ORIGINAL PAPER

Plant regeneration via somatic embryogenesis in Drimia robusta

Ponnusamy Baskaran • Johannes Van Staden

Received: 27 March 2014 / Accepted: 9 June 2014 / Published online: 20 June 2014

� Springer Science+Business Media Dordrecht 2014

Abstract A simple efficient in vitro plant regeneration

system was developed by direct and indirect somatic

embryogenesis of Drimia robusta, a medicinal plant

extensively used in South African traditional medicine.

Different developmental stages of somatic embryos (SEs:

globular embryos, partial pear-shaped embryos and club-

shaped embryos), club-shaped cotyledon initiation, plu-

mule initiation and plantlets were directly obtained from

leaf explants on Murashige and Skoog (MS) medium

containing 3.5 % (w/v) sucrose and different plant growth

regulators (PGRs). In MS medium containing 3.5 % (w/v)

sucrose and supplemented with 10 lM picloram, 1 lM

thidiazuron (TDZ) and 20 lM glutamine, a higher number

of SEs and plantlets were achieved. These were established

onto half-strength MS medium followed by successful

acclimatization (100 %) in the greenhouse. Liquid somatic

embryo medium (SEML) containing 500 mg of friable

embryogenic callus on MS medium supplemented with

different concentrations and combinations of PGRs and

organic elicitors produced different stages of SEs. Somatic

embryo production was enhanced by 0.5 lM picloram,

1 lM TDZ and mebendazole treatment. The highest

number of plantlets (9.0 ± 0.70) was obtained in SEML

containing 0.5 lM picloram, 1 lM TDZ and 25 mg l-1

haemoglobin. All the cotyledon and plumule embryos

germinated on half-strength MS medium, however 90 % of

SEs germinated on half-strength MS medium containing

0.5 lM naphthaleneacetic acid. All plantlets were suc-

cessfully acclimatized in the greenhouse. This first report

of D. robusta somatic embryogenesis provides an oppor-

tunity to control extinction threats, ensure germplasm

conservation and provides a system for analysis of bioac-

tive compounds and bioactivity.

Keywords Cell suspension � Drimia robusta � Medicinal

plant � Organic elicitors � Picloram � Somatic

embryogenesis � Thidiazuron

Abbreviations

2,4-D 2,4-Dichlorophenoxyacetic acid

BA 6-Benzyladenine

FEC Friable embryogenic callus

HB Haemoglobin

MS Murashige and Skoog medium

MBZ Mebendazole

NAA a-Naphthaleneacetic acid

OEs Organic elicitors

PGRs Plant growth regulators

PPF Photosynthetic photon flux

SEs Somatic embryos

SCV Settled cell volume

SEML Liquid somatic embryo medium

TDZ Thidiazuron

Introduction

Drimia robusta Baker (Hyacinthaceae) is an important

southern African medicinal plant, especially in KwaZulu-

Natal province (Eloff 1998). It contains the medicinally

important bufadienolide, proscillaridin A (PsA). This plant

is used to treat expectorant, emetic, diuretic, heart tonic and

feverish colds (Hutchings 1989; Pujol 1990; Van Wyk and

Gericke 2000). The leaves and bulbs of D. robusta exhibit

P. Baskaran � J. Van Staden (&)

Research Centre for Plant Growth and Development, School of

Life Sciences, University of KwaZulu-Natal Pietermaritzburg,

Scottsville 3209, South Africa

e-mail: rcpgd@ukzn.ac.za

123

Plant Cell Tiss Organ Cult (2014) 119:281–288

DOI 10.1007/s11240-014-0532-2

antibacterial, anti-inflammatory, anti-hypertensive and

anticancer activities (Luyt et al. 1999; Fouche et al. 2008).

D. robusta wild populations are over-exploited. Recently,

this species has been included given Data Deficient–

Taxonomically Problematic (DDT) status in the Red List of

South African plants (2013). In vitro plant regeneration of

D. robusta is potentially a valuable method for conserva-

tion, mass propagation and production of bioactive com-

pounds of this species. Although D. robusta

micropropagation has been described (Ngugi et al. 1998;

Baskaran et al. 2013), there is no report of its in vitro plant

regeneration by somatic embryogenesis.

There is growing interest in developing somatic

embryogenesis systems for medicinal plants (Martin

2004a; Baskaran and Van Staden 2012) as a fast and effi-

cient method for clonal mass propagation (Shoyama et al.

1997), production of artificial seeds (Manjkhola et al.

2005), genetic transformation (Parimalan et al. 2010),

cryopreservation (Hua and Rong 2010), and production of

bioactive compounds (Jeong et al. 2005). The technique is

also useful for germplasm conservation, the selection of

genetic variants with desirable characters, the generation of

somaclonal variants and for performing cellular genetic

manipulations (Vasil 1988). Recently, organic elicitors

have been used in improving somatic embryogenesis in

many plants (Martin 2004b; Jayabalan et al. 2004; Bask-

aran and Van Staden 2012) and were useful in enhance-

ment of secondary metabolites for commercial application

(Savitha et al. 2006). Therefore, the development of a

somatic embryogenetic system for plant regeneration

would be helpful for D. robusta in vitro programmes.

The aim of the present study was to develop simple and

efficient protocols for efficient plant regeneration rates via

direct and indirect somatic embryogenesis from leaf

explants of D. robusta, by investigating the effects of plant

growth regulators, and organic elicitors (OEs: haemoglo-

bin, glutamine and mebendazole) on the processes.

Embryogenic suspension cultures can be ideal vehicles for

further research on somatic mutation, protoplast culture,

somatic hybridization and genetic transformation.

Materials and methods

Plant regeneration by direct somatic embryogenesis

Plant material and explant preparation were done as

described previously (Baskaran et al. 2013). Leaf explants

(approximately 10 9 4.0 mm) of D. robusta were excised

from 20-day-old in vitro germinated seedlings and used as

initial explants for both direct and indirect somatic

embryogenesis. Leaf explants were cultured on MS (Mu-

rashige and Skoog 1962) solid [0.8 % (w/v) agar, Sigma,

USA] medium with 3.5 % (w/v) sucrose and different

concentrations and combinations of plant growth regulators

[PGRs: 2,4-dichlorophenoxyacetic acid (2,4-D), picloram,

benzyladenine (BA) and thidiazuron (TDZ)] and 20 lM

glutamine for production of direct somatic embryos (SEs)

and plantlet development. The PGR treatments are indi-

cated in Table 1. The SEs and degree of plantlet induction

were recorded after 10 weeks of culture. All the plantlets

(approximately 10–15 mm) were transferred into half-

strength MS medium for 4 weeks for plant growth and

development. Thereafter, plantlets (approximately

40–60 mm) were acclimatized successfully as described

previously (Baskaran et al. 2013).

In all experiments, the medium lacking plant growth

regulators served as control. The chemicals used were of

analytical grade (Biolab, South Africa; Oxoid, England and

Sigma, USA). All media were adjusted to pH 5.8 with

0.1 N NaOH and/or 0.1 N HCl before gelling with 0.8 %

(w/v) agar and autoclaved at 121 �C for 20 min. The cul-

tures were maintained at a temperature of 25 ± 2 �C and

light intensity of 40 lmol m-2 s-1 provided by cool white

fluorescent light (ORAM L 58 W/740, South Africa) under

a 16 h photoperiod.

Plant regeneration by suspension culture

For production of indirect somatic embryogenesis, the leaf

explants were cultured on MS solid [0.8 % (w/v) agar]

medium and 3.5 % (w/v) sucrose, supplemented with 2,4-

D or picloram in combinations with TDZ and glutamine, as

specified in Table 2. After 4 weeks, friable embryogenic

callus (FEC) from each medium were transferred to liquid

somatic embryo medium (SEML) containing 20 ml liquid

MS medium, 3.5 % (w/v) sucrose and supplemented with

different concentrations and combinations of PGRs (2,4-D,

picloram and TDZ) and organic elicitors [OEs: glutamine

(GM), haemoglobin (HB) and mebendazole (MBZ: methyl

5-benzoyl-1H-benzimidazol-2-yl carbamate] in 100 ml

erlenmeyer flasks. The precise concentrations and combi-

nations are outlined in Table 2. Suspension culture of D.

robusta was performed as previously described (Baskaran

and Van Staden 2012).

Different developmental stages of SEs (globular embryo,

partial pear-shaped, club-shaped, torpedo-shaped, and coty-

ledon and plumule stages) and plantlets per flask of culture

was determined and were recorded after 6 weeks of culture.

All embryo stages were photographed under a Leica M Stereo

Microscope (JVC-Digital Camera: KY-F 1030U type; 0.5X,

Wayne, NJ, USA). All stages of embryos were cultured on

plant induction media [half-strength solid MS medium; half-

strength MS medium plus 0.5 lM a-naphthaleneacetic acid

(NAA)]. The cultures were maintained in darkness for 3 days

and then incubated under 40 lmol m-2 s-1 light intensity

282 Plant Cell Tiss Organ Cult (2014) 119:281–288

123

and a 16 h photoperiod. Embryo germination percentage was

calculated after 8 weeks (number of germinated SE/total

number of SE 9100). The plantlets (approximately

40–60 mm) were successfully acclimatized in a greenhouse

(Baskaran et al. 2013).

Statistical analysis

All experiments were repeated at least three times with 25

replicates for direct somatic embryogenesis and FEC for-

mation, and 5 replicates for SE formation by suspension

culture and germination per treatment. Data were statisti-

cally analyzed using analysis of variance (ANOVA), and

are presented as mean ± standard error of three indepen-

dent experiments. Treatment means were separated using

Duncan’s multiple range test at the 5 % probability level

and analyzed using SPSS Windows version 11 (SPSS Inc.,

Chicago, IL, USA).

Results and discussion

Direct somatic embryo production and conversion

into plantlets

Direct somatic embryogenesis was achieved from leaf

explants on MS medium, 3.5 % (w/v) sucrose and various

plant growth regulators (PGRs) alone or in combination.

Friable embryogenic calli (FEC) formed at wounding sites

of leaf explants after a week in culture and later white

globular embryoids developed directly from leaf explants

in all treatments except the control after 4 weeks (Fig. 1a).

Lincy et al. (2009) have also reported a similar type of

embryo development in Zingiber officinale. SEs develop

either from callus or directly from the explants without an

intermediate callus stage (Jayanthi et al. 2011). In this

study, globular embryos were improved significantly in all

treatments after 8 weeks of culture (Table 1; Fig. 1b).

However, SEs [globular and other different stages of

embryos (partial pear-shaped embryo, club-shaped embryo,

club-shaped cotyledon initiation and the plumule initia-

tion)] and plantlet number were varied after 10 weeks in

treatments (Table 1; Fig. 1c, d). Similar patterns of embryo

development stages are reported for other plant species

(Lincy et al. 2009). Different patterns of developmental

stages of monocotyledon SEs were reported previously

(Shah 1982; Lincy et al. 2009; Raju et al. 2013). Combi-

nation of picloram or 2,4-D, TDZ and glutamine was more

effective in production of SEs and plantlet development

(Table 1). Picloram, TDZ and glutamine induced higher

rates of somatic embryogenesis in other bulbous plants

species (Tribulato et al. 1997; Bakhshaie et al. 2010;

Baskaran and Van Staden 2012). In contrast, cytokinins

alone are considered as the best inducers of SEs in other

Hyacinthaceae species such as Muscari armeniacum

(Wang et al. 2013). In this study, addition of glutamine in

Table 1 Development of direct somatic embryogenesis and plantlets originating from somatic embryos in D. robusta leaf explants

Growth regulators (lM) in solid MS

medium containing 3.5 % (w/v) sucrose

Frequency of

embryo formation (%)

Number of embryo stage/explant (mean ± SEM)

Developmental stage

Globular Different stages of embryo* Plantlets

Control 0 0 0 0

10 2,4-D 64.0 ± 0.58 f 4.6 ± 0.51 cd 2.6 ± 0.48 e 1.8 ± 0.32 de

10 2,4-D ? 1 BA 92.0 ± 0.40 c 8.6 ± 1.94 ab 9.0 ± 1.00 bc 5.4 ± 0.81 c

10 2,4-D ? 1 TDZ 88.0 ± 0.40 d 3.2 ± 0.37 d 4.8 ± 0.58 de 3.4 ± 0.51 cd

10 picloram 86.0 ± 0.40 d 3.6 ± 0.51 d 2.2 ± 0.37 e 1.2 ± 0.20 e

10 picloram ? 1 BA 90.0 ± 0.24 c 7.8 ± 1.98 ab 6.0 ± 0.70 bc 4.2 ± 0.73 c

10 picloram ? 1 TDZ 96.0 ± 0.20 b 9.4 ± 0.98 a 10.6 ± 1.50 b 8.4 ± 0.92 b

10 2,4-D ? 1 BA ? 20 glutamine 100 ± 0.00 a 9.2 ± 1.68 a 7.0 ± 1.30 bc 4.8 ± 0.86 c

10 2,4-D ? 1 TDZ ? 20 glutamine 100 ± 0.00 a 5.2 ± 0.37 bc 6.8 ± 0.86 bc 4.2 ± 0.58 c

10 2,4-D ? 5 BA ? 20 glutamine 100 ± 0.00 a 5.2 ± 0.86 bc 7.0 ± 1.30 bc 5.4 ± 1.03 c

10 2,4-D ? 5 TDZ ? 20 glutamine 84.0 ± 0.37 e 2.8 ± 0.58 d 6.4 ± 1.63 bc 4.0 ± 0.70 cd

10 picloram ? 1 BA ? 20 glutamine 100 ± 0.00 a 10.8 ± 2.17 a 7.6 ± 1.07 bc 3.6 ± 0.51 cd

10 picloram ? 1 TDZ ? 20 glutamine 100 ± 0.00 a 11.2 ± 1.63 a 14.0 ± 1.30 a 11.6 ± 1.03 a

10 picloram ? 5 BA ? 20 glutamine 100 ± 0.00 a 4.8 ± 0.58 bc 5.4 ± 1.03 de 4.0 ± 0.70 cd

10 picloram ? 5 TDZ ? 20 glutamine 92.0 ± 0.40 c 3.4 ± 0.51 d 7.4 ± 1.43 bc 5.0 ± 0.84 c

Results are mean ± SEM of % of embryo from 25 replicate per treatment. Mean values followed by same letters in each column are not

significantly different according to the Duncan’s multiple range test at 5 % level

* Different stages of embryo = partial pear-shaped, club-shaped, club-shaped cotyledon initiation and the plumule initiation

Plant Cell Tiss Organ Cult (2014) 119:281–288 283

123

media influenced effective somatic embryogenesis in D.

robusta. However, media containing 10 lM picloram,

1 lM TDZ and 20 lM glutamine produced significantly

higher frequency (100 %), SEs (11.2–14.0) and number of

plantlets (11.6) compared to other treatments (Table 1;

Fig. 1e). The present study revealed that a combination of

auxin, cytokinin and glutamine is important for significant

SEs production in D. robusta. In this study, all matured

embryos produced plantlets which grew well on half-

strength MS medium. Thereafter, all the plantlets were

successfully established in the greenhouse with a survival

rate of 100 %.

Induction of embryogenic callus

Different PGRs alone or in combinations were used to test

their effect on induction of embryogenic callus (data not

shown). It was observed that combinations of 10 lM

picloram or 2,4-D, 1 lM TDZ and 20 lM glutamine

played an important role in production of D. robusta

embryogenic calli (100 %). These two different combina-

tions promoted fast growing FEC after 2 weeks. In this

study, addition of TDZ and glutamine with picloram was

even more effective in FEC induction (Fig. 2a). Similar

phenomena were also found in other Hyacinthaceae

Table 2 Effect of growth regulators and organic elicitors on somatic embryo (SE) development from embryogenic suspension cells of D.

robusta

FEC induction

medium PGR (lM)

PGR (lM) and organic

supplement in SEML

Frequency of

embryo

formation (%)

Number of embryo stage/explant (mean ± SEM)

Developmental stage

Globular Different

stages

of embryo*

Plantlets

10 picloram ? 1 TDZ ? 20

glutamine

Control (no PGR and supplement) 48.0 ± 0.24 k 2.2 ± 0.37 h 1.2 ± 0.20 f 0.0 ± 0.00

0.5 picloram 72.0 ± 0.40 h 4.2 ± 0.58 fg 3.0 ± 0.54 f 1.0 ± 0.32 ij

0.5 picloram ? 1 TDZ 84.0 ± 0.37 e 5.4 ± 0.86 ef 8.0 ± 1.05 e 2.6 ± 0.24 hi

0.5 picloram ? 2 TDZ 92.0 ± 0.67 c 2.8 ± 0.37 gh 10.6 ± 1.03 cd 8.0 ± 0.92 ab

0.5 picloram ? 1 TDZ ? 20

glutamine

100 ± 0.00 a 3.8 ± 0.32 fg 8.2 ± 1.35 e 4.8 ± 0.37 ef

0.5 picloram ? 1 TDZ ? 40

glutamine

100 ± 0.00 a 4.6 ± 0.51 fg 12.6 ± 1.28 cd 7.2 ± 0.58 ab

0.5 picloram ? 1 TDZ ? 25

haemoglobin

100 ± 0.00 a 10.6 ± 0.92 bc 12.0 ± 1.68 cd 9.0 ± 0.70 a

0.5 picloram ? 1 TDZ ? 50

haemoglobin

76.0 ± 0.37 g 5.6 ± 0.51 ef 9.6 ± 1.36 de 3.8 ± 0.34 fg

0.5 picloram ? 1 TDZ ? 15

mebendazole

88.0 ± 0.40 d 14.0 ± 1.30 a 13.4 ± 1.66 cd 6.0 ± 0.72 bc

0.5 picloram ? 1 TDZ ? 30

mebendazole

80.0 ± 0.44 f 9.0 ± 0.70 cd 19.6 ± 2.01 a 4.6 ± 0.60 ef

10 2,4-D ? 1 TDZ ? 20

glutamine

Control (no PGR and supplement) 56.0 ± 0.58 j 3.0 ± 0.76 gh 1.2 ± 0.37 f 0.0 ± 0.00

0.5 2,4-D 74.0 ± 0.52 g 5.0 ± 0.70 fg 3.0 ± 0.54 f 0.8 ± 0.37 ij

0.5 2,4-D ? 1 TDZ 82.0 ± 0.24 e 6.2 ± 0.58 de 8.4 ± 1.28 e 3.0 ± 0.70 gh

0.5 2,4-D ? 2 TDZ 76.0 ± 0.37 g 4.8 ± 0.37 fg 8.6 ± 0.92 e 7.0 ± 0.64 ab

0.5 2,4-D ? 1 TDZ ? 20 glutamine 96.0 ± 0.20 b 5.8 ± 0.86 ef 9.8 ± 1.24 de 3.4 ± 0.58 gh

0.5 2,4-D ? 1 TDZ ? 40 glutamine 88.0 ± 0.40 d 4.0 ± 0.70 fg 11.8 ± 1.65 cd 4.2 ± 0.51 ef

0.5 2,4-D ? 1 TDZ ? 25

haemoglobin

72.0 ± 0.24 h 8.0 ± 0.92 cd 10.2 ± 1.06 de 7.8 ± 0.58 ab

0.5 2,4-D ? 1 TDZ ? 50

haemoglobin

68.0 ± 0.51 i 8.4 ± 1.20 cd 11.6 ± 1.36 cd 5.0 ± 0.74 de

0.5 2,4-D ? 1 TDZ ? 15

mebendazole

84.0 ± 0.34 e 12.8 ± 1.98 ab 14.8 ± 1.98 bc 5.2 ± 0.92 cd

0.5 2,4-D ? 1 TDZ ? 30

mebendazole

92.0 ± 0.24 c 8.2 ± 1.46 cd 18.6 ± 2.71 ab 5.6 ± 1.36 cd

Values with the mean ± SEM derived from 5 replicate (each 500 mg FEC) with 500 ll settled cell volume of embryogenic suspension cells per

replicate. Mean values followed by same letters in each column are not significantly different according to the Duncan’s multiple range test at

5 % level

* Different stages of embryo = torpedo-shaped, cotyledon-stage and plumule-stage embryos. Haemoglobin and mebendazole = mg l-1

284 Plant Cell Tiss Organ Cult (2014) 119:281–288

123

species (Baskaran and Van Staden 2012). The formation of

embryogenic tissues from somatic cells can be stimulated

by altering either the levels of exogenously applied auxins

or the ratio of auxin to cytokinin (Baskaran and Van Staden

2012). Many reports confirmed that somatic embryogenesis

is influenced by glutamine (Baskaran and Jayabalan 2009;

Deo et al. 2010; Baskaran and Van Staden 2012). The FEC

did not proliferate continuously but differentiated into

somatic embryogenesis after 6 weeks of culture (Fig. 2b).

In the present study, two different combinations of PGRs

that produced calli after 4 weeks were used in SEs for-

mation via cell suspension culture (Table 2).

Somatic embryos formation in cell suspension culture

Plant regeneration via somatic embryonic cell suspension

culture of D. robusta was successfully achieved with SEML

containing various treatments of PGRs alone or in

combination (Table 2). Fast growing FEC from two dif-

ferent combinations were transferred to SEML containing

PGRs (Table 2). FEC units enlarged and produced small

cell aggregates (approximately 0.5–1.0 mm diameter) after

2 weeks in SEML. Thereafter, it produced dense cell

aggregates which were white and soft, or white but watery

callus. However, proliferation rate of cell lines and pro-

portion of cell aggregates varied in settled cell volume

(SCV) of FEC in each treatment. Whitish-soft globular-

shaped, partial pear-shaped, club-shaped and torpedo-

shaped embryos developed from dense cell aggregates after

3 weeks of culture initiation (Fig. 2c). Whitish-green-rigid

globular, torpedo-shaped, cotyledon-stage and plumule-

stage embryos and plantlets were produced and varied in

number from each treatment after 6 weeks (Table 2;

Fig. 2d). Production of globular embryos increased sig-

nificantly on media containing 0.5 lM picloram or 2,4-D

and 1 lM TDZ, while induction of different stages of

Fig. 1 In vitro plant regeneration by direct somatic embryogenesis

form leaf explants of D. robusta: a induction of white globular

embryoids on MS medium plus 10 lM 2,4-D, 5 lM BA and 20 lM

glutamine. b Proliferation of globular embryos in MS medium

containing 10 lM picloram, 1 lM TDZ and 20 lM glutamine.

c Microscopic photograph of developmental stages of somatic

embryos (910). d Proliferation of different stages of embryos and

plantlets on MS medium plus 10 lM picloram, 1 lM TDZ and

20 lM glutamine. e Formation of plantlets on MS medium plus

10 lM picloram, 1 lM TDZ and 20 lM glutamine. Bar (a, b, d and

e) 10 mm

Plant Cell Tiss Organ Cult (2014) 119:281–288 285

123

embryos and plantlets was enhanced in 2 lM TDZ

(Table 2). These embryos were thick and covered with

friable calli. The conversion of the embryos to plantlets

was low (\50 %). Therefore, lower concentration of TDZ

is essential for healthy SEs production.

SEML containing PGRs and organic elicitors (OEs: hae-

moglobin, glutamine and mebendazole) combinations sig-

nificantly increased proliferation of SEs in D. robusta.

Organic additives contains minerals, essential nutrients and

vitamins and have been reported to be effective in somatic

embryogenesis in several plant species (Jayabalan et al. 2004;

Baskaran and Jayabalan 2009; Deo et al. 2010; Al-Khayri

2013; Baskaran and Van Staden 2012). SEs were relatively

large and green in colour in all treatments (Fig. 2e). Meben-

dazole (MBZ) significantly improved somatic embryo

production (globular to cotyledon-stage) after 6 weeks in

culture (Table 2; Fig. 2e). MBZ is a synthetic anthelmintic

drug which acted as a growth regulator on shoot and root

development in M. plumbea and D. robusta micropropagation

(Baskaran et al. 2012, 2013). However, in this study, hae-

moglobin (25 mg l-1) produced a higher number of plantlets

(9.0 ± 0.70 per SCV) from matured embryos (Table 2;

Fig. 2f). A similar effect was also observed with other Hya-

cinthaceae species such as M. plumbea (Baskaran and Van

Staden 2012). Haemoglobin plays a role in enhancement of

mitotic division (Azhakanandam et al. 1997) and cellular O2

supply in vitro (Anthony et al. 1997). Recently, two plant

hemoglobin (Hb) genes (ZmHb1 and ZmHb2) that regulate

the cell survival/death decision that influences somatic

embryogenesis through their cell-specific localization

Fig. 2 In vitro plant regeneration from somatic embryos on cell

suspension culture. a Induction of FEC on MS medium containing

10 lM picloram, 1 lM TDZ and 20 lM glutamine. b Formation of

cotyledon and plumule stages of embryos. c Different developmental

stages from dense cell aggregates. d Production of different stages of

embryos and plantlets on SEML containing 0.5 lM picloram and

2 lM TDZ. e Formation of somatic embryos (globular to cotyledon-

stage) on SEML containing 0.5 lM picloram, 1 lM TDZ and

30 mg l-1 mebendazole. f Formation of plantlets on SEML containing

0.5 lM picloram, 1 lM TDZ and 25 mg l-1 haemoglobin. g Germi-

nation of embryos and plantlet development on half-strength MS

medium supplemented with 0.5 lM NAA. h Well-developed plantlets

from germination medium. i Acclimatized plants of D. robusta in the

greenhouse after 6 months. Bar (a–g) 10 mm

286 Plant Cell Tiss Organ Cult (2014) 119:281–288

123

patterns have been reported (Huang et al. 2014). In this study,

increasing the concentration of haemoglobin significantly

decreased the frequency, number of SEs and plantlets

(Table 2). In contrast, a high concentration of haemoglobin

was effective in formation of SEs in M. plumbea, cotton and

peanut (Jayabalan et al. 2004; Ganesan and Jayabalan 2004;

Baskaran and Van Staden 2012). The medium containing

40 lM glutamine showed a significant improvement in pro-

duction of SEs (Table 2). Improvement of SEs by addition of

glutamine in the medium has also been reported for other

plant species (Baskaran and Jayabalan 2009; Deo et al. 2010;

Al-Khayri 2013; Baskaran and Van Staden 2012). The pres-

ent study indicated that SEs production in D. robusta is

dependent on the type and concentration of OEs. Similar

phenomena have also been reported in other plant species

(Deo et al. 2010; Al-Khayri 2013; Baskaran and Van Staden

2012).

Conversion of plantlets

Successful plant regeneration was achieved from post

somatic embryogenetic developmental stages; globular,

torpedo, cotyledon and plumule on the plant induction

medium. All mature embryos germinated to produce

complete plantlets after 2 weeks in culture. Mostly, glob-

ular embryos failed to germinate on half-strength solid MS

medium, but only rooting occurred with half-strength MS

medium plus 0.5 lM NAA. Half-strength solid MS med-

ium produced 65 % germination, whereas half-strength MS

medium supplemented with 0.5 lM NAA produced a

higher frequency of germination (90 %) and well rooted

plantlets (Fig. 2g). The beneficial effect of auxins and

cytokinins on somatic embryo germination has been

reported in many other monocotyledon plant species

(Kackar et al. 1993; Guo and Zhang 2005; Lincy et al.

2009). Successful germination and plant regeneration via

SEs has been reported with combinations of BA and NAA

in ginger and buffel grass (Lincy et al. 2009; Carloni et al.

2014). After germination, NAA has been required for plant

development in buffel grass (Carloni et al. 2014). Well-

developed plantlets were separated (Fig. 2h) and then

transferred to plastic pots containing a 1:1 (v/v) vermicu-

lite:sand mixture. The plantlets were successfully accli-

matized in a greenhouse (Fig. 2i) with 100 % survival rate.

The present work is a first report for plant regeneration

via direct and indirect somatic embryogenesis from leaf

explants of D. robusta. Promising plant regeneration from

SEs via direct and indirect somatic embryogenesis was

influenced markedly by PGRs combined with OEs (gluta-

mine and haemoglobin). The system will be helpful for

conservation, mass clonal propagation, production of bio-

active compounds and genetic transformation studies.

Acknowledgments The financial support by National Research

Foundation (NRF), Pretoria and the University of KwaZulu-Natal,

Pietermaritzburg is gratefully acknowledged. The authors are grateful

to the Microscopy & Microanalysis Unit (MMU), UKZN, Pieterma-

ritzburg for microscopic assistance.

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