Introduction

Catharanthus roseus is commercially valued for its >130 terpenoid indole alkaloids (TIAs). Among these, two of the leaf-derived antineoplastic bisindole alkaloids—vinblastine and vincristine—have no alternate synthetic substitute(s) [1]. These two bisindole alkaloids are used in several standard cancer chemotherapies for metastatic malignancy associated with acute lymphoblastic leukemias and Hodgkin’s/non-Hodgkin’s lymphomas [1]. The extremely low in-plant occurrence of TIAs in C. roseus plants, coupled with their high commercial demand and exorbitant market price, has brought them in focus of intense scientific scrutiny in the last 30 years with sole intention of increasing their production [2]. As a consequence, research in C. roseus has proceeded in two major directions: firstly, towards understanding the regulation of TIAs biosynthesis at the level of corresponding pathway genes and enzymes and, secondly, towards exploring their cell/tissue culture-based in vitro production [3]. The knowledge gathered in the last two decades has made TIAs biogenetic pathway as one of the most well-dissected and understood metabolic route in plants [2]. The entire pathway is characterized by at least 35 intermediates synthesized via 30 enzymatic reactions occurring in four different types of tissues (epidermis, internal phloem parenchyma, idioblasts, and laticifers) and also five subcellular compartments (cytosol, vacuole, thyllakoid membrane, nucleus, and endoplasmic reticulum) [1–6]. The TIA pathway in C. roseus proceeds with the condensation of tryptophan-derived indole moiety tryptamine and a plastidal nonmevalonate pathway-derived terpenoid moiety secologanin to form strictosidine that acts as a central precursor for all the downstream monomeric indole alkaloids like catharanthine, ajmalicine, tabersonine, vindoline, serpentine, etc. [1]. Dimerization of two monomeric alkaloids catharanthine and vindoline in the cell vacuole ultimately leads to the synthesis of most valuable dimeric alkaloids vincristine and vinblastine in the aerial tissues, particularly the leaves. In vitro cell, tissue and organ culture (including transgenic hairy roots) studies, so far carried out in C. roseus, have indicated that, depending upon the level of their morphogenic differentiation and set of specific culture conditions, these cultures are capable of biosynthesizing only the monomeric TIAs like ajmalicine, serpentine, catharanthine leurosine, lochnericine or horhammericine, etc. [2]. The synthesis of dimeric bisindole alkaloids, like vincristine and vinblastine in Catharanthus cultures, has still not been documented because such undifferentiated cultures fail to synthesize vindoline under their heterotrophic growth condition that do not allow the expression of a vindoline pathway enzyme N-methyltransferase for which a functional chloroplast system is required [7–9]. Extremely low occurrence of TIAs in C. roseus plant and its cell/tissue cultures can also be a consequence of poor availability of two precursor molecules tryptophan and secologanin drawn from the primary metabolic pools [10, 11]. While overexpression of the gene tryptophan decarboxylase (TDC), which codes for the enzyme that converts tryptophan into tryptamine, has alone not found effective for a concomitant increase in TIAs accumulation [12], the overexpression of strictosidine synthase (STR), either alone or along with TDC, was found more conducive for enhanced TIAs synthesis [13]. Exogenous feeding of STR overexpressing cell lines with tryptophan and/or terpenoid intermediates like geraniol or loganin, etc. has also resulted in higher TIAs production [11], indicating that it is not only the availability of tryptophan and secologanin, but their actual utilization which is more limiting in guiding the metabolic flux towards TIAs metabolism [10, 11]. Our group has earlier argued [14] that one of the reasons for the ambiguity, in results of such metabolic engineering efforts in C. roseus [2, 4], might be the fact that these studies were carried out with cell or tissue types wherein the availability of tryptophan or geraniol/loganin itself might be limiting for TIAs biosynthesis. Exogenous feeding of tryptophan in such cells/tissues also failed to produce desired results because tryptophan accumulation in them might have downregulated several other metabolic routes essential for cell survival and growth due to its strong feedback inhibition mechanism [15, 16]. Our group has earlier shown that callus and cell suspensions cultures of C. roseus resistant to growth inhibitory influence of 5-methyltryptophan (5-MT) (an analogue of tryptophan) could provide an ideal physiological environment for such pathway modulation efforts [14, 17]. It was successfully demonstrated that such 5-MT-resistant variant cell cultures were able to overcome the analogue stress by hyperaccumulating tryptophan in their free amino acid pool. The increased tryptophan availability could facilitate better biomass accumulation as well as TIAs productivity in them. It is pertinent to recall that tryptophan is least abundant amino acid in a plant cell, and its synthesis is strongly regulated by the feedback inhibition of its biogenetic enzyme anthranilate synthase [16]. The 5-MT resistance in several earlier studies has been traced to the appearance of a mutant form of the enzyme anthranilate synthase which was less sensitive to feedback inhibition [15–20]. Since tryptophan is the sole donor of an indole ring for a large number of cellular metabolites like auxins, phytoallexins, etc, it is always in high demand for maintaining primary cellular homeostasis. Its availability and diversion towards TIAs synthesis is, therefore, likely to be limiting in the background of concomitant growth penalties. The 5-MT-resistant callus and cell lines of C. roseus selected earlier by us [14, 17], however, failed to push the TIAs pathway flux beyond catharanthine accumulation because of the lack of requisite state of cellular/organ differentiation necessary for entire pathway expression.

The present study was, therefore, undertaken to select 5-methyltryptophan-resistant multiple shoot cultures of C. roseus to assess if increased availability of tryptophan in such variant tissue with higher state of tissue/organ differentiation could favorably advance the TIAs biogenesis beyond catharanthine under in vitro and in vivo conditions.

Experimental

Plant Material and Screening

Multiple shoot cultures of three genotypes of C. roseus (cv. Nirmal, Prabal, and Dhawal; National Gene Bank accession numbers 0865, 0862, and 0859, respectively) were raised and multiplied on a Murashige and Skoog medium [21] supplemented with 1.0 mg/l 6-benzylaminopurine and 0.1 mg/l α-naphthaleneacetic acid. The sensitivity of these multiple shoots to 5-methyltryptophan inhibition was tested by culturing their nodal explants on this shoot multiplication medium fortified with a graded series 10–50 mg/l 5-MT. Explant survival, axillary bud emergence, and elongation of shoots were taken as the major parameters for screening. Explants cultured on 5-MT-free medium served as control for comparison. The LD50 dose of 5-MT was ascertained in terms of explants survival and axillary shoot growth under analogue stress in comparison to nontreated controls. The 5-MT concentration, where explants survival or bud emergence failed to occur, was considered as lethal dose. The cultures, showing at least 50 % or more shoot growth than the nonstressed control cultures, were screened and advanced to four to five repeat screening cycles under the same intensity of analogue stress to eliminate escapees. The putative 5-MT-tolerant variants capable of growing in presence of LD50 to sublethal doses were subcultured on medium with or without respective dose of 5-MT for another three to four cycles to generate their stocks. The best growing shoots retrieved under varying levels of 5-MT stress were subsequently shifted to medium fortified with two levels higher and one level lower 5-MT concentrations for five to six subcycles to ascertain their threshold limits of analogue tolerance. A total of ten differentially 5-MT-resistant shoot lines, namely, P30, P40, P50, P70, D20, D30, D40, N30, N40, and N50 were thus screened (Table 1). Variants, showing stable survival and shoot growth comparable with nonselected shoots maintained under stress-free environment, were then characterized on the basis of the stability of acquired trait in the presence and absence of the 5-MT stress-free tryptophan levels and alkaloid profiles.

Table 1 Free tryptophan, total alkaloid, and vindoline contents of 5-MT-tolerant shoot culture lines of C. roseus under in vitro conditions. Medium was fortified with different concentrations of 5-MT
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All cultures were incubated under the photoperiod cycle of 10 h dark and 14 h light exposures provided by white fluorescent tube lights (15 μE/m/s). The temperature and humidity in the culture room were maintained at 26 ± 2 °C and 70–80 % RH, respectively.

Glasshouse Acclimatization of 5-MT-Tolerant Shoot Culture-Derived Plantlets

For assessing their in vivo performance, the 5-MT-tolerant shoots were transferred to a rooting medium consisting of a MS basal medium supplemented with 3.0 mg/l indole-3-butyric acid. Thirty-day-old-rooted plants were then taken out from the culture tubes, washed with running tap water to remove the adhered phytagel, and transplanted in earthen pots containing sterilized soil and vermicompost (1:1). These are being maintained under glasshouse conditions for more than 2 years now.

Postharvest in vivo Elicitation of 5-MT-Tolerant Plants

Flowering twigs with four to five pairs of leaves from 1-year-old glasshouse-grown 5-MT-resistant plants of selected variant lines were given a postharvest elicitation treatment by immediately dipping them in a solution of either 30 mg/l 5-MT or 5 mg/l tryptophan in a glass tube for 24 or 48 h. The tubes were kept under sunlight for 6–8 h to allow uptake of the elicitors via transpiration pull. The treated twigs were oven dried at 50 °C for alkaloid profiling. Similar postharvest elicitation treatments were also given to excised leaves of 5-MT-resistant plants by submerging them into 30 mg/l 5-MT or 5 mg/l tryptophan solutions for 24 or 48 h under room conditions. The treated tissues were then analyzed for their tryptophan, total alkaloids, and vindoline contents. Tissues treated in similar manner with distilled water (dH2O) alone were used as controls.

Tryptophan Estimation

Tryptophan in the free amino acid pool was estimated as per the procedure reported earlier [17]. For this, 2.0 g of oven-dried shoot tissue was grounded and extracted twice with 10 ml methanol/chloroform/water (MCW) mixture (12:5:3) and thrice with 10 ml 80 % ethanol at 12,000–15,000 rpm for 15 min each. To combined MCW extract, 7.0 ml distill water and 5.0 ml chloroform were added and centrifuged to separate phases. Top layer was taken and mixed with pooled ethanolic extract. The volume was reduced to 10 ml under vacuum, and the concentrate was loaded onto a Dowex 50 × 2 column (15 cm) and allowed to stand for 30 min before fractionating out at 1.0 ml/min for complete adsorption on the resin. To remove soluble carbohydrates, the column was then washed with 25 ml dH2O. Tryptophan was eluted from the column by 4 × 25 ml washing with 0.3 % ammonia solution. The pooled ammonia fractions were evaporated to dryness on a water bath at 80 °C. The residue was redissolved in 2.0 ml dH2O. For colorimetric quantification of tryptophan according to the procedure of Dalby and Tsai [22], 0.5 ml of the test solution prepared as above was mixed with the 2.0 ml of a reagent freshly made by mixing equal volume of reagents A and B (reagent A, 270 mg of FeCl3·6H2O dissolved in 0.5 ml water mixed with 1 l of 2 % acetic anhydride in glacial acetic acid; reagent B, 30 N sulfuric acid). The reaction mixture was vortexed and incubated at 60 °C on a water bath for 15 min. The reaction was stopped in an ice bath, and the absorption was read at 575 nm. The free tryptophan was calculated using a standard curve of l-tryptophan prepared in the range of 0–50 μg/0.5 ml.

Chemical Analysis

For determining the total alkaloids content in the in vitro and in vivo grown tissues, 1.0 g oven-dried (50–60 °C) tissue was extracted thrice with HPLC grade methanol (3 × 30 ml; 12 h each) at room temperature. The MeOH extracts were pooled and dried in vacuum to 10 ml, mixed with 10 ml dH2O, acidified with10 ml of 3 % HCl, and washed thrice with hexane (3 × 30 ml). The aqueous portion was basified with ammonia (till pH 8.0), extracted with chloroform (3 × 30 ml), dried over anhydrous sodium sulfate, concentrated in vacuum, and weighed. TLC analysis of the total alkaloid extract was carried out using activated silica glass plates (Merck; 20 × 20 cm; silica gel 60 F254). The mobile phase was consisted of 100 ml ethyl acetate with two to three drops of ammonia and 12 % methanol in chloroform [23]. Spots were resolved following spraying with Dragendorff’s reagent. For comparison, the commercially available reference samples of catharanthine, vindoline, vincristine, and vinblastine (Sigma-Aldrich, USA, and Tauto Biotech, China) were also spotted. For HPLC analysis of the alkaloid extracts, a modular HPLC apparatus (Waters Corporation, Milford, MA, USA) equipped with a 600E multisolvent delivery system and a 2,996 photodiode array detector was used. Data was processed using Empower Pro (Waters Corporation) chromatographic software. On-line degassing of solvents was done with helium, and 10 μl of the extract was loaded onto a RP-18e reversed-phase Chromolith Performance HPLC column (100 × 4.6 mm ID.). A constant flow rate of 1.2 ml/min was used for all the analysis. The mobile phase used was consisted of 21:79 (v/v) acetonitrile and 0.1 M phosphate buffer containing 0.5 % glacial acetic acid (pH 3.5). The detection was done at 254 nm. Standard mixture of vindoline, catharanthine, vincristine, and vinblastine (0.25 mg each/ml in methanol) was used for peak resolution based on their respective retention times [24].

Statistical Analysis

All initial 5-MT sensitivity and screening experiments were carried out with a minimum of 25 replicated nodal explant cultures per treatment, and the experiment was repeated twice. For quantification of tryptophan and total alkaloids, tissue was harvested from a minimum of five representative cultures of each 5-MT-tolerant lines and pooled before further processing in duplication. In vivo elicitation experiments were performed using tissues harvested from four glasshouse-grown plants of each tested variant lines with two replications. Data are expressed as mean ± SD of all the replicates.

Results

Selection of 5-MT-Tolerant Shoots

The sensitivity response of nodal explants to gradually increasing concentration of 5-MT stress in C. roseus was found to be genotype specific. The shoots of genotype Dhawal were most sensitive to analogue inhibition with a LD50 value of 15 mg/l 5-MT, followed by cv. Nirmal and cv. Prabal with a LD50 value of 20 and 25 mg/l, respectively. Repeated screening under sustained 5-MT selection pressure finally led to the isolation of three stable variant shoot lines in genotype Dhawal (D20, D30, and D40), four lines in Prabal (P30, P40, P50, and P70), and three lines in Nirmal (N30, N40, and N50). Hence, a total of ten lines displaying differential tolerance limit to 5-MT stress (20–70 mg/l) could be isolated. In general, the variants with a threshold tolerance limit of more than 40 mg/l 5-MT showed poor growth, limited shoot proliferation, pale green leaves, and compressed internodes. In comparison, the lines isolated in presence of 40 mg/l 5-MT were best growing and most stable with shoot proliferation rate comparable to nonselected wild-type shoots grown on the control medium (Fig. 1a–c). All selected variant lines registered two-to-threefold increase in their tryptophan content in the free amino acid pool when grown in the presence of 5-MT stress. This increment was more pronounced in selected lines of Prabal and Nirmal genotypes (Table 1). Highest tryptophan accumulation occurred in lines P40, P30, D40, and N40, i.e., 296.5, 241.0, 200.6, and 202.0 μg/g dry wt., respectively. A concomitant increase in the total alkaloid content (2.3–3.8 % dry wt.) under the analogue stress was also noticed in these lines when compared to 1.0–1.58 % dry wt. level in the respective wild-type shoot maintained in a stress-free environment. The major pharmaceutically important TIAs detected in HPLC analysis of alkaloid extracts of the selected lines was vindoline. The flux of the pathway towards vindoline synthesis under 5-MT was best represented in P40, D30, D40, N30, and N40 lines (Table 1). Under in vitro conditions, maximum vindoline was detected in N40 line (0.046 % dry wt.), followed by D40, D30, (0.034 % dry wt.), and P40 (0.022 % dry wt.).

Fig. 1
figure 1

Best growing 5-MT-tolerant lines of C. roseus and their TLC and HPLC-based detection/quantitation of vindoline (a) N40, (b) D40, (c) P40, (d) glasshouse-acclimatized shoot lines, (e) in vivo elicitation in 5-MT solution, (f) total alkaloid extracts of elicited leaf, and twigs of 5-MT-tolerant (P40, D40, and N40) and nonselected wild line (W) plants of C. roseus [tryptophan (24 h) treated leaf (L) and twig (T) samples under UV]; (g) HPLC chromatogram of D40L (48-h 5-MT) extract showing vindoline peak (RT = 22.719; arrow)

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In vivo Performance of 5-MT-Tolerant Plantlets Under Glasshouse Condition

The rooted plants of selected 5-MT-tolerant lines showed 70–80 % survival in the glasshouse (Fig. 1d). New axillary shoots started appearing in them within third week of transplantation. The plants could fully adapt to glasshouse environment within 30–35 days of growth. All the acclimatized plants flowered and set seed were set under in vivo conditions. Three of the best performing 5-MT-tolerant lines, namely, N40, P40, and D40, were subsequently analyzed for their tryptophan, total alkaloid, and vindoline contents after 1 year of in vivo growth (Table 2). The maximum tryptophan concentration of 190.45 μg/g dry wt. was recorded in P40 which was more than two folds than in the wild-type plants of Prabal genotype. It was followed by 180.32 and 165.75 μg/g dry wt. tryptophan in N40 and D40 lines, respectively. In comparison, the tryptophan concentration in the shoots of respective wild-type plants of Nirmal and Dhawal genotypes was found to be 120.12 and 94.10 μg/g dry wt., respectively. The in vivo performance of these three selected lines with respect to their total alkaloid and vindoline contents was more or less in accordance with that of their respective in vitro-grown shoots on a 5-MT-free media. The plants of Prabal wild-type line were least alkaloid producer (0.66 % dry wt.), with only trace amount of vindoline. In comparison, the plants of P40 lines registered 2.35 % dry wt. content of alkaloids with 0.071 % dry wt. vindoline. Lines N40 and D40, on the other hand, had a total alkaloid content of 2.26 and 2.73 % dry wt. respectively, in contrast to 1.41 and 1.28 % dry wt. in their respective wild-type plants. Vindoline accumulation in these two 5-MT-tolerant lines was, however, two times less (0.025–0.037 % dry wt.) than in the plants of P40 line (Table 2).

Table 2 Free tryptophan, total alkaloid, and vindoline contents in tissue of selected 5-MT-tolerant shoot lines and their wild-type counterparts grown under glasshouse conditions
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Postharvest Elicitation of 5-MT-Tolerant Plants by Tryptophan and 5-MT

Since plants of 5-MT-tolerant variant lines could be successfully reared in the normal soil under glasshouse conditions, it was considered worthwhile to test if they have retained their acquired trait at the whole plant level in vivo. For this, flowering twigs or excised leaves of plants of P40, N40, and D40 lines were given a postharvest treatment by dipping them in an aqueous solution of 30 mg/l 5-MT or 5.0 mg/l tryptophan for 24 and 48 h. The treated tissues upon their chemical analysis showed that postharvest elicitation treatment with 30 mg/l 5-MT could dramatically enhanced the vindoline content by nine to ten folds in them in comparison to nonelicited tissues of the selected or wild-type plants, treated with water alone (Table 3). The increment in vindoline content upon 5-MT elicitation was best depicted by flowering twigs of line D40 (0.31 % dry wt.) and leaves of P40 (0.74) and N40 (0.091) lines in contrast to 0.017, 0.008, and 0.002 % dry wt. vindoline levels in tissues treated with water alone, respectively. In general, exposure of tissues to 5-MT for 24 h was more effective than 48-h treatments. Tryptophan treatments could also positively influence the vindoline concentration in the harvested tissue, but to a limited extent (three to four folds) only.

Table 3 Effect of elicitation treatment of 5-MT (30 mg/l) and tryptophan (5.0 mg/l) on leaf and intact twigs of selected 5-MT-tolerant plants reared in vivo
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Discussion

Availability of free tryptophan in a plant cell is generally low due to its high metabolic demand in several growth sustaining pathways [15, 16, 25, 26]. Reports emanating from our laboratory, including the results presented here, have convincingly demonstrated that selection of cell/tissue types tolerant to growth inhibitory stress imposed by 5-methyltryptophan (an analogue of tryptophan) can be an effective strategy to increase the tryptophan concentration for its availability for the synthesis of useful secondary metabolites, such as terpenoid indole alkaloids in C. roseus [14, 17, 27]. Need for generating such tissue types has been repeatedly emphasized in the literature by many earlier Catharanthus researchers [10, 11, 13, 20, 28–31]. Overaccumulation of tryptophan in 5-MT-tolerant variants in several earlier studies on some food crops has been suggested as a possible mechanism to reverse the 5-MT stress through a dilution effect of the analogue [32–34]. Attempts to push the flux of exogenously fed tryptophan in the normal wild-type cells generally do not yield desired results and often proved inhibitory for cell survival and growth due to its strong downregulating influence on many other metabolic routes [15, 18, 20, 26, 35]. The 5-MT-resistant shoot lines of C. roseus isolated in the present work, therefore, assume importance from three perspectives. Firstly, the selected variants cannot only accumulate more tryptophan under the selection pressure of 5-MT, but also effectively divert the flux of the accumulated amino acid towards TIAs pathway up to vindoline biogenesis due to higher level of tissue differentiation in them. Secondly, the 5-MT tolerance trait, selected in vitro, could not only persist in the absence of the selection pressure in vitro, but also in the plants regenerated from such variant shoot lines and grown in vivo in normal soil for more than 2 years. Thirdly, the harvested leaves and flowering twigs of glasshouse-grown plants of these selected lines could display eight to ten folds more postharvest enhancement in vindoline content within 24–48 h of an elicitation treatment with 5-MT or tryptophan. In our earlier reports on 5-MT-tolerant callus and cell suspension lines of this plant system, the in vitro diversion of overaccumulated amino acid towards TIAs pathway was found restricted up to the level of catharanthine formation only due to lesser cellular differentiation [17]. It is likely that higher level of cellular/organelle differentiation in 5-MT-tolerant multiple shoots or their resultant plants must have allowed the expression of the enzyme N-methyltransferase in their chloroplast for vindoline synthesis—a situation which usually do not exist in callus or cell suspensions [2, 36]. Though we could not advance our work to the extent of assaying and characterizing anthranilate synthase (AS) in our 5-MT-tolerant lines, but we are inclined to presume that the variants must have had an alteration in AS-α subunit of the enzyme to avoid its feedback inhibition by tryptophan as suggested by many previous workers [20, 37, 38]. Further, since a lot of attention is being paid these days to identify ideal storage and processing conditions for maximum metabolite recovery from medicinal crops [39, 40], our findings that leaves and flowering twigs of glasshouse-grown 5-MT-tolerant plants were highly responsive to postharvest elicitation with 5-MT or tryptophan for enhanced vindoline accumulation may constitute an important strategy in redefining the postharvest processing of Catharanthus herb to boost TIAs recovery.