm explants were maintained on various concentrations of 2,4-D for 20 days followed by transfer to various concentrations of TDZ

Values with means ± SEM are derived from 10 replicate Petri dishes with 8–11 explants per replicate. Within a column, means followed by the

same letters are not significantly different (P \ 0.05)

Table 2 Further refinement of 2,4-D and TDZ concentrations required for callus initiation and regeneration in taro (Colocasia esculenta var.

esculenta) cv. CPUK

Plant growth regulator (mg/l) Number of explants

inoculated


callus


embryogenic callus


organogenic callus2,4-D TDZ

1 0.25 108 97 17.5 ± 3.1 b 61.3 ± 5.0 a

0.5 120 100 27.1 ± 4.9 a 64.2 ± 4.5 a

1 130 96.2 13.5 ± 3.4 b 56.9 ± 5.7 a

2 115 96.4 10.3 ± 3.4 b 38.5 ± 4.9 c

2 0.25 110 100 9.7 ± 2.3 b 55.3 ± 5.7 b

0.5 110 88.2 2.2 ± 1.5 c 50.3 ± 9.2 b

1 100 100 31.8 ± 2.4 a 52.9 ± 7.6 b

2 100 95.5 3.7 ± 2.5 c 42.7 ± 6.8 c

Corm explants were maintained on various concentrations of 2,4-D for 20 days followed by transfer to various concentrations of TDZ


same letters are not significantly different (P \ 0.05)

Plant Cell Tiss Organ Cult (2009) 99:61–71 63

123

Statistical analysis

Each callus induction experiment comprised 10 replicate

plates, with each plate containing 8–16 explants. All cul-

tures were observed fortnightly and the final results were

recorded 100 days after initiating the cultures. Data was

recorded as the percentage of explants producing callus per

Petri dish. For regeneration experiments, seven replicate

Petri dishes were used with 7–10 callus pieces per repli-

cate. Data were analyzed by one-way analysis of variance

(ANOVA) using a 95% confidence interval. When

P \ 0.05, significant differences between individual treat-

ment means were determined using Fisher’s Least Signif-

icant Difference (LSD) test. All data were analyzed by

SPSS for Windows, version 11.

Results

Since no previous information on the development of

organogenic callus from taro corm slices has been reported,

the effect of factors such as hormone and media concen-

tration, duration of treatment and light/darkness on callus

initiation from previously used taro explants (meristems

and leaves) was initially investigated to determine which

factors warranted further investigation. Explants were

meristems, consisting of the apical dome and two sur-

rounding leaf primordial, and the youngest three leaves

covering the growing point with petioles and the lamina

from each leaf cultured separately. Of these, the most

promising results were obtained using petiole explants

from the third youngest leaf incubated in the dark in

medium containing 2,4-D for various periods (3, 6, 9, 12,

and 15 days) followed by transfer onto TDZ-containing

medium (results not shown). Based on this preliminary

observation, the same regime was trialed on corm sections

where the use of an increased culture period on 2,4-D

(20 days) resulted in a high frequency of explants pro-

ducing callus. As such, all subsequent callus initiation

experiments were conducted using corm explants cultured

in the dark.

Effect of 2,4-D and TDZ on callus initiation

The effect of various 2,4-D and TDZ concentrations on

callus formation and regeneration was examined. Callus

was initiated from corm explants in two stages: culture on

CIM-I containing various concentrations of 2,4-D for

20 days followed by transfer to CIM-II containing various

concentrations of TDZ. Transfer of the explants from

medium containing 2,4-D to medium without TDZ or vice

versa resulted in the formation of non-regenerable callus.

In contrast, regenerable callus, which later underwent dif-

ferentiation, formed when explants were cultured on media

containing 2,4-D followed by media containing TDZ

(Table 1).

After 15 days on all 2,4-D concentrations, approxi-

mately 50% of the explants swelled in the mid portion,

after which a soft watery callus developed (Fig. 1a). Upon

subsequent transfer to any of the TDZ-containing media,

callus formation became more rapid with the callus

appearing friable, cream/white and consisting of cells that

were large and vacuolated (Fig. 1b). After 45 days on

TDZ, smooth, white compact callus developed upon which

structures resembling adventitious shoots were identified

(Fig. 1c). After 75 days, yellow-cream, nodular callus

developed upon which, somatic embryo-like structures

formed (Fig. 1d). The consistency of this yellow-cream

callus was not such that cells could easily be separated

(friable) nor their association so tight that nodules would

fracture (compact) when manipulated with forceps. Rather,

nodule consistency was intermediate and malleable.

Histological analysis was subsequently carried out to

determine whether the callus was embryogenic or orga-

nogenic. The yellow-cream nodular callus, from which

translucent, globular embryo-like structures were pro-

duced, contained two cell types; (1) small, isodiametric

cells with large nuclei and dense cytoplasm typical of

embryogenic cells, and (2) large, vacuolated parenchymal

cells containing few plastids typical of non-embryogenic

cells. The embryogenic cells were generally located on the

periphery of the callus and had a clustered distribution,

interspersed with large parenchymal cells (Fig. 2a, b).

Histological study of the embryo-like structures indicated

they were true somatic embryos as both shoot and root

meristems were present in individual structures (Fig. 3).

Based on these analyses, yellow-cream nodular callus was

termed embryogenic. Histological analysis of the glossy,

compact, white callus, from which elongated opaque

structures were produced, was not done. Hereafter, this

type of callus is referred to as organogenic to distinguish it

from embryogenic callus.

In general, individual explants produced both types of

callus. Initially, only very few (2–3) explants formed

embryogenic callus on 1.0 mg/l TDZ but by 100 days, the

frequency increased (10–20). By 100 days, a few explants

(2) on 2.0 mg/l TDZ also produced a callus similar in

appearance to that formed on 1.0 mg/l. After approxi-

mately 110 days on TDZ-containing media, there was no

further development of either organogenic or embryogenic

callus. However, when maintained on the same medium for

over 120 days, the adventitious shoots and somatic

embryos began to develop into plantlets (Fig. 4a, c). By

this time, some of the white organogenic callus became

green and formed adventitious roots (Fig. 4b). There was


123

also increased production of cream, friable non-regenerable

callus with large vacuolated cells, which eventually over-

grew the cultures. The percentage of explants producing

embryogenic callus ranged from 2.4 to 34.8% with the

highest percentage observed from treatment with 2.0 mg/l

2,4-D followed by 1.0 mg/l TDZ (Table 1). In contrast, the

percentage of explants producing organogenic callus ran-

ged from 28.4 to 67.8% with the highest percentage being

from treatment with 1.0 mg/l 2,4-D followed by 2.0 mg/l

TDZ.

To further investigate the optimal conditions for

embryogenesis, a second experiment was conducted in

which the 2,4-D concentration was restricted to either

1.0 mg/l or 2.0 mg/l while four different TDZ concentra-

tions ranging between 0.25 and 2.0 mg/l were examined

(Table 2). The combination of 2.0 mg/l (2,4-D) followed

by 1.0 mg/l (TDZ) resulted in the highest percentage

(31.8%) of explants producing embryogenic callus. This

frequency was significantly higher than all other treatments

except 1.0 mg/l 2,4-D and 0.5 mg/l TDZ (27.1%). The

highest frequency of organogenic callus formation (64.2%)

occurred using a combination of 1.0 mg/l 2,4-D ? 0.5 mg/

l TDZ. However, the differences between treatments were

not significant.

Duration of exposure of explants to 2,4-D

To further investigate the regenerative response, explants

were incubated on 2,4-D for different time periods before

transfer to TDZ (Table 3). Exposure for durations of 10 or

40 days resulted in significantly fewer explants producing

embryogenic callus than 20 days (3.1, 11.9, and 30.8%,

respectively). Once again, the difference in the frequencies

of the organogenic response was not significant.

Fig. 1 Taro callus initiation and regeneration from corm explants cv.

THA-07. Callus on CIM-I was initially soft and watery (a) but after

transfer to CIM-II, cream friable callus formed (b). After 45 days on

CIM-II what appeared to be adventitious shoots formed on compact

white (organogenic) callus (c) and after 75 days what appeared to be

somatic embryos formed on yellow-cream nodular (embryogenic)

callus (d). Scale bar = 2 mm (a); 2 mm (b); 3 mm (c); 2 mm (d).

(Color figure online)


123

Fig. 2 Different cell types in embryogenic callus in taro cv. CPUK.

Cells thought to be embryogenic (EC) were small, isodiametric cells

with a large nucleus and dense cytoplasm while non-embryogenic

cells (NEC) appeared to be parenchymal cells which were large

vacuolated and contained distinct plastids (a). The embryogenic cells

are in clusters, which are separated by large parenchymal cells (b).

Scale bar = 50 lm (a); 200 lm (b)

Fig. 3 Histology of taro somatic embryos. (a) Mature somatic

embryo showing shoot apical meristem (SAM) and root apical

meristem (RAM) with vascular tissues (VT) (blue arrows). Higher

magnification view of SAM (b), RAM (c), and VT (d). Secondary

thickening in VT cells with distinct xylem-like pattern can be seen.

Scale bar = 200 lm (a and d), 50 lm (b and c). (Color figure online)


123

Effect of plant genotype on embryogenic callus

The effect of genotype was examined by comparing the

response of three esculenta cultivars, CK-07, CPUK and

THA-07, on two hormone regimes (Table 4). There was no

significant difference in the frequency of embryogenic callus

formation in the different genotypes when treated with

1.0 mg/l 2,4-D and 0.5 mg/l TDZ. However, when treated

with 2,4-D (2.0 mg/l) and TDZ (1.0 mg/l), the frequency of

the explants undergoing embryogenesis in THA-07 was

significantly lower than CK-07 and CPUK. A similar pattern

was also observed in the organogenic response.

Fig. 4 Taro organogenesis in the form of adventitious shoots (a) and adventitious roots (b) on white compact callus and somatic embryogenesis

on yellow nodular callus (c) cv. CPUK. Scale bar = 4 mm (a), 2 mm (b) and (c). (Color figure online)

Table 3 The effect of 2,4-D exposure period on callus initiation and regeneration in corm explants of taro (Colocasia esculenta var. esculenta)

cv. CPUK

Duration of exposure to

2,4-D (days)

Number of explants

inoculated

% Explants

producing callus


embryogenic callus


organogenic callus

10 100 99 3.1 ± 1.6 c 32.6 ± 7.4 b

20 130 99.2 30.8 ± 4.3 a 63.8 ± 3.6 a

40 160 100 11.9 ± 2.5 b 51.9 ± 5.2 a

Explants were exposed to 2,4-D (2.0 mg/l) followed by transfer to TDZ (1.0 mg/l)


same letters were not significantly different (P \ 0.05)


123

Regeneration, plant development and recovery

Embryogenic callus was transferred to half-strength MS

medium containing either no hormones, 1.0 mg/l ABA or

0.2 mg/l NAA plus 0.1 mg/l kinetin and cultured in darkness

for 2 weeks before transfer to low light. The use of hormone-

free medium resulted in the highest frequency of callus

pieces producing somatic embryos (47%) and the greatest

number of embryos per callus clump (10 ± 1.2) (Table 5).

Approximately 70% of callus pieces on ABA became

necrotic while *30% turned green and formed globular

structures which later developed into plantlets. In contrast,

while the majority of callus pieces on medium containing

NAA and kinetin remained healthy, only 6% produced

somatic embryos. Somatic embryos forming on callus first

appeared as small translucent globular structures. When left

on regeneration medium without subculture, embryos

enlarged and became opaque and irregular in shape.

Non-synchronous germination commenced spontaneously

on the same medium 30 days after embryo formation and

continued for another 50 days. The germination rate on

hormone-free medium was 70% compared with only *30%

using the other two media.

Further evidence to support the claim that the globular

structures previously observed in this study were somatic

embryos was provided by the observation that these

structures formed both shoots and roots simultaneously

when germinating (Fig. 5a). Since multiple somatic

embryos were left intact on callus pieces, germinated

embryos tended to be fused into clumps of plantlets

(Fig. 5b). Individual plantlets were separated, and trans-

ferred to individual culture vessels containing the basic

culture medium for further development (Fig. 5c). A total

of 120 CPUK plantlets were generated which continued to

grow and spontaneously form roots in half MS. Upon

reaching a height of 4–5 cm, plantlets were acclimatized in

a screen house. Plants from the same CPUK accession, but

which had not undergone the callus and regeneration

Table 4 The effect of genotype on callus initiation and regeneration in taro (Colocasia esculenta var. esculenta)

Cultivar Hormone regime

(2,4-D/TDZ) (mg/l)

Total number of

explants inoculated

% Of total explants

producing callus

Mean % of explants producing

embryogenic callus

Mean % of explants

producing organogenic callus

THA-07 1.0/0.5 160 100 21.9 ± 4.4 a 67.3 ± 3.9 a

2.0/1.0 160 100 13.2 ± 3.8 b 59.4 ± 3.4 b

CK-07 1.0/0.5 140 97.9 31.9 ± 5.7 a 66.7 ± 6.3 a

2.0/1.0 140 94.3 30.0 ± 3.8 a 77.9 ± 3.9 a

CPUK 1.0/0.5 150 100 18.7 ± 3.4 a 60.7 ± 7.0 a

2.0/1.0 140 100 28.6 ± 2.6 a 73.6 ± 2.2 a

Corm explants were maintained on medium containing 2,4-D (2.0 and 1.0 mg/l) for 20 days followed by transfer to medium containing TDZ (1.0

and 0.5 mg/l, respectively)


same letters are not significantly different (P \ 0.05). Data for the two different hormone regimes were analysed in two separate ANOVA tests

Table 5 The effect of growth regulators on plantlet regeneration from embryogenic callus of taro (Colocasia esculenta var. esculenta) cv.

CPUK

Regeneration

medium

Total

number

inoculated

callus pieces

% Callus

pieces

producing

somatic

embryos

Total number

of somatic

embryos

produced

Mean number of

somatic embryos per

embryogenic callus

piece

Total

number of

plantlets

produced

%

Conversion

Total

number of

plantlets

acclimatized

% Plantlets

surviving

acclimatization

� MS 49 47 160 10 ± 1.2 a 113 71.8 a 70 100

� MS ? ABA 56 25 52 3.9 ± 0.6 b 20 33.3 b 20 100

� MS ?

NAA ? KIN

70 5.7 50 6.1 ± 3.2 a 38 32.4 b 30 100

Embryogenic callus was transferred to various regeneration media and maintained in darkness for 2 weeks then transferred to low light

conditions for embryo formation, maturation and germination. Germinated embryos were then separated and transferred to hormone-free � MS

for further development prior to acclimatizing

Concentrations of ABA, NAA and KIN in medium were 1.0, 0.2, and 0.1 mg/l, respectively

Values with means ± SEM are derived from seven replicate Petri dishes with 7–10 callus pieces per replicate

Within a column, means followed by the same letters are not significantly different (P \ 0.05)


123

process, were also acclimatized for comparison. All 120

embryo-derived plantlets survived the acclimatization

process, continued to grow and, in comparison to the ori-

ginal accession, appeared phenotypically normal after

2 months (Fig. 5d). Phenotypic assessment was based on

visual inspection for abnormal morphology.

Discussion

In this study, a two step-protocol was developed to initiate

embryogenic callus from corm explants of taro. Initially,

the explants were cultured on half-strength MS medium

containing 2.0 mg/l 2,4-D (CIM-I) and maintained in

darkness for 20 days at 25�C followed by transfer to half-

strength MS containing 1.0 mg/l TDZ (CIM-II) while

maintaining the same light and temperature conditions.

The events leading to the formation of embryogenic

callus occurred in succession. Initially, soft, watery callus

formed which was followed by white-cream, friable callus,

both of which were non-regenerable. Glossy, compact,

white callus subsequently formed from which early shoot

development was observed. Finally, yellow–cream nodular

callus developed which produced translucent globular

structures. The sequential formation of different types of

callus observed in this current study may have been due to

changing conditions within culture medium over a long

period on CIM-II without subculture. Such changes may

have been hormone degradation, nutrient depletion and

osmotic stress. Alternatively, there may have been suc-

cessive formation of cell types each dependent on the

previous type.

The translucent globular structures with a distinct epi-

dermis which developed on the yellow-cream nodular

callus were assumed to be somatic embryos since these

features are highly characteristic (Vasil et al. 1984). The

embryogenic nature of these structures was further con-

firmed by histological analysis which showed clusters of

Fig. 5 Regeneration of taro plants from somatic embryos cv. CPUK.

a Early germinating somatic embryos showing shoot and root poles,

b clump of fused somatic embryos germinating, c germinated somatic

embryos after transfer to individual culture vessels for further

development, and d acclimatized somatic embryo-derived plants.

(Scale bar = 2 mm)


123

embryogenic cells located mainly on the periphery of the

callus mass which were interspersed with non-embryogenic

parenchymal cells. Similar embryogenic cell clusters have

been reported to form the ‘pre-embryogenic units’ (glob-

ular embryos) in maize (Samaj et al. 2003), while the large

parenchymal cells may act as ‘nurse cells’ (Ogita et al.

2001). The presence of two meristems in the globular

structures, most likely root and shoot poles, were an indi-

cator that these structures were true somatic embryos

(Thompson et al. 2001).

The concentration of 2,4-D had a significant effect on the

percentage of taro corm explants forming embryogenic

callus, with a lower percentage of explants forming

embryogenic calluses using low (1.0 mg/l) and high

(4.0 mg/l) concentrations of 2,4-D but a high frequency

using 2.0 mg/l. Previous studies have indicated that 2,4-D

induces somatic embryogenesis by influencing endogenous

IAA in explant tissues. For example, embryogenic carrot

cells grown in the presence of 2,4-D contained high levels of

IAA while a loss in embryogenic competency of the calli

during prolonged culture occurred concomitantly with a

reduction in the IAA levels (Ribnicky et al. 1996). The

induction of embryogenesis by modulation of endogenous

hormone levels may also be the case with TDZ. Though the

mode of action is currently unclear, it has been hypothesized

that TDZ either directly promotes somatic embryogenesis

due to its own biological activity (Visser et al. 1992) or

affects the endogenous ratios of endogenous auxins and

cytokinins (Visser et al. 1992; Panaia et al. 2004; Thomas

and Puthur 2004). TDZ has been shown to induce the

accumulation of both endogenous auxins and cytokinins in

legumes and herbaceous plants (Bhuiyan and Adachi 2002).

In this current study, TDZ had a significant effect on the

percentage of corm explants forming embryogenic callus;

high (2.0 mg/l) and low (0.5, 1.0 mg/l) concentrations

exhibited inhibitory and promoting effects, respectively.

Low concentrations of TDZ (0.5–1.0 mg/l) have been

reported to promote callus induction, somatic embryo for-

mation and germination in other monocots such as Colocasia

esculenta var. antiquorum (Thinh 1997), banana (Srangsam

and Kanchanapoom 2003) and bamboo (Lin et al. 2004).

The duration of exposure of the explants to 2,4-D was

also found to be critical in taro somatic embryogenesis,

since exposure for 10 and 40 days did not favour

embryogenesis whereas exposure for 20 days significantly

increased the percentage of explants producing embryo-

genic callus. It would appear that explants require a

threshold of auxin concentration and exposure time to

attain embryogenic competence but that an upper limit of

concentration or exposure could be exceeded. Conse-

quently, a 20 day treatment with 2,4-D was required for

cells to become competent for embryogenesis but transfer

to TDZ was required for embryogensis to occur.

Various explant, including petioles, shoot-tip, meris-

tems, young leaves and axillary buds, have been utilized in

taro tissue culture (Abo El-Nil and Zettler 1976; Jackson

et al. 1977; Yam et al. 1990, 1991; Chng and Goh 1994).

Although Chng and Goh (1994) used corm slices to gen-

erate shoots for micropropagation, such explants have not

been used for callus induction. In the present study, both

embryogenic and organogenic callus was initiated from

corm explants. Meristematic tissues have proved to be

the most suitable explant for somatic embryogenesis

(Lakshmanan and Taji 2000) and the meristematic cells

present in the axillary buds on the periphery of the taro

corms may be contributing to the formation of regenerable

callus. In adequate culture conditions, the meristematic

tissues/cells are competent to undergo an embryogenic

developmental program thus resulting in the production of

somatic embryos (Guerra et al. 2001).

Although genotypic variation in the somatic embryo-

genic response has been observed in other species (Kim

et al. 2004; Kayim and Koc 2006), only moderate effects

were observed for the three cultivars of taro examined in

this study. Further studies using additional cultivars are

required to determine the full extent of such effects. The

fact that three taro cultivars did not differ significantly in

their response to embryogenesis when treated with 1.0 mg/l

2,4-D and 0.5 mg/l TDZ, but did when treated with 2.0 mg/l

2,4-D and 1.0 mg/l TDZ highlights the possibility that other

cultivars may have a significantly different response.

The taro callus initiation media used in this study is

similar to media used in other taro studies and for many

other species, the difference being growth regulators type,

concentration and timing. Regeneration in C. esculenta var.

antiquorum has been achieved using medium containing

NAA and kinetin (Abo El-Nil and Zettler 1976) and in C.

esculenta var. esculenta using (1) taro corm extract (TE)

plus 2,4,5-T and (2) taro corm extract plus coconut water

(CW) for shoot and root formation, respectively (Yam et al.

1990). In this study, the presence of NAA and kinetin or

ABA in the regeneration medium appeared to interfere

with embryo formation, maturation and germination pro-

cesses which instead occurred in a single step on hormone

free half-strength MS medium without the addition of

complex organic additives such as TE and CW.

In summary, a highly efficient and reproducible protocol

for the initiation of embryogenic and organogenic callus

from corm slices has been established for Colocasia

esculenta var. esculenta by modifying and optimizing

various in vitro parameters. This represents a significant

advancement on previously published protocols and may

lead to a method for proliferating regenerable callus on

solid media or liquid culture as a source of tissue for large-

scale regeneration and a source of embryogenic cells for

genetic transformation.


123

Acknowledgments The authors wish to thank New Zealand’s

International Aid and Development Agency, University Research

Committee-The University of the South Pacific, Faculty of Science,

Technology and Environment-The University of the South Pacific for

all financial assistance and Centre for Tropical Crops and Bio-

commodities-Queensland University of Technology, Secretariat of

the Pacific Community and The University of the South Pacific for

technical support during this project. PCD was a PhD candidate at

The University of the South Pacific.

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