ORIGINAL PAPER

Liquid overlaying improves somatic embryogenesisin Catharanthus roseus

Zahid H. Siddiqui • A. Mujib • Mehpara Maqsood

Received: 31 May 2010 / Accepted: 16 August 2010 / Published online: 27 August 2010

� Springer Science+Business Media B.V. 2010

Abstract Somatic embryos were induced from in vitro

germinating seed-hypocotyls of Catharanthus roseus. The

process of embryogenesis has been categorized into a few

distinct stages (induction, proliferation, maturation and

germination) in which liquid overlaying at varying levels

0 ml (T0), 0.25 ml (T1), 0.5 ml (T2), 0.75 ml (T3) and

1.0 ml (T4) was applied on solid medium. It was found that

liquid overlaying improved proliferation; maturation, ger-

mination of embryos in C. roseus. In proliferation stage,

particularly in T2, torpedo embryo number increased sig-

nificantly (i.e. 129.6%) as compared to control. Liquid

overlaying (T2, T3 and T4) also improved embryo matu-

ration and showed early germination even in maturation

medium. It also accelerated normal embryo germination

frequency particularly in treatment with T2 and shortened

‘embryo—plantlet’ recovery time. Biochemical analyses

revealed more proline, protein and amino acid with

increasing level of liquid overlaying as it improved embryo

induction, development and faster germination.

Keywords Somatic Embryogenesis � Liquid overlaying �Catharanthus roseus

Abbreviations

ANOVA Analysis of variance

BA N6-Benzyladenine

2,4-D 2,4-Dichlorophenoxyacetic acid

GA3 Gibberellic acid

KIN Kinetin

NAA Naphthalene acetic acid

MS Murashige and Skoog (1962) medium

TCA Trichloroacetic acid

Introduction

The family Apocynaceae consists of several genera, and

Catharanthus roseus is one of the important members of

this family. It contains more than 130 alkaloids (Van der

Heijden et al. 2004), out of which vincristine and vin-

blastine are very precious (Miura et al. 1988; Moreno et al.

1995). Both these compounds are well known anticancer

drugs and are administered against different kinds of car-

cinomas (leukemia, Hodgkin’s disease, Wilm tumor, neu-

roblastoma, rhabdosarcoma and reticulum cell sarcoma).

Beside antileukemic properties, the total alkaloids and

chloroform fractions of crude drug also showed sedative

and tranquilizing properties (Wealth of India 1992).

C. roseus is also a well known ornamental plant with pink

and white flowers, it is commonly known as Madagascar

periwinkle or ‘‘Sadabahar’’. C. roseus has been studied

extensively for several years in order to synthesize anti-

cancerous compounds in culture and to improve the yield

particularly vincristine and vinblastine content. (Moreno

et al. 1995; Mujib et al. 2003).

Somatic embryogenesis (SE) is an important biological

process in which embryos are formed in culture. Although

this in vitro embryogenesis is known for quite some times,

the technique is relatively new in this genus and was

described recently (Junaid et al. 2006). Various novel and

innovative ideas like optimization of plant growth regulator

levels, adjustment of osmotic conditions by using various

sugars (sucrose, mannitol, glucose, sorbitol), gelling

agents, media condition (solid, liquid, immersion, liquid

Z. H. Siddiqui � A. Mujib (&) � M. Maqsood

Cellular Differentiation and Molecular Genetics Section,

Department of Botany, Hamdard University,

New Delhi 110062, India

e-mail: amujib3@yahoo.co.in

123

Plant Cell Tiss Organ Cult (2011) 104:247–256

DOI 10.1007/s11240-010-9828-z

overlaying) have recently been trialed to improve the

embryogenesis efficiency in plants (Te-chato and Lim 1999;

Pullman and Skryabina 2007). Liquid overlaying can be

defined as a method in which liquid medium is put over solid

medium. Depending upon the need and requirement, dif-

ferent workers overlaid liquid medium which may be of

similar composition as that of solid medium as was observed

in Hevea brasiliensis (Sushamakumari et al. 2000), half-

strength liquid Murashige and Skoog (1962) medium (MS

medium) with plant growth regulators, used in Garcinia

mangostana (Te-chato and Lim 1999), in Anthurium, med-

ium without plant growth regulators was also added

(Nitayadatpat and Te-chato 2005). Pullman and Skryabina

(2007) used liquid overlaying treatment in medium 1,081 or

its modification with varying concentrations of NAA.

Anthony et al. (1995) reported using B5 medium as solid and

ammonium-free liquid MS medium for overlaying during

the protoplast culture of Mannihot esculenta. Mineral oil

overlaying was also applied by Mathur (1991) in Selenium

candolii which showed increased somatic embryo numbers

per gram of fresh weight. In Pseudotsuga menziesii,

embryogenic tissue initiation was successfully made by

gelled medium or in a gelled-liquid medium overlay system

(Pullman et al. 2009). Volume of liquid overlaying was,

however, varied in different plants, in Pinus taeda 0.25 to

0.75 ml was added (Pullman and Skryabina 2007) but in

Garcinia mangostana cultures, 10–15 ml of half- strength

liquid MS medium was used (Te-chato and Lim 1999). To

improve the selection of citrus transformation, a liquid

overlay system was used which decreased the growth of GUS

negative- shoots without inducing Agrobacterium over-

growth (Gutierrez-E et al. 1997; Yang et al. 2000). Liquid

overlaying also brings down the cost of plant production was

reported by Maene and Debergh (1985); temporary immer-

sion, another related technique involving liquid was earlier

estimated to reduce the cost by about 50–60% (Sluis 2006).

Gupta and Timmis (2005) noted a faster rate of growth in

liquid medium compared with gelled medium, where culture

growth was slow.

However, liquid medium also has a few limitations; the

transfer of the tissue to a semi-solid medium is often

essential at later stages, as continuous agitation hinders

plants’ development and induces vitrification, which is

considered to be a physiological disorder (Etienne et al.

1997). Semi-solid medium has some other disadvantages

too; some of the agars used in culture contain inhibitory

substances which prevent in vitro morphogenesis, and rates

of growth can be slow as the toxic exudates from explants

do not diffuse away quickly (Powell and Uhrig 1987). In

solid medium, explants are implanted above the medium

and do not require special means of aeration to proliferate,

shoots and roots grow in a more orderly fashion in a semi-

solid culture, even explants may be retrieved safely if not

responded in culture. Liquid overlay system in gelled

medium wisely utilizes the advantages of solid and liquid

medium and eliminates the disadvantages present in the

two systems. In this present communication, the impor-

tance of liquid overlaying in C. roseus in vitro embryogeny

has been analyzed and discussed.

Materials and methods

In vitro germination of seeds and culture condition

The basic process of somatic embryogenesis that was

described earlier in Catharanthus roseus cv. Nirmal (Junaid

et al. 2006) was followed. In short, seeds were collected from

in vivo grown plants of Jamia Hamdard (Hamdard Univer-

sity) herbal garden, which is located within the University

campus. Twelve to twenty-five surface disinfected seeds

were placed in Magenta 7 vessels containing 50 ml of MS

solid medium (Murashige and Skoog 1962) without any

growth regulator. Germinated seedlings were grown until

they had attained 2–4 cm length. Various parts (nodal stem,

leaf, root, and hypocotyl) were used and inoculated in test

tubes (Borosil, India) as explants. For embryogenic callus

initiation, MS medium was supplemented with 4.52 lM 2,4-

Dichlorophenoxyacetic acid (2,4-D). For somatic embryo

proliferation, medium was amended with 6.72 lM N6-

Benzyladenine (BA) and 5.37 lM Naphthalene acetic acid

(NAA); embryo maturation and germination medium con-

tained earlier optimized 2.60 lM Gibberellic acid (GA3) and

2.24 lM BA, respectively (Junaid et al. 2006). The pH of the

medium was adjusted to 5.6 before autoclaving at 121�C for

20 min. All cultures were incubated under a 16 h photope-

riod by cool white (Fluorescent F40 T12/CW/EG) lamp at a

photon flux density of 100 lmol m-2 s-1 at 25�C light/20�C

dark temperature.

Embryogenesis has been categorized into following

distinct stages induction and proliferation; maturation; and

germination or plantlet conversion. Liquid overlaying of

four different volumes i.e. 0.25 ml (T1), 0.50 ml (T2),

0.75 ml (T3) and 1.0 ml (T4) was applied to tissues with an

average area of 30-40 sq.mm and a control (T0) was also

maintained for the comparative study. The composition of

liquid medium is the same as that of MS solid medium,

which was used throughout the developmental stages of

embryogenesis. Morphogenetic and biochemical studies

were conducted after 4 weeks of culture. For estimation of

fresh—and dry weight, 4 weeks old calli were used. For

estimation of fresh weight, calli were weighed immediately

after isolation of culture media. Calli were dried at 60�C

for 18 h, their dry weights were measured and finally the

absolute dry mass was calculated using the following for-

mula (Winkelmann et al. 2004).

248 Plant Cell Tiss Organ Cult (2011) 104:247–256

123

Absolutedry mass %ð Þ ¼ Dry weight=Fresh weight� 100:

Estimation of total sugar

Estimation of total sugar was made according to Dey

(1990). Different stages of developing embryos (0.5 g)

were extracted twice with 90% ethanol, and the extracts

were pooled. The final volume of the pooled extract was

made up to 25 ml with double distilled water. To an aliquot

of 1.0 ml, 1.0 ml 5% phenol and 5.0 ml of concentrated

analytical-grade sulphuric acid were added, and cooled in

air. The optical density was measured at 485 nm. A solu-

tion containing 1.5 ml of 55% glycerol, 0.5 ml ninhydrin

and 4.0 ml double distilled water was used as a calibration

standard.

Estimation of proline

For measurement of proline content, 0.2 g of specific

stages of embryos were homogenized in 5 ml 3% aqueous

sulfosalicylic acid and filtered by Whatman filter paper

(No. 1). To 1 ml extract, 1 ml acid ninhydrin and 1 ml of

glacial acetic acid were added and the reaction mixture

incubated at 100�C for 1 h. The reaction mixture was

placed on ice and extracted with 2 ml toluene. Proline

content in the extract was subjected to the spectrophoto-

metric assay of Bates et al. (1973).

Estimation of protein

Protein was estimated by Bradford method (1976), 0.5 g

tissue was ground in a pre-cooled mortar and pestle with

1.5 ml (0.1 M) phosphate buffer (pH 7.0), placed on ice

and centrifuged at 5,000 rpm for 10 min. With 0.5 ml

Trichloroacetic acid (TCA), the sample was again cen-

trifuged at 5,000 rpm for 10 min. The supernatant was

discarded, and the pellet was washed with chilled acetone

and dissolved in 1.0 ml of 0.1 N Sodium hydroxide

(NaOH). Then 0.5 ml aliquot was added with 5.0 ml of

Bradford reagent, the optical density was measured at

595 nm.

Estimation of free amino acids

Free amino acids were estimated by the method developed

by Lee and Takahashi (1966). In short, 0.1 g tissue was

incubated overnight in 70% ethanol followed by washing

with double distilled water. Then 1.5 ml of 55% glycerol

and 0.5 ml ninhydrin solution were added, boiled at 100�C

for 20 min and cooled down. The final volume was made

up to 6 ml with double distilled water, and the optical

density was measured at 570 nm.

Statistical analysis

The data on the effects of plant growth regulators and

liquid overlaying on different stages of embryo and other

biochemical parameters were analyzed by one-way analy-

sis of variance (ANOVAs). Values are means of five rep-

licates from two experiments, and the presented mean

values were separated using Duncan’s Multiple Range Test

(DMRT) at P B 0.05.

Results

Overlaying improves induction of embryogenic tissue

The hypocotyls of in vitro grown plantlet were placed in

medium containing 4.52 lM 2,4-D, callus was friable, light

yellow and fast growing; and proved to be embryogenic.

Calli from other sources were non-embryogenic, therefore

was discontinued in future sets of experiments. Hypocotyl-

calli was routinely maintained on the same medium or in

combination with cytokinins, BA 2.22–8.9 lM or KIN

2.32–9.30 lM. We observed that in liquid overlaying

condition the embryogenic callus was more faster com-

pared to control (Fig. 1); the calli appeared to be more

friable and white (Fig. 1b) specially in treatment with T1

and T2. Higher volumes of liquid overlaying i.e. T3 and T4

(Fig. 1c) were less responsive, callus became compact and

light brown and showed poor growth.

Overlaying induced somatic embryo proliferation

Embryogenic cultures with developing embryos were

placed on medium supplemented with 5.37 lM (NAA) and

6.72 lM BA with different volumes of liquid overlaying

(Fig. 2a, b, c). It was found that the embryogenic calli

differentiated into embryos, and maximum number of

embryos was formed in T2 with an increase value of

23.89% (Table 1) and in the same treatment, the number

of torpedo embryo (129.61%) was more as compared to

control. After T2, in T1 treatment, the number of embryo

was also improved with an increase value of 14.17%, in T3,

the number of cotyledonary stage embryo was increased

with a value of 80.74%. With different volumes of liquid

overlaying, the dry weight and absolute dry mass level

increased, specially in treatment with T2 (Table 2); fresh

weight, however, was decreased.

Overlaying improves embryo maturation

White cotyledonary somatic embryos (5–6 mm) were

placed on medium supplemented with 2.60 lM GA3 with

different volumes of liquid overlaying. It was found that in

Plant Cell Tiss Organ Cult (2011) 104:247–256 249

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T1 embryos were well developed and in T2 embryos were

greener in colour as compared to control showing early

maturation (Fig. 3a, b). In T3 and T4, however, embryo

development was poor and several embryos turned light

brown to brown, few remained in the cotyledonary stage,

and those embryos that reached to maturity were thin,

showed poor growth with low pigmentation (Fig. 3c). After

4 weeks, shoot length was increased; which was maximum

in T2 stage (9.85 mm). Whereas in T4 it was decreased

(8.15 mm) as compared to control. Embryos in the T2, T3

and T4 showed germination on maturation medium

(Table 3). A decrease in fresh weight was observed on

enhanced volume of liquid overlaying. In T2, dry mass was

increased by 2.47% and an increase of 26.76% absolute dry

mass was also observed in the same treatment.

Overlaying enhances embryo germination ability

Matured green somatic embryos were placed on medium

supplemented with 2.24 lM BA with different volumes of

liquid overlaying (Fig. 4a, b, c). Embryos were germinated

in almost all the treatments, but compared to control and

Fig. 1 Embryogenic cultures grown in MS medium, contained 4.52 lM 2,4- D with different volumes of liquid overlaying a T0 (control)

b T2 and c T4 (Bar = 1 mm)

Fig. 2 Embryo on proliferation medium, MS medium contained 6.72 lM BA and 5.37 lM NAA with different volumes of liquid overlaying

a T0 b T2 and c T4 (showing advance stage embryos with patches of dead embryos) (Bar = 2 mm)

Table 1 Proliferation of somatic embryos with different levels of liquid overlaying

Treatment Globular Heart Torpedo Cotyledonary Total no. of SE

T0 39.33 ± 0.67a 11.67 ± 0.88c 8.00 ± 1.15c 10.33 ± 0.88b 82.33 ± 2.19 cd

T1 39.67 ± 1.20a 16.00 ± 1.15b 27.33 ± 1.45b 8.67 ± 0.88bc 94.00 ± 1.00b

T2 23.00 ± 1.53b 21.00 ± 1.53a 41.33 ± 0.88a 11.00 ± 1.53b 102.00 ± 2.65a

T3 20.33 ± 1.20b 17.00 ± 1.73b 19.33 ± 0.88c 18.67 ± 0.88a 88.33 ± 0.88bc

T4 17.00 ± 1.15c 16.33 ± 0.88b 18.67 ± 0.88c 6.33 ± 0.88c 76.33 ± 2.33d

Different overlaying levels used: 0 ml (T0), 0.25 ml (T1), 0.5 ml (T2), 0.75 ml (T3) and 1.0 ml (T4), MS medium contained 6.72 lM BA and

5.37 lM NAA; data were scored after 4 weeks of culture. Values are means ± standard errors of at least 5 replicates. Within each column,

means followed by the same letter are not significantly different at P B 0.05 according to Duncan’s multiple range test

250 Plant Cell Tiss Organ Cult (2011) 104:247–256

123

other treatments, T2 showed maximum germination

(55.55%), and in T4 minimum germination was minimum

(11.11%). In T2, there were plantlets showing shoots and

roots, shoots without roots and embryos with roots without

any visible shoot (Table 4). Length of shoot was found

maximum in T2 (10.00 ± 0.29 mm). In T2, fresh weight

increase was 76.15% and absolute dry mass increased by

11.69% in the same treatment.

Biochemical profiles during embryogenesis stages

as influenced by overlaying

Sugar content was observed to be maximum in induction

stage which was decreased during proliferation, maturation

and germination stage of embryos. Opon increasing the

volume of liquid overlaying, sugar level declined in all

growing stages. Maximum amount of sugar was however,

Table 2 Growth indexes of

somatic embryos in

proliferation, maturation and

germination stages with varying

levels of liquid overlaying

Different overlaying levels

used: 0 ml (T0), 0.25 ml (T1),

0.5 ml (T2), 0.75 ml (T3) and

1.0 ml (T4), and data were

scored after 4 weeks of culture.

Values are means ± standard

errors of at least 5 replicates.

Means within a column for each

of proliferation, maturation and

germination with differentletters are significantly different

at P B 0.05 according to

Duncan’s multiple range test

Treatment Fresh weight (g) Dry weight (g) Absolute dry mass (%)

Proliferation T0 1.39 ± 0.13c 0.118 ± 0.011c 8.54 ± 0.56b

T1 1.79 ± 0.11b 0.151 ± 0.011bc 8.61 ± 0.17b

T2 2.68 ± 0.14a 0.282 ± 0.032a 10.47 ± 0.64a

T3 1.97 ± 0.16b 0.183 ± 0.019b 9.26 ± 0.23b

T4 1.18 ± 0.09c 0.101 ± 0.010c 8.38 ± 0.25b

Maturation T0 1.43 ± 0.016c 0.128 ± 0.009c 9.01 ± 0.38a

T1 1.64 ± 0.11c 0.153 ± 0.003c 9.40 ± 0.72a

T2 2.53 ± 0.09a 0.250 ± 0.016a 9.87 ± 0.43a

T3 2.19 ± 0.11b 0.199 ± 0.010b 9.10 ± 0.14a

T4 2.06 ± 0.10b 0.186 ± 0.017b 9.02 ± 0.47a

Germination T0 1.30 ± 0.15b 0.18 ± 0.03b 13.26 ± 0.38ab

T1 1.63 ± 0.10b 0.22 ± 0.02b 13.78 ± 0.40a

T2 2.29 ± 0.26a 0.33 ± 0.03a 14.81 ± 0.43a

T3 1.54 ± 0.10b 0.22 ± 0.02b 14.36 ± 0.41a

T4 1.56 ± 0.08b 0.19 ± 0.00b 12.18 ± 0.35b

Fig. 3 Embryos on maturation

medium, MS medium contained

2.60 lM GA3 with different

volumes of liquid overlaying

a T0 (showing matured green

embryos) b T1 (showing

germination on maturation

medium) and c T4 (brown mass

of dead embryos with a few

mature embryo) (Bar = 5 mm)

Table 3 Maturation of somatic embryos with different levels of liquid overlaying

Treatment Initial length After 2 weeks After 4 weeks Germination (%)

T0 5.67 ± 0.08a 7.74 ± 0.22a 8.81 ± 0.08b 0.00 ± 0.00c

T1 5.56 ± 0.06a 7.01 ± 0.20b 8.80 ± 0.05b 0.00 ± 0.00c

T2 5.68 ± 0.03a 7.88 ± 0.23a 9.85 ± 0.12a 2.67 ± 0.33a

T3 5.73 ± 0.06a 7.00 ± 0.20b 8.16 ± 0.33c 1.33 ± 0.33b

T4 5.70 ± 0.04a 6.63 ± 0.19b 8.15 ± 0.27c 0.33 ± 0.33c

Different overlaying levels used: 0 ml (T0), 0.25 ml (T1), 0.5 ml (T2), 0.75 ml (T3) and 1.0 ml (T4), MS medium contained 2.60 lM GA3, data

scored after 4 weeks of culture. Values are means ± standard errors of at least 5 replicates. Within each column, means followed by the sameletter are not significantly different at P B 0.05 according to Duncan’s multiple range test

Plant Cell Tiss Organ Cult (2011) 104:247–256 251

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observed in the induction stage particularly in T2 treatment

(20.58 mg g-1) but maximum % increase (7.21%) in sugar

content was found in the germination stage (Fig. 5a). The

amount of proline accumulation showed a decreasing trend

with the growth and maturity of embryos. In one specific

embryo stage like proliferation or maturation, the proline

accumulation always increased from T1 to T4. The maxi-

mum amount of proline was found in the induction stage

(i.e. T4, 9.88 mg g-1). In T2 treatment, all developing

stages of embryos showed a moderate increase in proline

content except in proliferation stage where the amount of

proline was decreased by 1.12% (Fig. 5b).

Total soluble protein was less during induction stage

and was found to be maximum during germination stage.

However, with the increase in the volume of liquid

overlaying, protein accumulation also increased (Fig. 6a).

The amount of protein accumulation was increased in

germination stage (4.87 ± 0.080 mg g-1) but maximum

% increase was observed in T2 stage (41.03%) whereas

in T4 and at induction stage it was minimum (2.78 ±

0.1 mg g-1) and similarly maximum % decrease was

observed in T3 (0.22%). Amino acids showed a similar

trend as that of protein, their content increased during the

different morphogenetic stages of embryo and were

maximum in germination stage (T2, 1.75 ± 0.04 mg g-1).

Amino acids were minimum in induction stage, particu-

larly in T0 (0.96 ± 0.03) (Fig. 6b).

Discussion

In our earlier reports, somatic embryogenesis in C. roseus

and the importance of various factors’ role in influencing in

vitro embryogenesis has been described (Junaid et al. 2006,

2008). This study also reconfirms that liquid medium and

liquid overlay improved embryogenic tissue initiation in

plant cell culture as was earlier reported (Pullman and

Skryabina 2007). The use of liquid medium for in vitro

culture has many advantages and has been the subject of

several studies in recent times (Kevers et al. 1984; Anthony

et al. 1995; Hvoslef-Eide and Preil 2005; Pullman et al.

2009). The importance of liquid media lies in its ability to

improve the plant growth and development by increasing

shoot proliferation, microtuberization, microcuttings,

rootings and somatic embryogenesis (Fennel and Ascough

2004; Hvoslef-Eide and Preil 2005). Liquid culture is

believed to be much more efficient as replenishment of

medium or change of medium is easy. It also reduces the

production cost as gelling agent is absent (Gupta and

Timmis 2005; Rizvi et al. 2007). Berthouly and Etienne

(2005) reported that by optimizing the volume of liquid

Fig. 4 Embryo on germination medium, MS medium supplemented

with 2.24 lM BA a T0 showing germination; b T2 showing regenerated

plantlet; c T4, embryo turned brown, without germination; (Bar a and

c = 2 mm; b = 0.5 cm

Table 4 Germination of somatic embryo with different levels of liquid overlaying

Treatment No. of embryos germinated % Germination Shoot length (mm) Root length (mm) *ORN *OSN *LN

T0 2.33 ± 0.33ab 38.89 ± 5.56ab 3.33 ± 0.10c 4.37 ± 0.61b 0.00 ± 0.00b 9.80 ± 0.35b 3.33 ± 0.88b

T1 1.67 ± 0.33b 27.77 ± 5.56b 7.53 ± 0.22b 0.80 ± 0.06c 0.00 ± 0.00b 12.57 ± 1.31b 4.33 ± 0.67ab

T2 3.33 ± 0.88a 55.55 ± 14.70a 10.00 ± 0.29a 7.67 ± 1.45a 5.67 ± 0.88a 20.67 ± 4.67a 6.00 ± 1.15a

T3 1.00 ± 0.58b 16.66 ± 9.62b 1.73 ± 0.05d 2.00 ± 0.58bc 6.33 ± 0.88a 0.00 ± 0.00c 2.33 ± 0.33b

T4 0.67 ± 0.33b 11.11 ± 5.55b 0.83 ± 0.02e 0.47 ± 0.24c 0.47 ± 0.24b 0.47 ± 0.24c 1.00 ± 0.58b

Six matured embryos were placed on germination medium (MS) that contained 2.24 lM BA. Different overlaying levels used: 0 ml (T0),

0.25 ml (T1), 0.5 ml (T2), 0.75 ml (T3) and 1.0 ml (T4); data scored after 4 weeks of culture. *ORN = Embryo showing mean number of root

without shoot; *OSN = Embryo showing mean number of shoot without root; *LN = Mean number of leaf per germinated embryo. Values are

means ± standard errors of at least 5 replicates. Within each column, means followed by the same letter are not significantly different at

P B 0.05 according to Duncan’s multiple range test

252 Plant Cell Tiss Organ Cult (2011) 104:247–256

123

medium shoot proliferation efficiency improved substan-

tially in plant tissue culture system. In our study, T2 (0.5 ml

of liquid medium) was found to be most effective as

compared to T1, T3 and T4. It has been suggested that

liquid overlaying as temporary immersion, generally

improved plant tissue quality, and also promoted the

number of somatic embryos as well (Berthouly and Etienne

2005). We also observed that the number of embryos

increased significantly in T2 treatment during proliferation

and that the induced embryos were more distinct and

showed fast growth and development. Liquid overlaying in

this case was considered to be temporary because a film of

medium was retained on the tissue, which was earlier

reported not to hinder gaseous exchange. However, within

3–4 days, growing tissues absorbed the nutrient mist that

prevented plant tissue from hyperhydricity (vitrification).

This is a common incidence in tissue culture employing

liquid culture (Kevers et al. 1984; Gasper et al. 1987;

Debergh et al. 1992). In rubber plant, embryogenic pro-

toplasts showed fairly good growth on KPR semi-solid

medium when overlaid with same liquid medium

(Sushamakumari et al. 2000). Similar results were obtained

by Bell and Reed (2002) in Pyrus on solid medium with

two- phase liquid overlay or intermittent liquid immersion

techniques that increased shoot proliferation markedly.

Etienne et al. (1997) reported that somatic embryo devel-

opment was considerably improved when temporary

immersion was done, and frequency of germination was

0

5

10

15

20

25

T0 T1 T2 T3 T4

Su

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nt

(mg

g-1

fre

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tiss

ue

afte

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ks

0

2

4

6

8

10

12

T0 T1 T2 T3 T4Pro

line

con

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t (

mg

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fre

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tiss

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Induction Prolifreation Maturation Germination

Induction Prolifreation Maturation Germination

b

a Fig. 5 (a) and (b) Sugar

content (top) and proline

content (bottom) content at

different stages of

embryogenesis after various

liquid overlaying treatments

Plant Cell Tiss Organ Cult (2011) 104:247–256 253

123

also enhanced by improving polarization of two distinct

cotyledons. In our studied plant material, we observed

that medium added with 2.89 lM GA3 facilitate embryo

maturation but with liquid overlaying the same medium

promoted early embryo germination, thus the use of

liquid overlaying improved embryogenesis by shortening

‘embryo—plantlet’ recovery period which was just over

3 months. Earlier, Pullman and Gupta (1994) reported that

an increased osmolality of media found to be effective for

development and maturation of embryos.

In this study, we found that the sugar content decreased

with increased level of liquid overlaying, being maximum in

induction stage and decreased later on during proliferation,

maturation and germination stages of embryos, which is in

agreement with earlier work (Junaid et al. 2007). The amount

of protein and amino acid gradually increased with the

developing stages of embryo and with the use of increasing

level of liquid overlaying. Association of specific protein

with different developing stages (i.e. induction, development

and germination) of embryos was earlier reported in some

studied materials (Fellers et al. 1997; Roja Rani et al. 2005).

In contrast, the content of proline decreased with the devel-

oping stages of somatic embryo while some recovery in

proline level was noticed with the increasing amount of

liquid overlaying, which might be due to the immersion

effect of the liquid overlaying. The analysis of proline has

often been conducted as to study cellular stress conditions in

plants and during somatic embryogenesis. Recently, Kiran

Ghanti et al. (2009) observed increased and steady proline

accumulation during early embryo development that

declined sharply at later embryo stages and in germination

time. Proline is considered to act as an osmotic balance

agent, a reservoir of nitrogen pool or the source of energy and

NADP?, necessary for the fast growing tissues like embryos

(Kiran Ghanti et al. 2009). How liquid overlaying improves

the performance of the embryos is still not known clearly but

0

1

2

3

4

5

6

T0 T1 T2 T3 T4

Pro

tein

co

nte

nt

(mg

g-1

fre

sh w

eig

ht)

o

f ti

ssu

e af

ter

4 w

eeks

Induction Prolifreation Maturation Germination

Induction Prolifreation Maturation Germination

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

T0 T1 T2 T3 T4

Am

ino

aci

d c

on

ten

t (m

g g

-1 F

resh

wei

gh

t)

of

tiss

ue

afte

r 4

wee

ks

b

a Fig. 6 (a) and (b) Protein

content (top) and Amino acid

content (bottom) content at

different stages of

embryogenesis after various

liquid overlaying treatments

254 Plant Cell Tiss Organ Cult (2011) 104:247–256

123

liquid overlaying has been proved to be very effective in

plant regeneration in other studied plant species (Te-chato

and Lim 1999). It also improved the growth and development

of plants like Anthurium (Nitayadatpat and Te-chato 2005).

We, therefore, believe that the liquid overlaying causes faster

embryogenic growth in C. roseus, which may be due to direct

contact with the medium that facilitate rapid and fast

absorption of nutrients by the growing tissues. Liquid over-

laying provides an excellent tool to adjust the pH, nutritional

and hormonal environments of tissues growing in vitro

(Pullman et al. 2009). Solid medium alone shows several

disadvantages which ultimately reduce growth as in agar-

added medium water potential reduces linearly (Ghashghaie

et al. 1991). Gelling agents also contribute elemental content

to medium (Van Winkle and Pullman 2003) and in some

cases Ca, Mg, S, Mn, Fe and Al are completely bound to agar

(Scholten and Pierik 1998). Similarly, it generates concen-

tration gradients for each nutrient in the zone of gel, next to

cell that slows cellular growth (Gupta and Timmis 2005).

Conclusion

Callus mediated embryogenesis shows a prominent possi-

bility to increase the propagation rate as embryogenic

cultures can easily be developed from fast growing cell

suspensions (Caligari and Shohet 1993). In C. roseus,

embryogeny was further improved by using liquid over-

laying technique where induced embryos showed fast

growth and development. Liquid overlaying also improved

early germination in C. roseus thus shortening the ‘embryo

to plantlet’ recovery time. Liquid overlaying based

embryogenesis may provide a valuable tool to enhance the

pace of genetic improvement. This technique could be

utilized in other potential applications of biotechnology

such as artificial seeds, micropropagation and gemplasm

conservation. The origin of embryos in overlaid condition

may also help in our understanding of the cellular switches

that keep the cell totipotent while undergoing cell division,

growth and reorganization.

Acknowledgments The first author is thankful to Council of Sci-

entific and Industrial Research (CSIR) for receiving Senior Research

Fellowship (SRF). Thanks are also due to Dr. Saumitra Banerjee for

his help with this manuscript.

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