Employing barcoding markers to authenticate selected endangered medicinal plants traded in Indian markets.

The high demand of medicinal plants and their unrestricted collection have rendered many of these as rare or endangered. The restrictions imposed on their collection and trade are difficult to implement because of the inability to identify them in fragmented form. The rarity of these plants in nature and lack of their cultivation raise doubt about the authenticity of the herbals sold in markets. Therefore, in the present investigation, ITS/ITS2, matK, rbcL and rpoC1 sequences of fourteen species of important medicinal plants, some of which are endangered, were generated and checked for their species-specificity (sequences having maximum similarity only with their own) by BLAST1 and/or BOLD identifications. ITS sequences of 12 species were species-specific. However, ITS2 of only 10 of these 12 species were species-specific. As for the chloroplast loci, rbcL and rpoC1 sequences of all 14 species could be obtained, while matK sequences of only 10 of these could be generated. Of the retrieved sequences, rbcL, rpoC1 and matK sequences of 7, 11 and 7 species, respectively, were species-specific. The sequences of the targeted loci from the herbal samples of these species were difficult to retrieve because of failure in the amplification or sequencing. Nevertheless, based on ITS2 and/or one or more of the chloroplast loci targeted, the botanical identities of 22 herbal market samples were checked by phylogenetic tree, BLAST1 and BOLD identification methods. Of these 22 samples, only one of each of Rauvolfia serpentina and Picrorhiza kurroa were found to be authentic.

Evaluating five different loci (rbcL, rpoB, rpoC1, matK, and ITS) for DNA barcoding of Indian orchids.

Orchidaceae, one of the largest families of angiosperms, is represented in India by 1600 species distributed in diverse habitats. Orchids are in high demand owing to their beautiful flowers and therapeutic properties. Overexploitation and habitat destruction have made many orchid species endangered. In the absence of effective identification methods, illicit trade of orchids continues unabated. Considering DNA barcoding as a potential identification tool, species discrimination capability of five loci, ITS, matK, rbcL, rpoB, and rpoC1, was tested in 393 accessions of 94 Indian orchid species belonging to 47 genera, including one listed in Appendix I of CITES and 26 medicinal species. ITS provided the highest species discrimination rate of 94.9%. While, among the chloroplast loci, matK provided the highest species discrimination rate of 85.7%. None of the tested loci individually discriminated 100% of the species. Therefore, multi-locus combinations of up to five loci were tested for their species resolution capability. Among two-locus combinations, the maximum species resolution (86.7%) was provided by ITS+matK. ITS and matK sequences of the medicinal orchids were species specific, thus providing unique molecular identification tags for their identification and detection. These observations emphasize the need for the inclusion of ITS in the core barcode for plants, whenever required and available.

Protocols for In Vitro Mass Multiplication and Analysis of Medicinally Important Phenolics of a Salep Orchid, Satyrium nepalense D.Don ("Salam Mishri").

Satyrium nepalense is a rare and threatened medicinal orchid, populations of which in its native habitats are dwindling because of indiscriminate collections and habitat destruction, thus necessitating the development of methods for its in situ and ex situ conservation. Because of non-endospermous nature of the seeds and the immature embryos at seed dispersal stage, orchids cannot be seed-propagated as other plants. Micropropagation, using plant tissue culture techniques, offers an effective method for the multiplication of orchids. In this chapter, a five-step efficient reproducible protocol for large-scale in vitro multiplication of Satyrium nepalense is described. The first step involves asymbiotic germination of seeds isolated from immature green pods and cultured on Mitra's medium (M) gelled with 0.8 % agar and supplemented with 2 % sucrose and 1 % peptone (hereafter referred to as basal medium, BM). On this medium, seeds start germinating after a week of culture. Protocorms developed from the seeds are sub-cultured on BM fortified with 4 µM kinetin (Kn) after 8 weeks, for shoot differentiation and multiplication. The shoots developed on Kn-supplemented medium are transferred to BM alone for their elongation for the same period. The elongated shoots are transferred to the rooting medium, comprising BM supplemented with 0.5 or 1.0 µM indole-3-butyric acid, for further 8 weeks. The regenerated plantlets are transferred to a potting mix of sand and vermiculite (1:1) for acclimatization. The tubers and leaves excised from both in vitro-developed plants and those from their native habitats are analyzed and compared for the contents and concentration of medicinally important phenolics using high-performance liquid chromatography (HPLC), details of which are provided in this chapter.

Evaluation of guar gum derivatives as gelling agents for microbial culture media.

Guar gum, a galactomannan, has been reported to be an inexpensive substitute of agar for microbial culture media. However, its use is restricted probably because of (1) its highly viscous nature even at high temperatures, making dispensing of the media to Petri plates difficult and (2) lesser clarity of the guar gum gelled media than agar media due to impurities present in guar gum. To overcome these problems, three guar gum derivatives, carboxymethyl guar, carboxymethyl hydroxypropyl guar and hydroxypropyl guar, were tested as gelling agents for microbial growth and differentiation. These were also evaluated for their suitability for other routine microbiological methods, such as, enumeration, use of selective and differential media, and antibiotic sensitivity test. For evaluation purpose, growth and differentiation of eight fungi and eight bacteria grown on the media gelled with agar (1.5%), guar gum (4%) or one of the guar gum derivatives (4%), were compared. All fungi and bacteria exhibited normal growth and differentiation on all these media. Generally, growth of most of the fungi was better on guar gum derivatives gelled medium than on agar medium. The enumeration carried out for Serratia sp. and Pseudomonas aeruginosa by serial dilution and pour plate method yielded similar counts in all the treatments. Likewise, the selective succinate medium, specific for P. aeruginosa, did not allow growth of co-inoculated Bacillus sp. even if gelled with guar gum derivatives. The differential medium, Congo red mannitol agar could not differentiate between Agrobacterium tumefaciens and Rhizobium meliloti on color basis, if gelled with guar gum or any of its derivatives However, for antibiotic sensitivity tests for both Gram-positive and -negative bacteria, guar gum and its derivatives were as effective as agar.

The loci recommended as universal barcodes for plants on the basis of floristic studies may not work with congeneric species as exemplified by DNA barcoding of Dendrobium species.

BACKGROUND: Based on the testing of several loci, predominantly against floristic backgrounds, individual or different combinations of loci have been suggested as possible universal DNA barcodes for plants. The present investigation was undertaken to check the applicability of the recommended locus/loci for congeneric species with Dendrobium species as an illustrative example. RESULTS: Six loci, matK, rbcL, rpoB, rpoC1, trnH-psbA spacer from the chloroplast genome and ITS, from the nuclear genome, were compared for their amplification, sequencing and species discrimination success rates among multiple accessions of 36 Dendrobium species. The trnH-psbA spacer could not be considered for analysis as good quality sequences were not obtained with its forward primer. Among the tested loci, ITS, recommended by some as a possible barcode for plants, provided 100% species identification. Another locus, matK, also recommended as a universal barcode for plants, resolved 80.56% species. ITS remained the best even when sequences of investigated loci of additional Dendrobium species available on the NCBI GenBank (93, 33, 20, 18 and 17 of ITS, matK, rbcL, rpoB and rpoC1, respectively) were also considered for calculating the percent species resolution capabilities. The species discrimination of various combinations of the loci was also compared based on the 36 investigated species and additional 16 for which sequences of all the five loci were available on GenBank. Two-locus combination of matK+rbcL recommended by the Plant Working Group of Consortium for Barcoding of Life (CBOL) could discriminate 86.11% of 36 species. The species discriminating ability of this barcode was reduced to 80.77% when additional sequences available on NCBI were included in the analysis. Among the recommended combinations, the barcode based on three loci - matK, rpoB and rpoC1- resolved maximum number of species. CONCLUSIONS: Any recommended barcode based on the loci tested so far, is not likely to provide 100% species identification across the plant kingdom and thus is not likely to act as a universal barcode. It appears that barcodes, if based on single or limited locus(i), would be taxa specific as is exemplified by the success of ITS among Dendrobium species, though it may not be suitable for other plants because of the problems that are discussed.

DNA barcoding of endangered Indian Paphiopedilum species.

The indiscriminate collections of Paphiopedilum species from the wild for their exotic ornamental flowers have rendered these plants endangered. Although the trade of these endangered species from the wild is strictly forbidden, it continues unabated in one or other forms that elude the current identification methods. DNA barcoding that offers identification of a species even if only a small fragment of the organism at any stage of development is available could be of great utility in scrutinizing the illegal trade of both endangered plant and animal species. Therefore, this study was undertaken to develop DNA barcodes of Indian species of Paphiopedilum along with their three natural hybrids using loci from both the chloroplast and nuclear genomes. The five loci tested for their potential as effective barcodes were RNA polymerase-ß subunit (rpoB), RNA polymerase-ß' subunit (rpoC1), Rubisco large subunit (rbcL) and maturase K (matK) from the chloroplast genome and nuclear ribosomal internal transcribed spacer (nrITS) from the nuclear genome. The intra- and inter-specific divergence values and species discrimination rates were calculated by Kimura 2 parameter (K2P) method using mega 4.0. The matK with 0.9% average inter-specific divergence value yielded 100% species resolution, thus could distinguish all the eight species of Paphiopedilum unequivocally. The species identification capability of these sequences was further confirmed as each of the matK sequences was found to be unique for the species when a blast analysis of these sequences was carried out on NCBI. nrITS, although had 4.4% average inter-specific divergence value, afforded only 50% species resolution. DNA barcodes of the three hybrids also reflected their parentage.

Transformation of tomato with a bacterial codA gene enhances tolerance to salt and water stresses.

Genetically engineered tomato (Lycopersicon esculentum) with the ability to synthesize glycinebetaine was generated by introducing the codA gene encoding choline oxidase from Arthrobacter globiformis. Integration of the codA gene in transgenic tomato plants was verified by PCR analysis and DNA blot hybridization. Transgenic expression of gene was verified by RT-PCR analysis and RNA blot hybridization. The codA-transgenic plants showed higher tolerance to salt stress during seed germination, and subsequent growth of young seedlings than wild-type plants. The codA transgene enhanced the salt tolerance of whole plants and leaves. Mature leaves of codA-transgenic plants revealed higher levels of relative water content, chlorophyll content, and proline content than those of wild-type plants under salt and water stresses. Results from the current study suggest that the expression of the codA gene in transgenic tomato plants induces the synthesis of glycinebetaine and improves the tolerance of plants to salt and water stresses.

Large-scale in vitro multiplication of Crataeva nurvala.

Conservation and propagation of species using biotechnologic tools-such as plant tissue culture-are relevant when natural propagation is hampered for various reasons. In vitro techniques allow mass multiplication and propagation under pathogen-free conditions but also override dependence on season for availability of plant material. Moreover, in vitro genetic manipulation of a species, invariably, requires a prestandardized tissue culture protocol for its multiplication.To fulfill these requirements, efficient, cyclic, two-step protocols for micropropagation of the medicinal tree-Crataeva nurvala-employing juvenile explants and those from mature trees, were developed. Both protocols can be employed at commercial scale. The seedling-derived explants (e.g., cotyledonary nodes, epicotyl nodes, hypocotyl segments, first pair of leaves, cotyledons, and root segments) developed shoots on Murashige and Skoog's (MS) or the same supplemented with different concentrations of 6-benzylaminopurine (BAP). The epicotyl and cotyledonary nodal explants developed shoots on MS basal medium. Other explants exhibited caulogenesis on BAP (0-2.0 mg/L) adjuvated media. The explants from in vitro regenerated shoots too exhibited a similar caulogenic capability. Nodal explants from a 30-yr-old-tree, when cultured on MS medium supplemented with 0.5 mg/L BAP, produced multiple shoots which elongated satisfactorily on the same medium. Similar to the microshoots developed from the seedling derived explants, nodal and leaf explants from the microshoots regenerated from the mature explants too developed shoots, thus making the process recurrent. Due to the recurrent nature of the protocol, over 5400 shoots may be produced from a single nodal explant of an adult tree over a period of six months. The addition of casein hydrolysate significantly increased the average number of shoots per explant. The regenerated shoots could be rooted on the medium supplemented with 0.02 mg/L or 0.1 mg/L NAA (alpha-naphthalene acetic acid). Regenerated plantlets were acclimatized and successfully transplanted to soil.

An efficient, in vitro cyclic production of shoots from adult trees of Crataeva nurvala Buch. Ham.

An efficient, cyclic, two-step protocol for micropropagation of medicinal tree, Crataeva nurvala has been successfully developed, which can be employed at a commercial scale. Nodal explants from 30-year-old tree when cultured on MS medium supplemented with 2.22 microM BAP produced multiple shoots, which elongated satisfactorily on the same medium. Nodal and leaf explants from in vitro regenerated microshoots too developed shoots, thus making the process recurrent. In 6-month duration, owing to the recurring nature of the protocol, over 5400 shoots could be produced from a single nodal explant from the adult tree. Addition of casein hydrolysate significantly increased the average number of shoots per explant. Maximum number of shoots regenerated on medium supplemented with 100 mg l(-1) casein hydrolysate. Shoots could be rooted on 1/2 MS supplemented with 0.11 and 0.54 microM NAA. Regenerated plantlets were acclimatized and successfully transplanted to soil.

Xanthan gum: an economical partial substitute for agar in microbial culture media.

Xanthan gum, microbial desiccation-resistant polysaccharide prepared commercially by aerobic submerged fermentation from Xanthomonas campestris, has been successfully used alone and in combination with agar for microbial culture media. As illustrative examples, eight bacteria and eight fungi were grown on media solidified with either agar (A, 1.5%), xanthan gum (X, 1%), or combinations of both (0.9% X + 0.1% A, 0.8% X + 0.2% A, 0.7% X + 0.3% A, 0.6% X + 0.4% A). All fungi and bacteria exhibited normal growth and differentiation in all these treatments. Rather, growth of most of the fungi was better on xanthan (alone) and xanthan + agar media than agar medium. As the media gelled with xanthan gum alone flow, it was not possible to incubate Petri plates in inverted position. Moreover, because of the softness, streaking of bacteria was difficult on such media. However, these problems could be overcome by partially replacing xanthan gum with 0.3% agar. Bacterial enumeration studies carried out for Serratia sp. and Pseudomonas sp. by serial dilution and pour-plate method on agar (1.5%), 0.7%/0.6% X + 0.3%/0.4% A yielded similar counts. Selective media, succinate medium for Pseudomonas sp., and MacConkey broth medium for Escherichia coli gelled with 0.7%/0.6% X + 0.3%/0.4% A did not support growth of other bacteria when inoculated along with the above-mentioned bacteria. Likewise, differential medium, CRMA (Congo red mannitol agar) gelled with xanthan-agar combination could differentiate between Agrobacterium tumefaciens and Rhizobium sp.

Regulatory role of iron and EDTA in the shoot development from the epicotyl explants of Syzygium cuminii.

Iron has proved to be an integral component of the culture medium supporting the caulogenic response of the epicotyl segments of S. cuminii. In the absence of both the iron and EDTA even the shoot buds failed to develop, while in the presence of either of these, though the shoot buds developed, their elongation was adversely affected. Among the three iron sources tested, ferrous sulphate proved to be the best, as the ferric chloride was not as effective as the former when used either alone or along with EDTA. Ferric citrate, on the other hand, when provided alone, elicited better response than that induced by ferrous sulphate alone. However, in combination with EDTA, the response declined significantly. The estimation of endogenous levels of iron in the explants further supported these results. The quantum of iron absorption was at a maximum during the first week of the culture and the explants, once deprived of iron during the first week, failed to catch up to the level of iron accumulated in the explants maintained continuously on complete medium, even after transfer to the complete medium. Likewise, the level of copper ions did not come up to comparable levels even if the explants were transferred to the complete medium after initial deprivation.

Transient existence of life without a cell membrane: a novel strategy of siphonous seaweed for survival and propagation.

Siphonous seaweeds, which constitute a vital component of coral reefs, are structurally simple, single-celled coenocytic macroscopic green algae. Kim et al.1 have recently shown the extraordinary wound-repair and propagation mechanism of one such siphonous green alga--Bryopsis plumosa. Nucleocytoplasmic aggregates, which are released after injury to this plant, are membraneless structures that can survive in seawater for 10-20 minutes, before they are surrounded by a gelatinous envelope. Subsequently, a cell membrane and cell wall are synthesized around each of these aggregates and the resulting individual cells, so formed, develop into new plants. This report represents a significant advance in our understanding of wound response and, more significantly, is probably the first example of transient survival of life without a cell membrane!