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 vinblastine, and the monomeric alkaloids, ajmalicine and serpentine, respectively, are already used for the treatment of several neoplastic, hypertensive, and arrhythmic disorders (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 understanding the biosynthetic pathways for these compounds and for bioengineering efforts (Zárate 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).

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 variation 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 × 1.0–1.5 cm) from 4–6-month-old glasshouse-grown plants or 6–8-week-old multiple 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-benzyladenine (BA), 0.1 mg/l α-naphthaleneacetic acid (NAA), 0.4 mg/l thiamine-HCl, and 4 g/l phytagel (designated 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.

Table 1 Morphogenic response of Catharanthus roseus leaf explants on single and double combinations of 6-benzyladenine (BA) and/or α-naphthaleneacetic acid (NAA) containing medium
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Pre-plasmolysis treatment

For improving upon the frequency of shoot bud morphogenesis in cultured leaf explants, the efficacy of a per-plasmolysis 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 experiment, 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 (10–15-μm) 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 photographing 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 containing 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 genotypes. 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. Interestingly, 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, however, 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 tested, 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 protuberances 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, referred to as shoot bud induction medium (SBM).

Fig. 1a–s
figure 1

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, s glasshouse and field establishment of regenerated plants

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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 meristematic 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-plasmolysed 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 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 organogenic development. Control explants treated with only CPW solution (without mannitol) also did not show any positive effect on the regeneration frequency.

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
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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 subtending leaves (Fig. 1h–n). The direct mode of regeneration 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 acclimatized 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 frequency 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 embryogenesis 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 development of an efficient regeneration protocol via shoot bud organogenesis in C. roseus has further substantiated that BA at a high level (7.0 mg/l) can efficiently break the recalcitrancy 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 treatment 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 regeneration. 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 lowering 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 differentiation. 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; Möllers 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 production 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 experiments (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.