ORIGINAL PAPER

Direct shoot bud organogenesis and plant regenerationfrom pre-plasmolysed leaf explants in Catharanthus roseus

Priyanka Verma • Ajay Kumar Mathur

Received: 10 November 2010 / Accepted: 12 February 2011 / Published online: 2 March 2011

� Springer Science+Business Media B.V. 2011

Abstract A protocol for high-frequency shoot bud

regeneration from the leaves of Catharanthus roseus is

reported here for the first time. A 60-min pre-plasmolytic

treatment of leaf explants in a cell protoplast washing

medium containing 13% (w/v) mannitol followed by their

plating on a half-strength Murashige and Skoog (MS)

medium supplemented with 7.0 mg/l 6-benzyladenine

(BA) and 3.0 mg/l a-naphthaleneacetic acid (NAA) resul-

ted in the de novo induction and development of adventi-

tious shoot buds in more than 75% of explants. Histological

observations revealed a direct origin of these shoot buds

from hypodermal tissue around the mid-rib. The rooting in

the regenerated shoots was obtained in the presence of

3.0 mg/l indole-3-butyric acid (IBA) and the rooted plants

could be successfully established in soil with a 70% rate of

success. The relevance of the developed protocol in ter-

penoid indole alkaloids pathway engineering at the whole-

plant level in C. roseus is discussed.

Keywords Catharanthus roseus � Direct regeneration �Adventive shoot bud organogenesis � Pre-plasmolysis

Introduction

Catharanthus roseus (Madagascar periwinkle) plant is

commercially valued for harboring[120 types of bioactive

terpenoid indole alkaloids (TIAs) in leaves and roots (van

der Heijden et al. 2004). Among the pharmacological

compounds derived from leaf and root tissues of C. roseus,

the anti-mitotic bisindole alkaloids, vincristine and vin-

blastine, and the monomeric alkaloids, ajmalicine and

serpentine, respectively, are already used for the treatment

of several neoplastic, hypertensive, and arrhythmic disor-

ders (Moreno et al. 1995). Plant-based production of these

molecules is expensive due to their low levels in plants and

high cost of extraction, thus, they are in short supply, even

with their high commercial value (Di Fiore et al. 2004).

Moreover, C. roseus serves as a model plant for under-

standing the biosynthetic pathways for these compounds

and for bioengineering efforts (Zarate and Verpoorte 2007;

Facchini and De Luca 2008). So far, all bioengineering

investigations in C. roseus have relied on cell suspension

and transformed hairy root cultures. These systems lack

those tissues and the level of cyto-differentiation that is

necessary for the expression of all TIA pathway genes and

enzymes, especially those involved downstream in the

latter steps of the pathway leading to the synthesis of

vindoline (Di Fiore et al. 2004; Pasquali et al. 2006; Shukla

et al. 2010). This problem is further compounded by the

recalcitrant nature of cultured cells and hairy roots of

C. roseus to regenerate into whole transgenic plants.

Hence, barring just one report on the regeneration of plants

from transformed hairy root cultures (Choi et al. 2004),

Catharanthus is still considered to be a genetically non-

tractable plant species because of the non-availability of an

efficient regeneration protocol for pathway engineering at

the whole-plant level (van der Fits et al. 2001; Di Fiore

et al. 2004). The necessity to develop a regeneration and

transformation protocol operative at the whole-plant level

has, therefore, been repeatedly emphasized to advance the

issue of TIAs pathway engineering in C. roseus (Mahroug

et al. 2006; Zhao and Verpoorte 2007).

P. Verma � A. K. Mathur (&)

Plant Tissue Culture Laboratory, Department of Plant

Biotechnology, Central Institute of Medicinal and Aromatic

Plants, Council of Scientific and Industrial Research,

PO CIMAP, Kukrail Picnic Spot Road, Lucknow 226015, India

e-mail: akmcathp@gmail.com; ak.mathur@cimap.res.in

123

Plant Cell Tiss Organ Cult (2011) 106:401–408

DOI 10.1007/s11240-011-9936-4

Direct plant regeneration via adventitious shoot bud

formation, preferably from leaf explants, is a key step in

the application of genetic transformation techniques for

metabolic pathway manipulation in medicinal plants where

the biogenesis of a desired metabolite is often closely

linked with the cellular architecture of the whole plant

(Koroch et al. 2002; Sharma et al. 2005; Opabode 2006;

Cheruvathur et al. 2010). Direct shoot bud organogenesis

also helps in avoiding the interference of somaclonal var-

iation in the transformation process when an intervening

callus interface also becomes involved in the regeneration

process (Annapurna and Rathore 2010; Ghimire et al.

2010; Liu et al. 2010). Though such direct de novo

regeneration of adventitious shoot buds and plants from

cultured tissue may frequently follow a multicellular and

multihistogenic pattern of origin, they normally allow the

recovery of more stable and uniformly transformed plants

in comparison to those arising from the cells of a pre-

formed meristem like axillary/apical shoot buds or somatic

embryos with pre-defined germ-lines (Newell 2000; Yan

and Wang 2007; Zhu et al. 2007; Wang et al. 2009). In case

of C. roseus, such an attempt is limited to just one report

wherein petiole explants were tested for adventitious shoot

bud regeneration (Lee et al. 2003). In this study, the shoot

buds originated indirectly from the callus and the response

was found to be highly genotype-specific.

In this communication, we describe, for the first time, an

efficient method for direct shoot bud regeneration from

pre-plasmolysed leaf explants of C. roseus. The method

described here was tested successfully with leaf explants

obtained from both in vitro-grown multiple shoot cultures

and glasshouse-grown plants of three released genotypes of

C. roseus and can be gainfully employed for TIAs pathway

engineering at the whole-plant level in this valuable

medicinal herb.

Materials and methods

Explant source and culture conditions

Leaves without petioles (2.0–3.0 9 1.0–1.5 cm) from 4–6-

month-old glasshouse-grown plants or 6–8-week-old mul-

tiple shoot cultures of three released varieties (cv. Nirmal,

Prabal, and Dhawal; National Gene Bank [CIMAP]

accession nos. 0865, 0862, and 0859, respectively) of C.

roseus were used as explants. The stock of multiple shoot

cultures of all the accessions were initiated from nodal

explants and maintained in vitro on MS (Murashige and

Skoog 1962) medium supplemented with 1.0 mg/l 6-ben-

zyladenine (BA), 0.1 mg/l a-naphthaleneacetic acid

(NAA), 0.4 mg/l thiamine-HCl, and 4 g/l phytagel (desig-

nated hereafter as MSM). All stock cultures were incubated

at 24 ± 2�C at 3,000 lux white florescent light illumination

under a 16-h light and 8-h dark photoperiod.

Direct organogenesis and plantlet formation

For inducing direct shoot bud regeneration, leaf explants

from in vitro-grown multiple shoots or glasshouse-grown

plants of cv. Dhawal, Nirmal, and Prabal genotypes were

used. Ten explants per 90-mm radiation-sterilized plastic

petri plates were cultured on full- or half-strength MS

medium fortified with 23 combinations of BA and NAA

(Table 1). Data was collected after 6 weeks of incubation

as the mean performance (±SE [standard error]) of four

replicated plates per treatment. Morphogenic responses are

expressed as the mean percentage of explants which

responded and the number of shoot buds induced per

responsive explants. For shoot elongation, the regenerated

shoot buds were serially shifted first onto a half-strength

basal medium for 2–3 weeks and then to MSM. These

adventitious shoots readily rooted on half-strength MS

medium supplemented with 3.0 mg/l indole-3-butyric acid

(IBA). Rooted plants were successfully acclimatized in

pre-sterilized soil with [70% survival under the normal

glasshouse conditions of 28 ± 4�C temperature and

60–70% RH.

Pre-plasmolysis treatment

For improving upon the frequency of shoot bud morpho-

genesis in cultured leaf explants, the efficacy of a per-plas-

molysis treatment was assessed. For this, the leaf explants,

prior to their plating on best responded BA/NAA containing

medium in the above plant hormones optimization experi-

ment, i.e., 3.0 mg/l NAA and 7.0 mg/l BA, were submerged

in a 13, 15, or 20% (w/v) mannitol containing cell protoplast

washing solution (CPW; Frearson et al. 1973). The duration

of the plasmolysis treatment varied from 15 min to 3 h, after

which the leaves were thoroughly washed with liquid MS

basal medium (MSO), blot dried, and implanted horizontally

onto the medium with their adaxial surface in contact with

the medium. Non-plasmolysed leaves or those treated with

only CPW solution without mannitol served as the control in

this experiment.

Histological studies

To trace the early ontogenic stages of direct shoot bud

regeneration, the leaf explants were periodically fixed in

70% ethanol:acetic acid:formaline solution (18:1:1 v/v) at

5-day intervals until the shoot buds became discernible to

the naked eye. The fixed tissues were dehydrated through a

graded ethanol-butyl alcohol series and embedded in

paraffin wax (Johansen 1940). Serial transverse sections

402 Plant Cell Tiss Organ Cult (2011) 106:401–408

123

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Plant Cell Tiss Organ Cult (2011) 106:401–408 403

123

(10–15-lm) were cut on a rotary microtome, deparaffinized

in a xylene series, stained with safranine (1% w/v), and

mounted in Canada balsam before viewing and photo-

graphing with an Olympus stereo-microscope (BH-2) fitted

with a C35AD-2 camera.

Results and discussion

Shoot bud organogenesis from leaf explants

Initially, the leaf explants from multiple shoot cultures of

all three genotypes maintained on MSM medium were

cultured on full- or half-strength MS basal medium con-

taining 2.5–15.0 mg/l BA or 2.0–7.0 mg/l NAA, either

alone or in combination (Table 1). Explants cultured on

medium prepared with half-strength MS basal salts showed

better survival and morphogenesis among all of the geno-

types. BA alone (2.5–5.0 mg/l) in the medium caused only

limited callusing from the cut margin of the explants. The

extent of callusing was further reduced when the BA

concentration was increased to 7.0–15.0 mg/l. Interest-

ingly, 10–30% of the explants cultured on these higher

levels of BAP showed prominent swelling of the tissue

around the mid rib (Fig. 1a). The extent of swelling was

greater in the proximal half of the mid rib towards the

petiolar end and the response was genotype-specific

(Dhawal[Nirmal[Prabal). No organized structure, how-

ever, appeared from these swellings until the completion of

the 8 weeks culture passage. The addition of NAA alone in

the medium, on the other hand, supported only a callusing

response, with 3.0 mg/l being the optimal dose (Table 1).

When double combinations of growth hormones consisting

of 2.5–15.0 mg/l BA along with 3.0 mg/l NAA were tes-

ted, the number of explants showing callus response

gradually increased until the BA concentration reached

7.0 mg/l. BA at the 7.0–10.0-mg/l level in combination

with 3.0 mg/l NAA again induced prominent mid rib

swelling with limited callusing. More than 80% of explants

across the three genotypes showed this morphogenic

response on half-strength MS medium fortified with

7.0 mg/l BAP and 3.0 mg/l NAA. About 30% of the

responded cultures on this medium also showed the direct

appearance of a few green and round protuberances on

swollen tissues around the mid rib. Some of these protu-

berances also organized into leafy shoot buds when the

culture cycle was extended to 9–10 weeks. The number of

responding explants and the intensity of callusing or

organogenesis again declined when the BA level was

increased to 10.0–15.0 mg/l in 3.0 mg/l NAA containing

medium. To further standardize the right combination of

these growth hormones, the concentration of NAA in the

7.0 mg/l BA containing medium was varied from 0.5 to

3.5 mg/l. The results again suggested a critical requirement

of 3.0 mg/l NAA and 7.0 mg/l BA for optimal shoot bud

morphogenesis response. This medium is, hereafter, refer-

red to as shoot bud induction medium (SBM).

Effect of pre-plasmolysis treatment of leaf explants

on direct shoot bud induction

Since Catharanthus leaves are the active site of synthesis

and accumulation of several cyto-toxic/anti-mitotic TIAs,

it was thought logical to presume that the presence of these

molecules in the explants at the time of culturing might

itself be causing a block in the complete expression of a de

novo organogenetic cycle in them. To test if the dilution/

removal of such toxic compounds from the site of meri-

stematic zones can help in overcoming such a block, the

leaf explants were given a pre-plasmolytic treatment prior

to their culturing on SBM. The leaves were pre-plasmol-

ysed for 15–180 min in a CPW salt solution containing 13,

15, or 20% mannitol (Table 2). An osmotic treatment of

1 h in CPW 13% mannitol solution prior to the culturing of

leaves on SBM was found to be very effective in allowing

the development of 7.1 ± 1.7 well organized shoot buds

from the green meristemoids formed on the swollen mid rib

portion of the leaf explants (Fig. 1b–e). The response was

evident in[75% of the cultures across the three genotypes

and the bud regeneration occurred directly from leaf tissue

without any intervening callus tissue. The exposure of

explants to CPW 13% mannitol stress also helped in

reducing the time period for shoot bud emergence to

30–35 days in comparison to 60–70 days in non-treated

controls. The induced buds grew into 1.0–1.5-cm-long

micro-shoots by the end of the 8 weeks culture cycle

(Fig. 1f, g). The treatment of explants at higher osmoticum,

i.e., 15% mannitol, was effective at 30-min durations of

pre-plasmolysis for both the percentage of explants which

responded, as well as the number of shoot buds regenerated

per responded explants when compared with the controls

(Table 2). The time required for shoot bud emergence was

further reduced to 25–30 days in this treatment when

compared with explants plasmolysed in 13% mannitol

containing solution. Longer exposures in this solution for

60–120 min, though, adversely affected the explant

Fig. 1a–s Direct shoot bud induction from pre-plasmolysed leaf

explants of Catharanthus roseus. a–e Early ontogenic stages of shoot

bud organogenesis around the swollen mid rib portion of the leaf

explants on shoot bud induction medium (SBM). f, g Elongation of

induced shoot buds on SBM. h–n Thin histological sections of

regenerating shoot buds showing their direct origin and vascular

connectivity with the parent tissue; (o–q) shoot elongation on half-

strength basal medium (o), multiple shoot formation on MSM (p), and

rooting on half-strength MS ? 3.0 mg/l IBA medium (q); r, sglasshouse and field establishment of regenerated plants

c

404 Plant Cell Tiss Organ Cult (2011) 106:401–408

123

viability and frequency of shoot bud organogenesis, and the

emergence of induced buds was again faster in comparison

to the controls or 13% mannitol treated explants.

Plasmolysis of leaves in 20% mannitol containing CPW

solution failed to support explants survival and any orga-

nogenic development. Control explants treated with only

Plant Cell Tiss Organ Cult (2011) 106:401–408 405

123

CPW solution (without mannitol) also did not show any

positive effect on the regeneration frequency.

Histological examination

Light microscopic examination of serial sections of leaf

explants pre-plasmolysed for 1 h in CPW 13% mannitol

solution and then cultured on SBM revealed that tissue

around the mid rib portion acquired intense meristematic

activity after 10–12 days of culturing. Organized shoot

primordia started appearing as small protuberances from

hypodermal tissues and grew further into well-defined

shoot buds comprising a growing apex, flanked by sub-

tending leaves (Fig. 1h–n). The direct mode of regenera-

tion of these shoot buds was evident by the absence of

intervening callus and continuity of their vascular strands

with that of the parent tissue.

Shoot elongation, rooting, and plantlet development

Shoots regenerated on SBM, when transferred to fresh

medium of similar composition, did not elongate further

and dried off within 3–4 weeks of culture. For optimal

growth and elongation of such shoots, a serial transfer, first

onto a half-strength basal medium for 2–3 weeks (Fig. 1o)

and then to full-strength MSM medium containing 1.0 mg/l

BA and 0.1 mg/l NAA (Fig. 1p), was found to be essential.

Rooting in these shoots occurred on half-strength MS

medium supplemented with 3.0 mg/l IBA (Fig. 1q). More

than 80% of shoots developed roots within 2–3 weeks of

culture. The thin elongated roots appeared in dense clusters

(20–40 roots/shoot) without any callusing at the root–shoot

junction. Rooted plantlets could be successfully acclima-

tized in soil with [70% survival (Fig 1r, s). The entire

plant regeneration protocol from explants-to-plants in the

field took about 4 months. Though the developed method

was found to be applicable to leaf explants obtained both

from in vitro multiple shoot cultures or glasshouse-grown

stock plants of all three genotypes of C. roseus, the fre-

quency of explants which responded to the regeneration

cycle was better when leaves from multiple shoot cultures

were used.

A positive effect of BA has been implicated in several

earlier tissue culture studies dealing with somatic embryo-

genesis and axillary bud growth in C. roseus (Ramawat

et al. 1978; Krueger et al. 1982; Lee et al. 2003). The

present study that constituted the first report on the devel-

opment of an efficient regeneration protocol via shoot bud

Table 2 Morphogenetic response from pre-plasmolysed leaf explants of C. roseus on 7.0 mg/l BA and 3.0 mg/l NAA containing SBM

Plasmolysis

treatment

Duration of

pre-plasmo-lysis

(min)

% of explants showing direct shoot bud

organogenesis (mean ± SE)

No. of shoot

buds/explants (mean ± SE)

Time required for

shoot bud emergence (days)

cv. Dhawal cv. Nirmal cv. Prabal cv. Dhawal cv. Nirmal cv. Prabal cv. Dhawal cv. Nirmal cv. Prabal

CPW 13% M 0 (control) 34.7 ± 3.8 36.3 ± 2.7 28.8 ± 1.9 1.6 ± 0.2 1.7 ± 1.0 1.2 ± 0.6 60–65 60–70 60–65

15 33.2 ± 2.8 31.5 ± 3.3 34.5 ± 2.8 1.8 ± 0.2 1.8 ± 0.6 1.3 ± 0.7 55–60 50–60 50–60

30 42.6 ± 3.5 38.7 ± 4.2 39.4 ± 3.0 3.2 ± 0.6 4.1 ± 1.9 2.7 ± 1.3 40–45 40–45 40–45

60 84.0 ± 5.7 78.3 ± 3.7 77.6 ± 2.9 7.1 ± 1.7 6.1 ± 2.9 5.9 ± 0.8 30–35 35–40 35–40

120 11.5 ± 1.4 12.8 ± 3.9 15.0 ± 3.7 2.5 ± 0.8 1.8 ± 1.1 1.8 ± 0.5 30–35 30–35 30–35

180 0 0 0 – – – – – –

CPW 15% M 15 23.6 ± 2.8 20.2 ± 1.3 19.9 ± 2.4 1.3 ± 0.27 1.5 ± 0.3 1.3 ± 0.3 30–35 30–35 35–40

30 38.3 ± 5.3 39.2 ± 3.8 33.1 ± 1.3 4.3 ± 0.91 2.1 ± 0.9 2.3 ± 0.9 28–30 25–30 25–30

60 44.6 ± 7.3 36.9 ± 3.2 33.4 ± 2.5 2.5 ± 1.03 2.1 ± 1.1 1.5 ± 1.0 28–30 28–30 28–30

120 10.5 ± 1.0 13.9 ± 1.0 11.3 ± 0.6 2.3 ± 1.11 1.9 ± 1.1 1.3 ± 1.1 30–32 30–35 30–35

180 0 0 0 0 0 0 0 0 0

CPW 20% M 15 0 0 0 – – – – – –

30 0 0 0 – – – – – –

60 0 0 0 – – – – – –

120 0 0 0 – – – – – –

180 0 0 0 – – – – – –

CPW 0% M

(control)

15 33.9 ± 2.8 34.9 ± 1.8 30.9 ± 2.1 1.9 ± 0.6 1.9 ± 0.5 1.1 ± 0.9 60–65 60–70 60–65

30 29.9 ± 3.8 31.9 ± 3.1 32.0 ± 3.1 1.1 ± 0.3 1.3 ± 0.7 2.1 ± 0.3 60–65 60–70 60–65

60 30.1 ± 1.0 30.1 ± 1.6 27.1 ± 1.6 1.8 ± 1.0 1.6 ± 1.1 1.8 ± 1.0 60–65 60–70 60–65

120 31.3 ± 2.9 30.3 ± 2.0 31.3 ± 2.4 0.9 ± 0.3 1.9 ± 0.5 1.2 ± 0.3 60–65 60–70 60–65

180 29.8 ± 0.8 30.0 ± 1.8 29.7 ± 1.8 1.6 ± 0.5 1.6 ± 0.3 1.1 ± 0.4 60–65 60–70 60–65

Each treatment consisted of ten explants with three replications. Data were recorded after 10 weeks of incubation

CPW cell protoplast washing medium (Frearson et al. 1973); M mannitol (w/v); SE standard error

406 Plant Cell Tiss Organ Cult (2011) 106:401–408

123

organogenesis in C. roseus has further substantiated that BA

at a high level (7.0 mg/l) can efficiently break the recalci-

trancy of leaf explants for shoot bud regeneration in this

plant system, as was observed in an earlier study by Lee

et al. (2003) using petiolar explants. Our findings also

highlight that a plasmolytic treatment of leaves in CPW

13% mannitol solution for 60 min could significantly

improve the complete expression of a direct shoot bud

regeneration cycle in them. Such a mild dehydration treat-

ment might have influenced the organogenesis either by

easy/rapid uptake of growth hormones by the explants

(Wetherell 1984) or by lowering the concentration of cyto-

toxic TIAs (through ex-osmosis) in and around dividing

cells involved in the process of de novo shoot bud regen-

eration. Recently, Roepke et al. (2010) have also shown that

catharanthine, which is one of the cytotoxic TIAs of

C. roseus, is almost solely accumulated in leaf epidermal

cells and, hence, it is likely that the plasmolysis treatment

given to leaf explants in the present study might have helped

in lowering its concentration and inhibitory influence on the

organogenesis. It also appears that the culturing of explants

in the presence of high BA concentration was necessary

only for the initial development of shoot buds and its low-

ering through a serial transfer to a BA-free and then to a low

BA-containing medium was essential for subsequent shoot

elongation and rooting.

The non-availability of a reliable plant regeneration

method in C. roseus has been frequently highlighted as a

major impediment in the metabolic engineering of TIAs

pathway at the whole-plant level in this herb. The reason is

obvious because the biosynthesis of these molecules

demands complex inter- and intra-cellular levels of dif-

ferentiation. Although a few reports pertaining to shoot/

plant regeneration in C. roseus from stem node, immature

or mature zygotic embryo, hypocotyl, petiole, or anther-

derived callus/cell suspensions do exist in the literature

(Abou-Mandour et al. 1979; Hirata et al. 1987; Miura et al.

1988; Mollers and Sarkar 1989; Furmanowa et al. 1994;

Kim et al. 1994; Kaur et al. 1996; Piovan et al. 2000; Lee

et al. 2003; Choi et al. 2004; Campos-Tamayo et al. 2008),

they primarily aimed to either explore the potential of such

cultured tissues for in vitro secondary metabolite produc-

tion or to produce disease-free plants. Moreover, the

regeneration rate in all of these studies was too low to be of

any use in Agrobacterium-mediated transformation exper-

iments (Dhandapani et al. 2008). The regeneration protocol

described here, therefore, assumes great relevance in the

light of these limitations. The method has been gainfully

employed to generate transgenic C. roseus plants (Verma

and Mathur 2011) and it is hoped that it will fill the long-

pending gap in advancing the metabolic engineering

research in C. roseus to the desired levels of cellular and

tissue differentiation.

Acknowledgments This work was financially supported by the

Council of Scientific and Industrial Research (CSIR), New Delhi,

India, through its Network Project COR-02. The help rendered by our

colleague Dr. G.D. Bagchi during the micro-photography is immen-

sely acknowledged. P.V. also thanks the CSIR for awarding a senior

research fellowship to her during the course of this investigation. We

are also grateful to the Director of the Central Institute of Medicinal

and Aromatic Plants (CIMAP) for providing the facilities and support.

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