Plant Biotechnol Rep DOI 10.1007/s11816-013-0273-4 Improved furanocoumarin production in Ruta graveolens L. regenerated via in vitro stem internode cultures Sagarika Bohidar, Suchismita Pattanaik & Manikkannan Thiruvanoukkarasu 1 3 Your article is protected by copyright and all rights are held exclusively by Korean Society for Plant Biotechnology and Springer Japan. This e-offprint is for personal use only and shall not be self-archived in electronic repositories. If you wish to self-archive your work, please use the accepted authors version for posting to your own website or your institutions repository. You may further deposit the accepted authors version on a funders repository at a funders request, provided it is not made publicly available until 12 months after publication. ORI GI NAL ARTI CLE Improved furanocoumarin production in Ruta graveolens L. regenerated via in vitro stem internode cultures Sagarika Bohidar
Suchismita Pattanaik
Manikkannan Thiruvanoukkarasu Received: 6 November 2012 / Accepted: 10 January 2013 Korean Society for Plant Biotechnology and Springer Japan 2013 Abstract A rapid and efcient in vitro propagation pro- tocol by enhanced multiple shoot proliferation from inter- node cultures of Ruta graveolens was established. Mean shoot number was maximum (55.83) in Murashige and Skoog (MS) basal medium fortied with 1.0 mg L -1 benzyl amino purine and 0.25 mg L -1 indole-3-acetic acid. The elongated shoots rooted within 1012 days in 1/2- strength MS medium supplemented with 2.0 mg L -1 indole 3-butyric acid. About 80 % of the rooted plantlets survived acclimatization and transfer to the eld. Phyto- chemical analysis revealed that micropropagated plants produced linear furanocoumarins, characteristic of the species, in greater quantities as compared to the in vivo- grown plants. The results will facilitate the conservation of this valuable medicinal plant and to obtain plants with improved phytochemical constituents. Keywords Auxins In vitro propagation Linear furanocoumarins Ruta graveolens Stem internode Introduction Ruta graveolens L. (Family: Rutaceae) has been reported to be a rich source of linear furanocoumarins (FCs), i.e. bergapten (5-methoxypsoralen), and xanthotoxin (8-meth- oxypsoralen), which have many therapeutic properties. Many pharmacological tests have revealed that couma- rins have antitumor, antioxidative, antimicrobial, anti- inammatory and antimutagenic effects, as well as inu- encing insect hormonal activities (Khatune et al. 2004; Bapat et al. 2005; Xiao et al. 2010; Jan et al. 2012). Owing to the high cost of chemical synthesis of bergapten, phar- maceutical industries use it as a by-product of the essential oil (bergamot oil) of Citrus bergamia. However, in recent times, there has been a sharp decline in the source plants due to over-exploitation and lack of simultaneous cultivation, thereby posing a threat to the pharmaceutical industries and compelling the search for new sources for bioproduction of furanocoumarins. R. graveolens was reported to be one of the most promising candidates (Poutaraud et al. 2000; Diwan and Malpathak 2008), as it contains high quantities of four linear, commercially important, FCs: psoralen, bergapten, xanthotoxin, and isopimpinellin. In spite of its well-known potential as a valuable medicinal plant, R. graveolens is not available in abun- dance in the wild and its cultivation is restricted to a few pockets in Orissa. The species is generally propagated through conventional vegetative methods. Propagation through seeds is an intricate task due to the physical dor- mancy caused by an impermeable seed coat resulting in poor germination (Diwan and Malpathak 2008). Moreover, the conventional propagation methods of this species are not only inadequate to cater to the needs but it is also time consuming. Therefore, development of a rapid mass propagation protocol of this medicinally important plant has become essential in order to reduce the existing pres- sure on the wild population as well as to bring about a cost reduction for commercial exploitation, with emphasis on the use of in vitro cultures. Earlier reports (Massot et al. 2000; Ekiert et al. 2001), described the use of in vitro germinated seedlings for raising shoot cultures. However, as Ruta is a cross-pollinated plant, shoots thus obtained would not be genetically identical to the parent plant and S. Bohidar S. Pattanaik M. Thiruvanoukkarasu (&) Bioresources Engineering Department, CSIR-Institute of Minerals and Materials Technology, Bhubaneswar 751013, Odisha, India e-mail: mtarasu@yahoo.com; arasu@immt.res.in 1 3 Plant Biotechnol Rep DOI 10.1007/s11816-013-0273-4 Author's personal copy the genetic makeup may vary with individual shoots. This may lead to variations in FCs production. Therefore, direct organogenesis from vegetative parts is preferred as there are fewer chances of somaclonal variation and the plants obtained are true to type (Diwan and Malpathak 2008). A large biomass of herbal raw material with fewer efcacies will not fulll the quality control requirement of the international market and will also not fetch high eco- nomic values for a sufcient time in the domestic market. In this scenario, a detailed comparative phytochemical evaluation of micropropagated and the mother plants becomes indispensable to select the right quality plant material for better quality of drugs. A plant may grow well in different situations but may fail to produce the same constituents, because active constituents present in the plant system are inuenced by the physiological and environmental factors. The advantage of developing plants through tissue culture is that they are grown under controlled environmental conditions, and hence, their phytochemical constituents are believed to be maintained. The overall objective of the current study was to develop an in vitro system for rapid propagation of R. graveolens plantlet cultures that could contain high levels of furanocoumarins. Materials and methods Source material and explants preparation Healthy shoots, with 45 nodes, were collected from 6-month-old plants of R. graveolens maintained in the experimental garden of the CSIR-Institute of Minerals and Materials Technology (IMMT), Bhubaneswar (2017 0 45 00 N, 8549 0 15 00 E; altitude 58 m), India. After trimming the leaves, the shoots were cut into pieces (5.07.0 cm long), washed thoroughly under running tap water, then disinfec- ted with 0.1 % HgCl 2 solution for 34 min, and nally washed with sterile double-distilled water under sterile condition. Stem internode segments measuring 2.53.0 cm were prepared from the sterilized shoots and used for in vitro culture. Culture medium and culture condition Murashige and Skoog (1962) basal media containing 30 g L -1 sucrose and 7 g L -1 agar with benzyl amino purine (BAP) either alone or in combination with auxins indole-3-acetic acid (IAA) or a-naphthalene acetic acid (NAA) was employed in the present study. Previous experiments on multiple induction in nodal segments cul- ture (Bohidar et al. 2008) and petiole cultures (unpublished) revealed that synthetic cytokinin BAP at 1.0 mg L -1 con- centration exerted the highest shoot formation. Hence, throughout the study, BAP 1.0 mg L -1 concentration was xed while auxin was used in varied concentrations (0.25, 0.5, 1.0, 2.0, and 3.0 mg L -1 ). For root induction, half-strength MS medium supplemented with different auxins, indole-3-butyric acid (IBA), NAA, and IAA, in concentrations ranging from 0.25 to 4 mg L -1 were used. The pH of the media was adjusted at 5.8 prior to autoclaving at 121 C and 108 kPa for 20 min. All chemicals used in the present work were of analytical grade (Sigma Chemical, USA; Merck; Hi-Media; and Qualigens India). All the cultures were maintained at 25 2 C and 60 5 % rel- ative humidity in a culture room under a 16-h photoperiod of 40 lmol m 2 s -1 light intensity provided by cool white uorescent tubes. Shoot induction, in vitro rooting and acclimatization Disinfected stem internode segments were inoculated onto the various shoot induction media compositions (Table 1) and maintained for 4 weeks and sub-cultured onto the same media compositions following expiry of a period of 4 weeks. When the shoots were more than 3.0 cm long, they were transferred to rooting media (Table 2) and cul- tured for 5 weeks. Regenerated plants with healthy root systems were washed (especially the root portions) under running tap water for about 15 min and the plantlets were then transferred to root trainers containing pre-soaked sterilized vermiculite medium and kept inside the mist chamber. The timer was set to 1 min on and 30 min off, and the relative humidity was set to 90 %. After 4 weeks, they were transferred to poly-bags containing soil ? sand ? farmyard manure (1:2:1) and shifted to a shade-net house for 6 weeks and subsequently transferred to the eld. The acclimatized plants were successfully transferred and established in the eld, and these plants are termed as ex vitro plants. The survival percentage was recorded after 4 weeks of transfer to the eld. Estimation of furanocoumarins Healthy, 5-month-old ex vitro plants of R. graveolens were evaluated for their furanocoumarin contents by HPLC and the values were compared with that of in vivo-grown plants. The plant materials were oven dried at 60 C for 8 h. The dried plant materials were powdered using a mixer grinder. Dried plant material weighing 200 mg was subjected to cold extraction with ethyl acetate overnight. The macera- tion process was repeated thrice and, at the end of the third day, the ethyl acetate extracts were pooled together. The solvent was removed under vacuum at temperature below Plant Biotechnol Rep 1 3 Author's personal copy 50 C in a rotary evaporator and the extracts were stored in a refrigerator at 4 C until further use. One milligram of each of the plant extracts was weighed separately and dissolved in methanol. The quantitative analysis of two FCs, i.e. xanthotoxin (xanthotoxin) and bergapten (5-MOP), was carried out by HPLC methods using Shimadzu Japan/LC-10Avp apparatus coupled to a photo diode array (DAD) on Xtimate TM C-18, 4.6 9 250 mm, 5 lm column. The isocratic solvent system was methanol: water 1:1.2 v/v. The ow rate was 1 mL min -1 and detection was performed at 310 nm. Identication and quantication of the secondary metabolites was carried out by comparing the retention time of the relevant peaks with their reference standards. Experimental design and statistical analysis At the end of the 6 weeks of the in vitro multiplication cycle, the following parameters were determined: the per- centage of explant response, number of shoots longer than 8.0 mm, and the length of the shoots produced by each explant. Rooting experiments were evaluated by deter- mining rooting percentage, root number, and root length. Length of shoots and roots were intended as a cumulative length of each organ on one plant. Each treatment consisted of 10 explants and all the experiments were repeated thrice. The data were analyzed statistically using analysis of var- iance ANOVA, and the means were compared by Duncans multiple range test at a 5 % probability level according to Gomez and Gomez (1984). Table 1 Shoot regeneration from stem internode segments of Ruta graveolens cultured in MS ? cytokinin (BA 1.0 mg L -1 ) along with auxins (IAA and NAA) supplements Cytokinin ? auxin (A) Conc. mg L -1 (B) % Response Mean shoot numbers Mean shoot length (cm) BAP ? IAA BAP ? NAA BAP ? IAA BAP ? NAA BAP ? IAA BAP ? NAA 0.25 96.66 76.66 55.83 a 8.73 f 3.29 a 3.20 a 0.5 90.00 70.00 43.00 b 7.50 f 3.43 a 2.17 b 1.0 83.33 66.66 33.50 c 6.50 f 3.62 a 1.70 c 2.0 83.33 76.66 27.00 d 5.67 f 2.44 b 1.67 c 3.0 93.33 76.66 21.57 e 4.37 f 2.04 b 1.27 d Mean shoot numbers Mean shoot length (cm) A B A B F value 759.74* 40.16* 194.29* 66.35* SEM 0.760 1.202 0.049 0.077 CD at 5 % 2.116 3.346 0.136 0.215 Means followed by the same letter are not signicantly different at P = 0.05 of Duncans multiple range test Observations were made 6 weeks after culture (n = 30) SEM standard error of mean, CD critical difference * P = 0.05 Table 2 Effect of auxins on rooting of in vitro shoots of R. graveo- lens in half-strength MS medium submitted with auxins Auxin concentrations (mg L -1 ) Rooting rate (%) Mean root number/shoot Mean root length/shoot (cm) IBA 0.25 63.33 4.57 k 6.82 a 0.5 76.66 5.63 j 5.15 b 1.0 86.66 5.73 j 4.95 b 2.0 73.33 8.40 g 4.85 b 3.0 73.33 7.57 h 4.14 c 4.0 73.33 6.30 i 3.97 c NAA 0.25 80.00 24.47 c 2.40 f 0.5 80.00 25.23 b 2.35 f 1.0 83.33 26.73 a 1.76 g 2.0 83.33 17.87 d 2.24 f 3.0 80.00 14.40 e 1.44 h 4.0 83.33 10.70 f 0.81 i IAA 0.25 80.00 4.03 l 3.41 d 0.5 86.66 6.30 i 3.00 e 1.0 83.33 7.67 h 4.52 c 2.0 70.00 8.20 g 4.34 c 3.0 73.33 6.40 i 4.03 4.0 70.00 5.03 j 3.96 c Observations were made after 4 weeks of culture Means followed by the same letter are not signicantly different at P = 0.05 of Duncans multiple range test Means obtained from 30 observations Plant Biotechnol Rep 1 3 Author's personal copy Results and discussion In vitro culture Variation in shooting response was observed due to the auxin concentration in the medium (Table 1). Initiation of multiple shoots in the stem internode explants observed on the both ends within 710 days of inoculation (Fig. 1a). Control treatments involving no plant growth regulators produced no shoots at all. Taking the data on shoot pro- duction from stem internode explants into account, increasing concentrations of auxin caused a dramatic decrease in terms of both the mean number of shoots per explant and the percentage of explants producing shoots. Between the two auxins IAA and NAA tested for efcacy in shoot induction, IAA showed superior to NAA. This is in line with the result obtained in Solanum nigrum in vitro culture (Sridhar and Naidu 2011). The highest number of shoots (55.83 shoots per explant at 96.6 % frequency) was obtained when 1.0 mg L -1 BAP was combined with 0.25 mg L -1 IAA (Table 1). In such combinations, shoots regenerated directly without the intervention of callus for- mation. Multiple shoot buds occurred as mass clusters from both cut ends. After a period of 4 weeks, a cluster of regenerating shoot buds covered the explant. Hence, the explants with shoot clumps were dissected into two pieces and sub-cultured on the same media composition (Fig. 1b, c) for further shoot elongation. The rate of mul- tiplication increased as the number of subcultures increased (every subculture was made at 4-week intervals). This was probably due to adaptation of the explants to in vitro con- ditions. Similar observations have been reported for Pic- rorhiza kurroa (Upadhyay et al. 1989), Plumbago zeylanica (Rout et al. 1999) and Clitoria ternatea (Rout 2004). Many authors report that cytokinin is required in optimal quantity for shoot proliferation in many genotypes, but that inclusion of a low concentration of auxin along with cytokinin increases the rate of shoot multiplication (Shas- any et al. 1998; Rout et al. 2000; Abdullah et al. 2003; Thirunavoukkarasu et al. 2006; Nayak et al. 2007). MS medium fortied with 1.0 mg L -1 BAP in combination with NAA (0.253.0 mg L -1 ) were less effective than IAA comprising media for multiple shoot induction (Table 1). Contrary to the present nding, Faisal et al. (2005) observed better shoot formation in the presence of NAA rather than IAA in nodal segment culture of R. graveolens. This contradiction may be attributed to the explant type and its source. Shoot inducing capacity was better when Fig. 1 Plantlet regeneration from stem internode culture of Ruta graveolens L. a Initiation of multiple shoots in the stem internode explants on the both ends, cultured in MS ? 1.0 mg L -1 BA ? 0.25 mg L -1 IAA (10 days old). b, c Shoot clumps developed on the stem internode were dissected and sub-cultured on the same medium for further shoot elongation (4 weeks old). d A single shoot rooted in 1/2- MS ? 2.0 mg L -1 IBA (4 weeks old). e Acclimatized plants showing normal growth Plant Biotechnol Rep 1 3 Author's personal copy explants were inoculated in BAP 1.0 mg L -1 ? NAA 0.25 mg L -1 . With a further increase in NAA concentra- tion, there was decline in the percentage of cultures with multiple shoots and the mean number of shoots per culture. A higher concentration (3.0 mg L -1 ) favored callus growth immediately after a week of culture. Auxin type and concentration used in combination with BAP profoundly affected shoot growth of the in vitro- proliferated shootlets. Plants gained higher overall shoot length in BAP ? IAA-fortied medium as compared to BAP ? NAA comprising the media. MS medium supple- mented with BAP 1.0 mg L -1 ? IAA 1.0 mg L -1 pro- duced shoots with highest shoot length (3.62 cm); however, a further increase in the IAA concentration had an antagonistic effect on shoot length. Likewise, in the case of BAP ? NAA-fortied media, maximum shoot length (3.20 cm) was obtained at 0.25 mg L -1 concentration which further diminished with increasing NAA concen- tration. Consequently, differential responses of explants to the auxin concentrations may be attributed to endogenous hormone levels of explants. Wernicke et al. (1986) opined that increased auxin concentrations prevented meristematic cell divisions, resulting in decreased reactions. Well-developed shoots with a length of ([3.0 cm) were excised and transferred to half-strength MS medium sup- plemented with different concentrations of auxins such as NAA, IBA and IAA (0.251.0 mg L -1 ). Although the highest number of roots per shoot was observed with 1.0 mg L -1 NAA (26.73 roots per shoot at 83.33 % fre- quency), the roots failed to elongate, attained mean root length of 1.76 cm, remained fused, and exhibited poor growth. The number of healthy roots produced per shoots was higher when the rooting medium contained IBA, especially at the 2.0 mg L -1 concentration which induced lengthy roots (Table 2; Fig. 1d). From the present ndings, auxin IBA proved to be better than IAA and NAA in terms of inducing healthy roots per shoot. This is in close conformity with the results obtained in Solanum nigrum (Sridhar and Naidu 2011) in vitro culture studies. Reports concerning in vitro rooting studies reveal that IBA has been widely used as a root-inducing hormone in difcult-to-root plants both under in vitro and in vivo conditions (Minocha 1987). The stimulatory effect of IBA on root formation has also been reported in many medicinal plants like Murraya koenigii (Bhuyan et al. 1997), Ocimum basilicum (Sahoo et al. 1997), and Clitoria ternatea (Barik et al., 2007). The control treatment involving no auxins did not produce any roots at all. Addition of any of these three root-inducing hormones was essential for rooting of in vitro shoots. Plantlets with well-developed root systems were sepa- rated from the culture tubes, washed, and transferred to polypots containing vermiculite for hardening. After 3 weeks of hardening, the plantlets were transferred to the polybags lled with a mixture of soil, sand, and FYM (2:1:1). The acclimatized plants showed normal growth (Fig. 1e). Finally, the hardened plantlets were transferred to eld conditions. The survival rate was 80 % after 1 month. Field-transferred plants were periodically watered and after 5 months growth period in the eld, a few plants were randomly selected and processed for fur- anocoumarin estimation. Furanocoumarin quantication The results of the analysis of concentrations of the FCs (xanthotoxin and bergapten) in root, shoot and fruit organs of in vitro- and eld-raised plants are shown in Fig. 2. The data revealed that the concentrations of both the FCs was always greater in ex vitro-derived plants, i.e. plants regenerated via in vitro culture, as compared to that of in vivo-grown plants. Xanthotoxin concentration in shoots of ex vitro-raised plants was 671 lg/g of DW which was about 3.4 times greater than that of in vivo plants (197 lg/g of DW). Roots of micropropagated plants contained maximum amounts of xanthotoxin (9,089 lg/g of DW) whereas the same extracts from in vivo plants had very negligible concentrations of the secondary metabolite, amounting to only 185 lg/g of DW. Likewise, fruit organs of ex vitro plants accumulated more xanthotoxin (209 lg/g of DW) than the in vivo fruit parts (87 lg/g of DW). Similar to xanthotoxin, bergapten concentrations were also strikingly higher in ex vitro shoot (10,076 lg/g of DW), root (10,656 lg/g of DW), and fruit organs (7,193 lg/g of DW) as compared to in vivo plants. The different levels of bergapten concentrations detected in in vivo plant parts were: shoots (143 lg/g of DW), roots (3,014 lg/g of DW), and fruits (3,107 lg/g of DW), respectively. Fig. 2 Bergapten (5-MOP) and xanthotoxin (8-MOP) production from different organs (fruit, root and shoot) in ex vitro and in vivo plants of R. graveolens Plant Biotechnol Rep 1 3 Author's personal copy Baskaran and Jayabalan (2008) reported higher psoralen contents in ex vitro plant parts as compared to in vivo plant parts. Similarly, Kang et al. (2004) reported enhanced production of tropane alkaloids in ex vitro-propagated plants than in the in vivo plants of Scopolia parviora. Enhanced accumulation of secondary metabolites in tissue culture cells typically occurs under specic conditions. Mulabagal and Tsay (2004) have established that optimi- zation of culture conditions results in increased accumu- lation of several secondary metabolites in cultured cells as compared to the native plants. Therefore, in the present study, increased furanocoumarin levels may be attributed to the in vitro stress conditions (incubation culture condi- tion). The change in secondary metabolite patterns with the introduction of plant material into in vitro conditions has been described earlier for several other plant species (Luczkiewicz and Glod 2003, 2005; Lucchesini et al. 2009). The hormonal composition of the culture media is known to be one of the main causes of morphological and physiological modication in regenerated plantlets (Van Staden et al. 2006), and in turn it could alter the plant biomass capacity to produce secondary metabolites. In summary, an efcient protocol for micropropagation of an important medicinal plant, R. graveolens, was developed in this study. An improved phytochemical constituent of in vitro-raised plants was observed. The results will make the enhanced production of furanocoumarin much easier. Acknowledgments The authors are grateful to the Director, CSIR- Institute of Minerals and Material Technology, Bhubaneswar for providing facilities. 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