Histochemical evaluation of catechins in PEG stressed transgenic tea plants using catechin-specific-diazotized sulfanilamide reagent.

We investigated the applicability of catechin-specific-reagent (CSR) for histochemical evaluation of catechins. The diazotized arylamine moiety in CSR reacts specifically with the A-ring of catechins to yield a golden yellow complex. This makes it highly specific for spectrophotometric quantification of catechins. Therefore, microtome cut sections of untransformed and osmotin-expressing transgenic leaves and stem of tea were stained with CSR. We found catechins in the form of golden yellow globules. The catechin globules increased in the structurally intact and highly turgid cells of osmotin expressing transgenic tea plants after stress treatment with 20% PEG; by contrast, the cells in non-transgenic plants accumulated fewer catechin globules. Spectrophotometric quantification of catechins also confirmed higher levels in transgenics compared to untransformed plants. We found elevated accumulation of catechins in stress tolerant cells of tea leaves.

AFLP-based genetic diversity assessment of commercially important tea germplasm in India.

India has a large repository of important tea accessions and, therefore, plays a major role in improving production and quality of tea across the world. Using seven AFLP primer combinations, we analyzed 123 commercially important tea accessions representing major populations in India. The overall genetic similarity recorded was 51%. No significant differences were recorded in average genetic similarity among tea populations cultivated in various geographic regions (northwest 0.60, northeast and south both 0.59). UPGMA cluster analysis grouped the tea accessions according to geographic locations, with a bias toward China or Assam/Cambod types. Cluster analysis results were congruent with principal component analysis. Further, analysis of molecular variance detected a high level of genetic variation (85%) within and limited genetic variation (15%) among the populations, suggesting their origin from a similar genetic pool.

p-Coumarate:CoA ligase as a key gene in the yield of catechins in tea [Camellia sinensis (L.) O. Kuntze].

Tea is an important crop known for its beverage and antioxidant polyphenols -- catechins and its derivatives. Catechins are synthesized through flavonoid (FL) pathway and stored in the vacuole. A metabolic flux for the operation of FL pathway is maintained through the supply of 4-coumaroyl-CoA of phenylpropanoid pathway. 4-Coumaroyl-CoA is synthesized through the catalytic activity of p-coumarate:CoA ligase (4CL) using 4-coumaric acid and acetyl-CoA as the substrates. The present manuscript reports the full-length cDNA cloning of 4CL from tea (Cs4CL accession number DQ194356) and its association with catechin yield. Cs4CL comprised of 2,165 bp with an open reading frame (ORF) of 1,764 nt, starting from 118 to 1,882 encoding 588 amino acids. Altering catechin content through a variety of environmental conditions such as drought stress (DS), abscisic acid (ABA) and gibberellic acid (GA(3)) treatments, and wounding established a strong positive correlation coefficient between catechins content and the expression of Cs4Cl. In addition, tea clones with high levels of catechins had higher expression of Cs4Cl whereas tea clones with lower catechins exhibited lower expression of this gene. Exposure of tea shoots to 50-100 microM catechins led to down-regulation of the expression of Cs4CL suggesting product-mediated feedback regulation and an important role for the phenylpropanoid pathway in determining catechin yield in tea.

Processing of apple pomace for bioactive molecules.

The growth of horticulture industries worldwide has generated huge quantities of fruit wastes (25%-40% of the total fruits processed). These residues are generally a good source of carbohydrates, especially cell wall polysaccharides and other functionally important bioactive molecules such as proteins, vitamins, minerals and natural antioxidants. "Apple pomace" is a left-over solid biomass with a high moisture content, obtained as a by-product during the processing of apple fruits for juice, cider or wine preparation. Owing to the high carbohydrate content, apple pomace is used as a substrate in a number of microbial processes for the production of organic acids, enzymes, single cell protein, ethanol, low alcoholic drinks and pigments. Recent research trends reveal that there is an increase in the utilization of apple pomace as a food processing residue for the extraction of value added products such as dietary fibre, protein, natural antioxidants, biopolymers, pigments and compounds with unique properties. However, the central dogma is still the stability, safety and economic feasibility of the process(s)/product(s) developed. This review is mainly focused on assessing recent research developments in extraction, isolation and characterization of bioactive molecules from apple pomace, along with their commercial utilization, in food fortification.

High level of genetic diversity among the selected accessions of tea (Camellia sinensis) from abandoned tea gardens in western Himalaya.

To revive cultivation of the tea unique to the western Himalayan region, it is important to evaluate the seed-derived bushes available in the area's abandoned gardens. This study used quantitative leaf characters, catechin content, and AFLP markers to assess these China cultivar type bushes. Compared with other China cultivar germplasm, these accessions showed a higher level of diversity among themselves. Among the quantitative morphological characters, leaf length is important in distinguishing the accessions studied, with a high loading value in the principal component analysis. The catechins and AFLP markers displayed the genetic makeup of the accessions. Other than total catechins, the trihydroxylated catechins showed a high loading value in differentiating the accessions. The genetic control of the ratio of dihydroxylated and trihydroxylated catechins is found to be based on a correlation with AFLP markers. The genetic similarity between Kangra Asha and Kangra Jat suggests that Kangra Jat must be descended from Kangra Asha. Kangra Jat is well adapted to local environmental conditions, as is evident from its high catechin content.

Rose protoplast isolation and culture and heterokaryon selection by immobilization in extra thin alginate film.

Somatic hybridization has been identified as one method for the genetic improvement of roses. The success of somatic hybridization programmes relies to a great extent upon efficient protoplast isolation and culture and selection of heterokaryons. This paper reports the isolation of rose cell suspension protoplasts by direct sucrose flotation and demonstrates their culture using extra thin alginate film. A comparative assessment of the efficiency of conventional culture techniques versus those with extra thin alginate film or thin alginate layer is also presented. A very high plating efficiency (80%) was obtained using thin alginate layer or extra thin alginate film techniques with improved media formulations. Protoplasts of Rosa damascena and R. bourboniana were fused by using polyethylene glycol as fusogen and later immobilized in the thin layer of alginate. The fused protoplasts were tracked on the basis of differential fluorescent staining, and the hybridity of heterokaryons following their development to callus was confirmed by molecular characterization. This novel selection strategy has general applicability and is faster and simpler to perform during somatic hybridization experiments.

Biology and chemistry of Ginkgo biloba.

Ginkgo biloba has been existing on earth since 200 million years and is considered as a "living fossil". It is among the most sold medicinal plants in the world. A number of secondary metabolites representing terpenoids, polyphenols, allyl phenols, organic acids, carbohydrates, fatty acids and lipids, inorganic salts and amino acids have been isolated from the plant. However, the main bioactive constituents are terpene trilactones and flavonoid glycosides which are considered responsible for the pharmacological activities of its standardized leaf extract. Scattered information is available on the extraction and analysis of these pharmacologically important constituents which have been compiled in the present review.

Validated high-performance thin-layer chromatography method for steviol glycosides in Stevia rebaudiana.

A high-performance thin-layer chromatographic (HPTLC) method was developed and validated as per ICH (International Conferences on Harmonization) guidelines for simultaneous quantification of three steviol glycosides, i.e. steviolbioside, stevioside and rebaudioside-A in Stevia rebaudiana leaves. For achieving good separation, mobile phase of ethyl acetate-ethanol-water (80:20:12, v/v/v) on pre-coated silica gel 60 F254 HPTLC plates were used. The densitometric quantification of steviol glycosides was carried out at lambda=510 nm in reflection-absorption mode after spraying with acetic anhydride:sulphuric acid:ethanol reagent. The calibration curves were linear in the range of 160-960 ng/spot for steviolbioside, 1-6 microg/spot for stevioside and 0.5-3 microg/spot for rebaudioside-A with good correlation coefficients (0.998-0.999). The method was found to be reproducible for quantitative analysis of steviol glycosides in S. rebaudiana leaves collected from ten different locations and will serve as a quality control indicator to monitor the commercial production of stevioside and its allied molecules during different stages of its processing.

Evaluation of rice and sugarcane SSR markers for phylogenetic and genetic diversity analyses in bamboo.

Simple sequence repeat (SSR) markers are valuable tools for many purposes such as phylogenetic, fingerprinting, and molecular breeding studies. However, only a few SSR markers are known and available in bamboo species of the tropics (Bambusa spp.). Considering that grass genomes have co-evolved and share large-scale synteny, theoretically it should be possible to use the genome sequence based SSR markers of field crops such as rice (Oryza sativa) and sugarcane (Saccharum spp.) for genome analysis in bamboo. To test this, 98 mapped SSR primers representing 12 linkage groups of rice and 20 EST-derived sugarcane SSR primers were evaluated for transferability to 23 bamboo species. Of the tested markers, 44 (44.9%) rice and 15 (75%) sugarcane SSR primers showed repeatable amplification in at least one species of bamboo and thus were successfully utilized for phylogenetic and genetic diversity analyses. Transferred SSR primers revealed complex amplification patterns in bamboo, with an average of 9.62 fragments per primer, indicating a high level of polyploidy and genetic variability in bamboo. Forty-two of these primers (34 rice and 8 sugarcane SSR primers) detected an average of 2.12 unique fragments per primer and thus could be exploited for species identification. Six bamboo SSR primers exhibited cross transferability, to varying degrees, to different bamboo species. The genetic similarity coefficient indicated a high level of divergence at the species level (73%). However, a relatively low level of diversity was observed within species (25% in 20 accessions of Dendrocalamus hamiltonii). Further, cluster analysis revealed that the major grouping was in accordance with the taxonomical classification of bamboo. Thus, the rice and sugarcane SSRs can be utilized for phylogenetic and genetic diversity studies in bamboo.

Radioprotective and antioxidant activity of fractionated extracts of berries of Hippophae rhamnoides.

Plants are an abundant source of medicinal compounds, some of which are useful in combating free radical-mediated oxidative stress. In the present study, initially two fractions designated REC-1001 (flavonoid-rich fraction) and REC-1002 (flavonoid-poor fraction) of Hippophae rhamnoides were screened on the basis of their reducing power in the aqueous phase. REC-1001 was selected for further study, since it exhibited 27.38 times higher antioxidant activity than REC-1002. REC-1001 also showed significant (P < .05) membrane protection potential at 50 microg/mL, which was attributed to its ability to scavenge peroxyl radicals (64.82 +/- 1.25% scavenging within 1,440 min). A significant (P < .05) difference of 67.02% in free radical scavenging activity at 1,000 ng/mL between REC-1001 and vitamin E demonstrated the extract fraction's worth in radiation protection. Such activities were attributed to the presence of quercetin, isorhamnetin, and kaempferol in this fraction. Further, REC-1001 was found to be nontoxic up to 200 mg/kg of body weight. This research suggests that the REC-1001 fraction of H. rhamnoides extract is a safe and effective antioxidant nutraceutical product.

Regulation of growth of Lilium plantlets in liquid medium by application of paclobutrazol or ancymidol, for its amenability in a bioreactor system: growth parameters.

Effect of growth retardants (paclobutrazol or ancymidol) was studied in Lilium plantlets growing in liquid culture. A significant increase in leaf chlorophyll, epicuticular wax, plant dry weight and bulb starch contents were found in plantlets treated with growth retardants. A similar increase in the number of leaves, roots and bulbs was also noted. However, total leaf area and the fresh weight increased only marginally. These features resulted in robust plantlets that showed significantly improved ex vitro survival. Based on these features, a comprehensive index (CI) was calculated as a measure of quality of the plantlets, and it correlated well with their ex vitro survival. Treatment of plantlets with 3.4 microM paclobutrazol was found to be the best and its carry over effects were also minimal.

Taxanes from in vitro cultures of the Himalayan yew Taxus wallichiana.

Culture conditions have been standardized for initiation of callus cultures of Himalayan yew (Taxus wallichiana) using young stem and needle explants from mature trees. Cultures were established on a modified Murashige and Skoog's medium supplemented with various levels of auxins (2.4-D, NAA) and cytokinin (kinetin). A medium containing 0.25 mg/l kinetin and 5.0 mg/l 2.4-D was optimal for stem callus growth whereas the presence of 0.25 mg/l kinetin along with 3.0 mg/l NAA in the medium supported optimal needle callus growth. Growth of stem callus was faster than needle callus growth. Supplementation of ascorbic acid (30 mg/l) amongst various anti-phenolic agents tested significantly reduced browning of initiated callus. Two taxanes (2-deacetoxytaxinine 1 and 2'-deacetoxyaustrospicatine) known to occur in stem bark, have also been isolated from undifferentiated tissue of T. wallichiana in equal or higher yields, for the first time.

Fertile somatic hybrid between sexually incompatible Hyoscyamus muticus and Hyoscyamus albus.

Somatic hybrid plants have been regenerated from fused protoplasts of a chlorophyll deficient mutant of H. muticus (2n=28) with wild type protoplasts of H. albus (2n=68). The inability of protoplasts of H. albus to regenerate was utilized in complementation with achlorophyllous, but regenerating, protoplasts of H. muticus for the selection of green somatic hybrid colonies and plants. The somatic hybrid plants showed intermediate morphological characters, and possessed 82-120 chromosomes, with a modal number of 96 which is also the amphidiploid complement of the two species. The isozyme patterns indicated the presence and expression of genes from both parents. The hybrid plants produced 33-78% viable pollen and set viable seeds upon selfing and backcrossing in a directional manner.

Propagation of Indian Rhubarh (Rheum emodi Wall.) using shoot-tip and leaf explant culture.

Shoot-tip explants of Rheum emodi Wall. (Polygonaceae) gave rise to multiple shoots when cultured on a Murashige and Skoog (1962) medium (MS) with 2.0 mg/l 6-benzylaminopurine (BAP) and 1.0 mg/l indole-3-butyric acid (IBA). Also, shoot buds developed from leaf explants using MS medium with 2.0 mg/l BAP and 0.25 to 1.0 mg/l indole-3-acetic acid (IAA) or IBA. Roots were induced when the resulting shoots were placed on MS medium with 1.0 mg/l IBA. Both regeneration procedures gave rise to healthy plantlets that were established in soil under glasshouse conditions at 80% frequency after hardening phase of two weeks. Regenerated plants showed a constant chromosome number of 2n=2x=22, same as the parent plant. The use of liquid shake cultures minimized the time and culture medium requirements for propagation. This procedure can be applied for the conservation and utilization of elite clones of R. emodi.

In vitro Propagation of Valeriana wallichii.

A tissue culture procedure has been developed for the rapid multiplication of VALERIANA WALLICHII D C. through shoot tip and axillary bud explants. MS medium containing Kn or BAP (5.0 mg/l (-1)) in combination with IAA (1.0 mg/l (-1)) induced an optimal growth of shoots within 6-8 days from both apical and axillary bud explants. The roots developed on the same medium within 2-3 weeks. Hardening of IN VITRO grown plantlets in pots under glass-house conditions was dependent upon the temperature and humidity. A cold-temperate climate favoured early establishment. Following the given procedure, a large number of plants have been established under field conditions at two locations. The method has implications in the early introduction of an elite population as well as its improvement.

Clonal propagation of Picrorhiza kurroa royle ex benth. by shoot tip culture.

A procedure has been developed for the clonal propagation of Picrorhiza kurroa Royle ex Benth. through shoot tip culture. Murashige and Skoog's medium (1962) supplemented with kinetin (3.0 to 5.0 mg/l) supported rapid proliferation of multiple shoots from the explants. Addition of indole-3-acetic acid (1.0 mg/l) to the kinetin containing medium showed marked improvement in the growth of regenerated shoots. However, presence of IAA in the medium did not alter the frequency of shoot multiplication. Rooting was readily achieved upon transferring shoots onto MS medium containing ∝-naphthaleneacetic acid (1.0 mg/l). Plantlets were successfully transferred to soil.

Morphogenetic potential of foliar explants in Duboisia myoporoides R.Br. (Solanaceae).

The effects of serial combinations of either indole-3-acetic acid, indole-3-butyric acid or α-naphthaleneacetic acid (0.5-10.0 mg/l) with either kinetin, 6-benzyl-amino-purine, zeatin or 6-methylaminopurine (0.5-5.0 mg/l) have been investigated to assess the morphogenetic potential of foliar explants of Duboisia myoporoides. Shoot buds developed either directly or via a callus interphase. Combinations involving indole-3-acetic acid with any of the cytokinins were more effective in inducing shoot bud formation compared to those containing indole-3-butyric acid or α-napthalenacetic acid as an auxin. Among cytokinins, zeatin, kinetin and 6-benzylamino-purine were equally effective for shoot formation. However, optimum response with zeatin could be achieved at low concentrations (0.5-2.0 mg/l), while kinetin and 6-benzylamino-purine exhibited comparable efficacy at higher levels (3.0-5.0 mg/l). 6-Methylaminopurine proved least effective in all concentrations and combinations tested. Rooting of the differentiated shoots was readily achieved with α-naphthaleneacetic acid alone (0.5 mg/l) after changing the physical form of the medium from gel to static liquid. Regenerated plantlets were transferred to pots and grown to maturity in the field with a high rate of survival (80-90%).

Protoplast-derived streptomycin resistant plants of the forage legume Onobrychis viciifolia Scop. (sainfoin).

Approximately 10(6) protoplast-derived cell colonies of sainfoin were stressed with streptomycin and two resistant colonies were recovered. Plants regenerated from these colonies could be recallused on streptomycin-containing medium three years after growth in the absence of the antibiotic.Ultrastructural studies showed cells of resistant callus grown in the presence of streptomycin to contain chloroplasts with internal thykaloids and grana. Such mutant plants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids.

Prolific plant regeneration from protoplast-derived tissues of Lotus corniculatus L. (birdsfoot trefoil).

Protoplasts isolated enzymatically from seedling roots, hypocotyls and cotyledons of Lotus corniculatus L. produced callus which underwent prolific shoot regeneration. The rapidity and ease of recovering plants from protoplast-derived tissues makes this forage legume an attractive experimental system for genetic manipulation.