Biotechnological advances in tea (Camellia sinensis [L.] O. Kuntze): a review.

KEY MESSAGE: This article presents a comprehensive review on the success and limitations of biotechnological approaches aimed at genetic improvement of tea with a purpose to explore possibilities to address challenging areas. Tea is a woody perennial tree with a life span of more than 100 years. Conventional breeding of tea is slow and limited primarily to selection which leads to narrowing down of its genetic base. Harnessing the benefits of wild relatives has been negligible due to low cross-compatibility, genetic drag and undesirable alleles for low yield. Additionally, being a recalcitrant species, in vitro propagation of tea is constrained too. Nevertheless, maneuvering with tissue/cell culture techniques, a considerable success has been achieved in the area of micropropagation, somatic embryogenesis as well as genetic transformation. Besides, use of molecular markers, "expressomics" (transcriptomics, proteomics, metabolomics), map-based cloning towards construction of physical maps, generation of expressed sequenced tags (ESTs) have facilitated the identification of QTLs and discovery of genes associated with abiotic or biotic stress tolerance and agronomic traits. Furthermore, the complete genome (or at least gene space) sequence of tea is expected to be accessible in the near future which will strengthen combinational approaches for improvement of tea. This review presents a comprehensive account of the success and limitations of the biotechnological tools and techniques hitherto applied to tea and its wild relatives. Expectedly, this will form a basis for making further advances aimed at genetic improvement of tea in particular and of economically important woody perennials in general.

High-performance liquid chromatographic quantification of plumbagin from transformed rhizoclones of Plumbago zeylanica L.: inter-clonal variation in biomass growth and plumbagin production.

An optimized protocol for induction and establishment of Agrobacterium rhizogenes-mediated hairy root cultures of Plumbago zeylanica L. was developed through selection of suitable explant type and the bacterial strain. The infection of internodal explants from an in vivo plant and leaves of in vitro origin with the A4 strain resulted in the emergence of hairy roots at a transformation frequency of 86.33 and 42.33 %, respectively. Independent transformed root somaclones (rhizoclones) capable of sustained growth were maintained under a low illumination in auxin-free agar-solidified Murashige and Skoog (MS) medium through subcultures at periodic intervals. The presence of pRi T L-DNA rolB or rolC genes and pRi T R-DNA mas2 gene in the transformed rhizoclone genome was ascertained by PCR amplification. Concentrations and type of carbon source, auxin and media strength were optimized for root biomass growth. Five independent rhizoclones each from A4- and LBA9402-transformed root lines were studied for their plumbagin accumulation at different growth phases, using HPLC analysis. The potential for plumbagin biosynthesis was expressed in all the tested rhizoclones, although distinct inter-clonal variations were noted. It was evident that maturation of hairy roots was more important for plumbagin accumulation; slow-growing and early-maturing rhizoclones accumulated more plumbagin compared to fast-growing and late-maturing rhizoclones. A4-induced rhizoclone HRA2B5 was identified as the most superior clone with a higher plumbagin yield potential in comparison with other tested hairy root clones, in vitro-grown non-transformed roots and in vivo roots of naturally occurring P. zeylanica.

Production of triterpenoid anti-cancer compound taraxerol in Agrobacterium-transformed root cultures of butterfly pea (Clitoria ternatea L.).

Independent transformed root somaclones (rhizoclones) of butterfly pea (Clitoria ternatea L.) were established using explant co-cultivation with Agrobacterium rhizogenes. Rhizoclones capable of sustained growth were maintained under low illumination in auxin-free agar-solidified MS medium through subcultures at periodic intervals. Integration of T(L)-DNA rolB gene in the transformed rhizoclone genome was verified by Southern blot hybridization, and the transcript expression of T(R)-DNA ags and man2 genes was ascertained by reverse transcription polymerase chain reaction analysis. The major compound isolated and purified from the transformed root extracts was identified as the pentacyclic triterpenoid compound taraxerol using IR, (1)H-NMR, and (13)C-NMR spectroscopy. The taraxerol yield in cultured hairy roots, as quantified by HPTLC analysis, was up to 4-fold on dry weight basis compared to that in natural roots. Scanning of bands from cultured transformed roots and natural roots gave super-imposable spectra with standard taraxerol, suggesting a remarkable homology in composition. To date, this is the first report claiming production of the cancer therapeutic phytochemical taraxerol in genetically transformed root cultures as a viable alternative to in vivo roots of naturally occurring plant species.

Tissue culture-mediated biotechnological intervention in pomegranate: a review.

The past 30 years have witnessed a series of systematic biotechnological advances made in pomegranate. These encompass optimization and establishment of in vitro culture techniques including micropropagation, somatic embryogenesis, synthetic seed production, plant regeneration via callus-mediated shoot organogenesis, adventitious shoot regeneration, anther culture, tetraploid induction and genetic transformation. This review attempts to provide a comprehensive account on the tissue culture-mediated biotechnological interventions made in pomegranate aimed at complementing conventional programmes for improvement of this nutraceutically important fruit crop.

Analysis of genetic diversity among selected grasspea (Lathyrus sativus L.) genotypes using RAPD markers.

Randomly amplified polymorphic DNA (RAPD) technique was applied to assess the genetic variability among five selected genotypes of grasspea. Out of 30 random decamer primers tested for the present investigation 20 showed reproducible DNA amplification. A total of 257 loci were amplified of which 159 were polymorphic including 57 genotype-specific unique bands. Amplicons had molecular weights ranging from 3.0 kb to 0.1 kb. Majority amplicons were shared by most of the genotypes which indicated a very narrow genetic gap between them. The dendrogram constructed on the basis of RAPD data showed two clusters. The local genotype collected from Nayagarh was grouped along with IC-120451 and IC-120453, sharing a common node at an 82% similarity level. The other genotypes, IC-120478 and IC-120487, were located in the second clade having a common node at 84% similarity level. The investigation showed that though all the genotypes of grasspea were of apparently similar morphology there exists polymorphism at the molecular level, which can be exploited in breeding programmes aimed at crop improvement.

Silver nitrate and aminoethoxyvinylglycine promote in vitro adventitious shoot regeneration of pomegranate (Punica granatum L.).

A protocol is presented for direct adventitous shoot organogenesis and complete plant regeneration from seedling-derived explants of pomegranate (Punica granatum L.), a tropical fruit tree. Murashige and Skoog (1962) (MS) medium enriched with 8.9 mumol/L benzyladenine (BA), 5.4 mumol/L naphthaleneacetic acid (NAA) and 10% coconut water (CW) induced adventitious shoot bud differentiation in axenic seedling-derived cotyledons as well as hypocotyl segments. The cotyledons were more responsive than the hypocotyls. Addition of ethylene inhibitors such as AgNO3 (10-40 mumol/L) and aminoethoxyvinylglycine (AVG) (5-15 mumol/L) to the medium markedly enhanced regeneration frequency as well as number of shoots obtained per explant. The promotive effect of AVG and AgNO3 on shoot organogenesis was observed only in cotyledon explants. The regeneration medium containing AgNO3 (20 mumol/L) or AVG (10 mumol/L) induced adventitious shoot buds from 57% or 53% of the cotyledon explants respectively. These shoot buds developed into shoots upon transfer to a regeneration medium without AgNO3 and AVG. The promotive effect of AVG on shoot regeneration was reversed by exogenous application of 20 mumol/L 2-chloroethylphosphonic acid (CEPA), an ethylene releasing compound. On the other hand, shoot regeneration stimulated by AgNO3 was relatively less affected by CEPA. Regenerated shoots were rooted in half-strength MS medium (1/2 MS) containing 0.54 mumol/L NAA. The well rooted plantlets were acclimatized and eventually established in soil.