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In Vitro Regeneration of Roots of Phyla Nodiflora and Leptadenia Reticulata, and Comparison of Roots from Cultured and Natural Plants for Secondary Metabolites

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In Vitro Regeneration of Roots of Phyla Nodiflora and Leptadenia Reticulata, and Comparison of Roots from Cultured and Natural Plants for Secondary Metabolites Indian Journal of Experimental Biology Vol. 40, December 2002, pp. 1382-1 386 In vitro regeneration of roots of Phyla nodiflora and Leptadenia reticulata, and comparison of roots from cultured and natural plants for secondary metabolites Trupti Bhatt, Vandana Jain, M G Jayathirtha, G Banerjee & S H Mishra* Pharmacy Department, The M S University of Baroda, Faculty of Technology and Engineering, Kala-bhavan, Vadodara 390 00 1, India Received 27 May 2002; revised 3 October 2002 Adventitious roots, generated using leaf explants of P. nodiflora. and meristem explants of L. reticulata. were cultured on Murashige and Skoog (MS) medium supplemented with nap thy lace tic acid (2 J.lM) and indole butyric acid (3J.lM) respec­ tively. After 30 days, subculturing of roots in liquid MS medium with napthylacetic acid (1.5 J.lM) for P. nodiflora and in­ dole butyric acid (3 J.lM) for L. reticulata afforded considerable increase in root mass. HPTLC profiles and microscopic ex­ amination of transverse sections of ill vitro and naturally grown roots provided information on secondary metabolite accu­ mulation vis-a-vis developmental stages of the root. Phyla nodifloraLinn. (Syn.) Lippia nodifloraMich. is (w/v) bavistin for L. reticulata followed by O.l % a runner plant with scanty roots and reported to pos­ (w/v) mercuric chloride and then they were thor­ sess a number of ethnobotanical uses in diseases like oughly washed with sterile distilled water. The steril­ 1 adenopathy, chronic indolent ulcers, etc . These acti­ ized leaf explants (1 1 cm) of P. nodiflora were 2 x vities are attributed to the presence of flavonoids like placed on static MS (Murashige and Skoog) medium6 lippiflorin-A and B. During preliminary studies on containing different ratios of naphthalene acetic acid this plant, it has been observed that aerial parts versus (NAA): benzyl aminopurine (BAP) ranging from 1: 0 root biomass ratio was 98:2 and flavonoids contents f..lM to 2.5: 5 f..lM. In another set of experiments, the in roots are more than aerial parts. Flavonoids are leaf explants were placed on static MS medium con­ known to exhibit wide range of pharmacological ac­ taining different concentrations of napthylacetic acid tions like antimicrobial, anticancerous and reduce the 3 (NAA) ranging from 1 to 16 f..lM. Based on the prior risk involved in cardiovascular disorders • Therefore, experiments, the explants were also inoculated on B5 the aim of present investigation was to establish an in (Gam borg 1968) and SH (Schenk and Hilderbrandt vitro system to generate roots in bulk, which may 6 1972) media containing napthylacetic acid (2 f.-lM). have more accumulation of bioactive compounds. Agar (0.75%) was used as a solidifying agent and su­ Leptadenia reticulata Wight and Am. has vitaliz­ crose (2% w/v) was used as a carbohydrate source in ing and rejuvenating propert . The roots are used in / 5 all the above-mentioned media. many ayurvedic/ herbal formulations . Due to com­ The sterilized meristeItl explants of L. reticulata mercial use of this plant, especially the roots, the spe­ were inoculated on MS, SH and B5 media with simi­ cies is suffering from over-exploitation and depletion lar hormonal combination and on MS medium con­ of natural resources. It was, therefore, thought of in­ taining different concentrations of indole butyric acid terest to develop an in vitro system to generate the (IBA) ranging from 1 to 50 f..lM. Agar (0.75%) was roots in bulk to meet the demand for this species. used as a solidifying agent and sucrose (3% w/v) was Materials and Methods used as a carbohydrate source. Leaves of Phyla nodiflora and meristem stems of The cultures were incubated at 25D± 2° C in dark Leptadenia reticu/ata were obtained from cultivated environment at pH 5.8. The fresh weight, dry weight plants at Guj arat Agriculture University, Anand. The and dimensions (length) of the roots were measured ex plants were thoroughly washed first with tap water after 35 days. The roots, so obtained, were used for and then surface sterilized by treating them with subculturing in the liquid medium for further sets of 0.15% (w/v) bavistin for P. nodiflora and with 0.25% experiments. In the next set of studies, after thirty days, the de­ *Correspondent author : veloped root tip explants of P. nodiflora (3-4 cm E-mail : [email protected] BHATT et al.: IN VITRO REGENERATION OF ROOTS OF PHYLA NODIFLORA AND LEPTADENIA RETICULA TA 1383 approx. length) were subcultured in MS liquid me­ medium, B5, SH, and MS media in case of L. reticu­ dium, which were supplemented with 1.5 and 1 /lM of lata were separately spotted by auto sampler napthylacetic acid. In case of L. reticulata, the subcul­ (CAMAG) on pre-coated basified (0.3 M, sodium ace­ turing of root tips (2- 2.5 cm approx.) was done into tate) silica plates for TLC. Chromatograms were de­ MS liquid medium, which was supplemented with 1, veloped using ethyl acetate: methanol: water (l0: 2, 3, 4 and 5 /lM of IBA. The pH of the medium was 1.35: 1) as mobile phase. The plates were first ob­ adjusted to 5.8. The cultures were maintained in the served at 254 nm and then at 366 nm under UV light same liquid medium without agar at 100 rpm in dark and subsequently sprayed with natural prod­ 8 at 25° ± 2°C. The growth parameters were measured uct/polyethylenglycol reagent (NP/PEG) . after 60 days by subculturing into fresh liquid me­ Comparative study of cellular architecture of both, 7 dium at the end of every month . naturally grown and propagated roots from cultured In case of both the plants 10 replicates for each explants of P. nodiflora and L. reticulata, was under­ concentration were used in all the above studies. taken by microscopic observation of roots in trans­ Preparation of ethanol extracts and their HPTLC verse sections. profile-One gram powder of naturally grown roots and that of cultured roots of P. nodiflora obtained Results and Discussion from static and liquid MS, static B5 and static SH Optimization of medium fo r in vitro root regenera­ media and roots of L. reticulata obtained from static tion MS medium, were extracted with ethanol (80%), and From leaf of Phyla nodiflora - Initiation of roots all the extracts were evaporated to dryness under in P. nodiflora took place in MS medium supple­ pressure and resuspended in ethanol ( 2ml). mented with NAA (1 /lM) and NAA (1 /lM)+BAP Ethanol extracts (10 IlL) of roots which were natu­ (0.5 /lM), revealing that BAP could be excluded from rally grown and of those grown on MS solid medium the combinations. Maximum root generation without in case of P. nodiflora and of those grown in liquid callus formation was observed when static medium Table I-Comparative account of dimensions of in vitro grown roots of Phyla nodiflora in different media [Values are mean ± SEM of 10 replicates] Dimensions *Static media **MS liquid medium NAA (2 1lM) Conc. of NAA (1lM) B5 SH MS 1.0 1.5 Fresh wt 0.030± 0.004 0.135± 0.03 0.355± 0.03 0.064± 0.0104 0. 160 ± 0.0104 (g) Dry wt 0.012± 0.0004 0.027± 0.002 0.033± 0.003 0.0237± 0.002 0.035± 0.002 (g) Length 0.0±1.5 0.5±1.5 M: 3±0.2 M: 4.5 ±0.32 M: 4±0.5 (cm) L: 1±O.18 L: 1.5±0.29 L: 1.5±0.03 M: Main root; L: Lateral root *Data taken after 35 days; and **after 60 days Table 2-Rf values of components of natural and in vitro root extract of Phyla Ilodijlora observed under UV light (366 nm) Natural root MS SH B5 extract Static medium Liquid medium Liquid medium Static medium Static medium (2 IlM, NAA) (1.5 IlM, NAA) (1.0 IlM, NAA) (2 IlM , NAA) (2 IlM, NAA) 0.3 0.03 0.02 0.03 0.03 0.D3 0. 11 0. 10 0. 10 0.11 0. 11 0.l5 0.14 0.16 0. 16 0.27 0.26 0.28 0.28 0.28 0.35 0.34 0.48 0.49 0.67 0.67 0.66 1384 INDIAN J EXP BIOL, DECEMBER 2002 was supplemented with NAA (2 IlM; Fig. 1a). Callus callus formation (Fig. lb) and was most suitable for formation along with the roots was observed in all further growth of biomass while subculturi ng in MS other combinations. A considerable amount of root liquid medium. The other media like B5 and SH with biomass was observed in MS medium as compared to different hormonal combination, however, did not other media. prove effective for induction of roots. Estimation of fresh and dry weight of roots con­ Comparative HPTLC profile of in vitro (artificial firmed that NAA (2 1lM)was better hormonal concen­ cultured) grown and in vivo (natural) grown roots of tration in static MS medium for initiation of root Phyla nodiflora and Leptadenia reticulata­ mass. The liquid MS medium having AA (1.5 j..lM) Methanol extracts of in vitro and in vivo grown roots was also found more suitable maintenance medium of P. nodiflora were subjected to screening for pres­ (Table 1). The subculturing in the liquid MS medium ence of fIavonoids, using HPTLC. The chroma­ containing NAA 0.5 j..lM) also increased the biomass tograms when observed under UV light at 366 nm production to a considerable amount. before spraying of NP/PEG reagent revealed that fIa­ From meristem Leptadenia reticulata-In case of vonoids accumulated in young roots, which did not L.
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