Potted 2-year-old lemon trees [Citrus limon (L.) Burm. fil, cv. Verna] grafted on sour orange (C. aurantium L.) rootstock were subjected to flooding for 3 d. Control plants were irrigated daily to field capacity. Continuously (sap flow, trunk diameter fluctuations) and discretely (predawn and midday leaf water potential, leaf conductance) measured plant-based water status indicators were compared. The sensitivity of the maximum daily trunk shrinkage signal intensity to flooding and its behaviour during the recovery period demonstrated that this indicator is more feasible than the others for use in automatic irrigation. The responses to flooding of continuously and discretely measured plant-based water status indicators were very similar to those observed in response to drought stress indicating that it necessary to use soil water measurement automatic sensors to detect the cause of the stress. The results underlined the robustness of the compensation heat-pulse technique for estimating instantaneous and daily transpiration rates on flooding stress and recovery.

Accumulation of extracellular chitinases in Brassica napus plants infected with Turnip yellow mosaic virus (TYMV) and fungal pathogen Leptosphaeria maculans was studied in both compatible and incompatible interaction. Analysis of apoplast fluid by means of non-denaturing anodic and cathodic PAGE followed by in-gel detection of chitinase activity revealed a number of chitinase isozymes. TYMV induced 8 acidic and 4 basic isozymes in a systemic way. Except for one acidic and one basic isozyme, all other chitinases were also constitutively present in low amounts in mock inoculated control. In TYMV systemically infected plants, chitinases were detected in leaves expressing symptoms as well as in symptomless ones. Both virulent and avirulent L. maculans isolates induced production of chitinase isozymes in cotyledons in a time dependent manner. Some of them were present in plants constitutively and their content increased after inoculation. Three of five acidic and two of three basic isozymes responded to L. maculans infection. Chitinases started to accumulate before symptom appearance. First two acidic isozymes were detected 24 h after inoculation. The difference between compatible and incompatibe interaction reflected two basic isozymes.

This paper describes the effect of NaCl on the respiration of Citrus cell suspensions namely on the induction of the alternative oxidase. The exposure of two Citrus (cvs. Carvalhal tangor and Valencia late) cell suspensions to 200 or 400 mM NaCl lead to a reduction on cell respiration rates. Under these conditions, the respiration rate decreased less in the presence of KCN indicating a stimulation of the capacity of the alternative oxidase (AOX). In addition, immunoblots showed an increase on the amount of AOX protein. Antibodies raised against the Sauromatum guttatum enzyme recognized the reduced form of the enzyme near the 35 kDa band. The protein accumulation was correlated with the significantly higher AOX capacity observed for cv. Carvalhal tangor.

Seed protein diversity of fourteen lentil cultivars grown in Turkey was studied by using sodium dodecyl sulfate polyacrylamide gel electrophoresis. A distance matrix was produced based on five polymorphic protein bands, scored for their absence as 0 and presence as 1. Seed protein distances among the cultivars ranged from 0.00 to 0.80. The dendrogram based on the distance matrix indicated two distinct clusters. The first cluster includes the cultivars Sultan 1, Meyveci 2001 and Kayi 91. The second cluster contains the cultivars Pul 11, Ozbek, Emre 20, Malazgirt 89, Ciftci, Seyran 96, AliDayi, Firat 87, Sazak, Erzurum 89 and YerliKirmizi.

We describe the optimized storage conditions of recombinant Potato virus A coat protein (ACP) carrying two different epitopes from Human papillomavirus type 16 (HPV-16). Epitope derived from minor capsid protein L2 was expressed as N-terminal fusion with ACP while an epitope derived from E7 oncoprotein was fused to its C-termini. The construct was cloned into Potato X potexvirus (PVX) based vector and transiently expressed in plants using Agrobacterium tumefaciens mediated inoculation. The effect of storage conditions on the serological activity of L2ACPE7 was studied by ELISA using IgG anti PVX, PVA and L2. Purified L2ACPE7 stored freeze-dried (at -20 °C), frozen at various temperatures (-20 °C, -70 °C) and at +4 °C were tested. Purified L2ACPE7 was most stable as lyophilized material stored at -20 °C. Our study demonstrates suitable way for the storage of plant material containing foreign viral epitopes for the purposes of edible vaccination.

Fucosylation in plants occurs in glycoproteins and polysaccharides but the function of fucosylation is largely unknown. We aimed to analyze the effects of over-expression of human fucosyltransferase III (hFucT III) on in vivo N-glycan accumulation in tobacco plant leaves and focused on comparing the amount of Lewis a (Le^a)-epitope accumulation in transgenic and in wild-type plants. Fucosyltransferase assays, Western blot and mass spectrometry were used to identify, quantify and analyse Le^a N-glycans. We found that constitutive overexpression of hFucT III activity had no effect on Le^a complex type N-glycans accumulation. Our results suggest that tobacco recombinant hFucT III acts more as a hydrolase than as a transferase.

Shoot buds were induced directly on either side of midrib from adaxial surface of immature leaf explants in Stevia rebaudiana Bertoni five weeks after culturing in Murashige and Skoog's nutrient medium supplemented with 8.88 µM of N ⁶-benzylaminopurine and kinetin ranging from 4.65 to 6.98 µM. Immature leaves of 0.6 to 1 cm were found to produce best response (93 %) with a highest number of 4.93 shoot buds per explant. For elongation of regenerated shoot buds, MS medium supplemented with 30 g dm^-3 sucrose and indole-3-butyric acid (IBA) ranging from 4.92 to 7.38 µM were found most suitable. The medium was further modified to suit bioreactor cultivation of regenerated shoots wherein the use of two-fold MS salts and 60 g dm^-3 sucrose resulted in a high biomass yield of 50.68 g dm^-3 (m/v) accounting for about 590 micro-cuttings in three weeks. Best rooting of micro-cuttings occurred in half strength MS medium supplemented with IBA ranging from 4.92 to 7.38 µM, 15 g dm^-3 sucrose and gelled with 0.8 % agar. Rooted plants were successfully established in substrate containing sand, Vermicompost and garden soil in equal proportions and grown in greenhouse. This is the first report on direct shoot regeneration from Stevia leaves.

Two selection systems for Agrobacterium tumefaciens mediated transformation of tomato and potato were compared. In the tomato (Lycopersicon esculentum cv. Moneymaker), the highest transformation rate, 4.2 %, of cotyledon explants on mannose-selection medium was obtained when mannose/sucrose concentration in the regeneration medium was 5/15 g dm^-3. The best transformation efficacy with the commonly used concentration of 100 mg dm^-3 kanamycin as a selection agent was 9 %. In the potato (Solanum tuberosum cv. Bintje), the highest transformation frequency was 53.3 % when mannose concentration in the regeneration medium was 5 g dm^-3 during the first 3 weeks after transformation and 10 g dm^-3 afterwards. The optimum concentration of sucrose was 20 g dm^-3. The transformation efficiency using kanamycin as a selection agent at a concentration 100 mg dm^-3 was 33.3 % with potato. Our results demonstrate that the transformation efficiency using mannose selection is 1.6-fold higher for potato and about 2 times lower for tomato comparing with the ordinary protocol using kanamycin.

Thirty five bands (alleles) from six enzyme systems and fifty seven random amplified polymorphic DNA (RAPD) fragments were selected to analyse the genetic diversity of 33 polyploid wheatgrasses (Triticeae) populations of species Thinopyrum junceiforme and Elytrigia pycnantha, and two hybrids, one pentaploid and one novel 9-ploid. Dice's similarity coefficient, the UPGMA-derived phenograms from RAPD, and allozymes markers showed that the clustering of wheatgrass populations was based on ploidy level. These markers had similar levels of diversity between populations, with high genetic similarity within the same ploidy-level and within population's individuals. The tetraploid Th. junceiforme populations are closely related, with a large similarity distances varied from 0.8 to 1. Based on the isozyme and RAPD analyses, diploid taxa are related to polyploids with similarity coefficients 0.4.

Inflorescence explants of two winter wheat cultivars, Triticum durum cv. Kiziltan-91 and T. aestivum cv. Bezostaja-01, were used to evaluate the effects of vernalization period of donor plants, callus age and medium composition on regeneration capacity. Donor plants were grown for 7 d and they were exposed to 4 °C for 1, 2, 3, 4, and 5 weeks. The maximum inflorescence formation was observed as 79 % at 4 weeks and 73 % at 5 weeks of vernalization period for Kiziltan-91 and Bezostaja-01, respectively. Among 6 different callus induction and regeneration mediums, I₁-R₁ and I₃-R₃ have to be the best responding mediums for Kiziltan-91 and Bezostaja-01, respectively. In Kiziltan-91, calli induced from donor plants, vernalized for 3 weeks, showed a significantly lower regeneration capacity than counterparts vernalized for 4 and 5 weeks. The highest regeneration capacity of 69 % was obtained from 6-week-old calli produced from 4 weeks vernalized Kiziltan-91 donor plants. In contrast to Kiziltan-91 cultures, the effects of vernalization period and callus age on regeneration capacity were not significant in Bezostaja-01 cultures. The maximum numbers of tillers were obtained from 6-and 15-week-old calli for Bezostaja-01 and Kiziltan-91, respectively. In contrast to vernalization period of donor plants, callus age had no effect on seed number.

Apple (Malus × domestica Borkh. cv. Golden Delicious) trees were inoculated with fungal pathogens, corresponding either to an incompatible strain of Venturia inaequalis or to non-host pathogens (Venturia pyrina, Alternaria brassicicola) in order to characterize the regulation of PR-10 genes in these different situations in relationship to symptom development. Macro-and microscopic observations of the plant-fungus interactions revealed typical symptoms of resistance with V. inaequalis and symptoms of hypersensitivity (HR) on around 5 % of leaves with V. pyrina. No HR was microscopically observed with A. brassicicola. In the non-host situations, the PR-10 expression in leaves at the transcriptional level was not (A. brassicicola) or very slightly (V. pyrina) activated. This strongly suggests that PR-10 is not a component of the non-host resistance in the interactions studied here. By contrast specific up-regulation of PR-10 was evident after inoculation with V. inaequalis.

Effects of exogenous salicylic acid (SA) on plant growth, contents of Na, K, Ca and Mg, activities of superoxide dismutase (SOD), guaiacol peroxidase (GPX), ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), glutathione reductase (GR) and catalase (CAT), and contents of ascorbate and glutathione were investigated in tomato (Lycopersicon esculentum L.) plants treated with 100 mM NaCl. NaCl treatment significantly increased H₂O₂ content and lipid peroxidation indicated by accumulation of thiobarbituric acid reactive substances (TBARS). A foliar spray of 1 mM SA significantly decreased lipid peroxidation caused by NaCl and improved the plant growth. This alleviation of NaCl toxicity by SA was related to decreases in Na contents, increases in K and Mg contents in shoots and roots, and increases in the activities of SOD, CAT, GPX and DHAR and the contents of ascorbate and glutathione.

The human papillomavirus type 16 (HPV 16) oncogene E7 fused with the gene for β-glucuronidase (gus) was used in plant transformation experiments. The E7 gene modified for lower cancerogenicity and fused with the 5' end of the gus in cassettes with cauliflower mosaic virus 35S promoter and transcription terminator produced high contents of fusion proteins in potato protoplasts. Expression vectors harbouring E7 fusion cassettes were used for Agrobacterium tumefaciens LBA4404 mediated transformation of either potato (Solanum tuberosum L. cv. Bintje) or tomato (Lycopersicon esculentum Mill. cv. Moneymaker). A fusion gene was found in all rooted regenerants using polymerase chain reaction with primers providing amplified fragments from E7 and gus genes. GUS activity was revealed in all regenerants obtained. Nevertheless, the level of GUS expression in different constructs varied much more than in transient expression experiments with potato protoplasts. Especially, expression level in plants carrying vectors with the whole E7 gene fused with gus was lowered by 2-3 orders of magnitude comparing with fusion of the first 41 codons of E7 and gus. Southern hybridisation of 18 tomato and 23 potato regenerants revealed mostly multiple tandem integration of T-DNA into the plant genome and Western blot proved the presence of the fusion protein in 9 tomato and 11 potato plants out of 41 tested individuals.

Mature apricot (Prunus armeniaca L. cv. Búlida) trees, growing under field conditions, were submitted to two drip irrigation treatments: a control (T1), irrigated to 100 % of seasonal crop evapotranspiration (ETc), and a continuous deficit (T2), irrigated to 50 % of the control throughout the year. The behaviour of leaf water potential and its components, leaf conductance and net photosynthesis were studied at three different times during the growing season, when they revealed a diurnal and seasonal pattern in response to water stress, evaporative demand of the atmosphere and leaf age. The deficit-irrigated trees showed, among other effects, a pronounced decrease in leaf water potential (ψ_w), decreased in leaf conductance (g_s) and no osmotic adjustment. For this reason, g_l and ψ_w can be considered good indicators of mature apricot tree water status and can therefore be used for irrigation scheduling.

The accumulation of steviol glycosides (SGs) in cells of Stevia rebaudiana Bertoni both in vivo and in vitro was related to the extent of the development of the membrane system of chloroplasts and the content of photosynthetic pigments. Chloroplasts of the in vitro plants, unlike those of the intact plants, had poorly developed membrane system. The callus cells grown in the light contained proplastids of almost round shape and their thylakoid system was represented by short thylakoids sometimes forming a little number of grana consisting of 2-3 thylakoids. In cells of the etiolated in vitro regenerants and the callus culture grown in the dark, only proplastids practically lacking the membrane system were observed. All the chloroplasts having developed thylakoids and forming at least a little number of grana were equipped with photochemically active reaction centers of photosystems 1 and 2. Leaves of in vivo plants accumulated greater amount of the pigments than leaves of the in vitro plants. In both the callus culture grown in the light and the etiolated in vitro regenerants, the content of the pigments was one order of magnitude lower than that in leaves of the intact plants. The callus tissue grown in the dark contained merely trace amounts of the pigments. Leaves of the intact and the in vitro plants did not exhibit any significant differences in photosynthetic O₂ evolution rate. However, photosynthetic O₂ evolution rate in the callus cells was much lower than that in the differentiated plant cells. The in vitro cell cultures containing merely proplastids did not practically produce SGs. However, after transferring these cultures in the light, both the formation of chloroplasts and the production of SGs in them were detected.

An efficient in vitro propagation of kava (Piper methysticum) was established. Utilizing 15-d-old tender shoots from dormant auxiliary buds as explants, significant induction of vigorous aseptic cluster shoots was achieved in Murashige and Skoog (MS) medium containing 0.5 mg dm^-3 6-benzyladenine (BA), 0.5 mg dm^-3 indole-3-acetic acid (IAA), and antibiotics after 30 d. In vitro rooting was achieved at 100 % efficiency in MS medium containing 0.75 to 1.00 mg dm^-3 IAA or indole-3-butyric acid and 3 % sucrose. The most robust and long roots were observed in medium with IBA. Moreover, the embryonic callus was induced from petioles in MS medium supplemented with 1.0 mg dm^-3 BA and 0.1 mg dm^-3 IAA, of which 70 % differentiated into shoots in the presence of 1.0 mg dm^-3 BA and 0.5 mg dm^-3 IAA.

The effects of Al, Cd and pH on growth, photosynthesis, malondialdehyde (MDA) content, and some antioxidant enzyme activities of the two soybean cultivars with different Al tolerance were determined using a hydroponic culture. There were six treatments as follows: pH 6.5; pH 4.0; pH 6.5 + 1.0 µM Cd; pH 4.0 + 1.0 µM Cd; pH 4.0 + 150 µM Al; pH 4.0 + 1.0 µM Cd + 150 µM Al. The results showed that the low pH (4.0) and Al treatments caused marked reduction in the growth (root and shoot length and dry mass), chlorophyll content (SPAD value) and net photosynthetic rate. Higher malondialdehyde content, superoxide dismutase (SOD) and peroxidase (POD) activities were detected in the plants exposed to both Al and Cd than in those exposed to Al treatment alone. An expressive enhancement of SOD and POD was observed in the plants exposed to 150 µM Al in the comparison with the control plants, especially in Al-sensitive cv. Zhechun 2 which had also significantly higher Al and Cd content than Al tolerant cv. Liao-1. Cd addition increased Al content in the plants exposed to Al + Cd stress, and cv. Zhechun 2 had relatively lower Al content. The present research indicated that Al and Cd are synergistic in their effects on plant growth and some physiological traits.

The DNA genetic diversity of 40 accessions of genus Leymus was analyzed by random amplified polymorphic DNA (RAPD) markers. A total of 352 products were amplified by 34 10-mer arbitrary primers, among which 337 products (95.74 %) were found to be polymorphic. 5-14 polymorphic bands were amplified by each polymorphic primer, with an average of 9.91 bands. The data of 352 RAPD bands were used to generate Jaccard's similarity coefficients and to construct a dendrogram by means of UPGMA. Great genetic diversity in genus Leymus was observed, the genetic diversity among the different species more abundant than that of the different accessions, and the different accessions in a species or the species from the same areas were clustered together.

Multiple shoots of Spilanthes acmella Murr. were induced from nodal buds of in vivo and in vitro seedlings on Murashige and Skoog (MS) medium containing 1.0 mg dm^-3 6-benzyladenine (BA) and 0.1 mg dm^-3 α-naphthalene-acetic acid (NAA). Adventitious shoots were successfully regenerated from the leaf explants derived from the above mentioned multiple shoots. The efficiency of shoot regeneration was tested in the MS medium containing BA, kinetin, or 2-isopentenyl adenine in combination with NAA, indole-3-acetic acid (IAA), or indole-3-butyric acid (IBA) and gibberellic acid. Maximum number of shoots per explant (20 ± 0.47) was recorded with 3.0 mg dm^-3 BA and 1.0 mg dm^-3 IAA. An anatomical study confirmed shoot regeneration via direct organogenesis. About 95 % of the in vitro shoots developed roots after transfer to half strength MS medium containing 1.0 mg dm^-3 IBA. 95 % of the plantlets were successfully acclimatized and established in soil. The transplanted plantlets showed normal flowering without any morphological variation.

Rapid shoot multiplication of Nyctanthes arbor-tristis L. was achieved from axillary meristems on Murashige and Skoog (MS) basal medium supplemented with 1.0-1.5 mg dm^-3 6-benzylaminopurine (BA), 50 mg dm^-3 adenine sulfate (Ads) and 3 % (m/v) sucrose. Inclusion of indole-3-acetic acid (IAA) in the culture medium along with BA + Ads promoted a higher rate of shoot multiplication. Maximum mean number of microshoots per explant (6.65) was achieved on the MS medium supplemented with 1.5 mg dm^-3 BA, 50 mg dm^-3 Ads and 0.1 mg dm^-3 IAA after 4 weeks of culture. The elongated shoots rooted within 13 to 14 d on half-strength MS medium supplemented with either indole-3-butyric acid (IBA), IAA or 1-naphthaleneacetic acid (NAA) with 2 % sucrose. Maximum percentage of rooting was obtained on medium having 0.25 mg dm^-3 IBA and 0.1 mg dm^-3 IAA. About 70 % of the rooted plantlets survived in the greenhouse. The in vitro raised plants were grown normally in the field.

The present work is focused on the possible relationship between nitric oxide and the induction of proline in response to salt stress. The plants were subjected to 100 mM NaCl and sodium nitroprusside (SNP; the donor of NO) at different concentrations. The plants showed lower NaCl-induced oxidative stress and proline accumulation after application of low concentrations of SNP together with the NaCl treatment. The reduction in the proline content was related to increased activity of proline dehydrogenase. These results suggest that the NO could be capable of mitigating damage associated with salt stress.

The observed inability of 6-d-old seedlings of spring wheat (Triticum aestivum L.) to tolerate the same water deficit as compared to the 4-d-old seedlings seems to be associated with the higher carboxypeptidase and lower aminopeptidase activities. Free amino acid pools differentiated also the 4-d-old seedlings from the older ones. Dehydration decreased the amino acid content in 4-d-old seedlings, increased it in 6-d-old seedlings and changed composition of amino acid pool. In tolerant phase of wheat seedling growth carboxypeptidase activity increased in response to water deficit and aminopeptidase activity increased in dehydrated seedlings, independently of their age.

Three constructs harbouring novel Bacillus thuringiensis genes (Cry1C, Cry2A, Cry9C) and bar gene were transformed into four upland cotton cultivars, Ekangmian10, Emian22, Coker201 and YZ1 via Agrobacterium-mediated transformation. With the bar gene as a selectable marker, about 84.8 % of resistant calli have been confirmed positive by polymerase chain reaction (PCR) tests, and totally 50 transgenic plants were regenerated. The insertions were verified by means of Southern blotting. Bioassay showed 80 % of the transgenic plantlets generated resistance to both herbicide and insect. We optimized conditions for improving the transformation efficiency. A modified in vitro shoot-tip grafting technique was introduced to help entire transplantation. This result showed that bar gene can replace antibiotic marker genes (ex. npt II gene) used in cotton transformation.

A new micropropagation system for Lycium barbarum (L.) was developed using root explants as starting material. Callus can be produced from root explants on Murashige and Skoog (MS) medium containing 0.2 mg dm^-3 2,4-dichlorophenoxyacetic acid. After three subcultures on the same medium, callus was then transferred onto the MS medium supplemented with 500 mg dm^-3 lactalbumin hydrolysate to induce somatic embryogenesis (SE). After 20 d, about 60 somatic embryos per 0.25 g(f.m.) of embryogenic callus were obtained but only about 10 % of embryos converted into plantlets. After acclimated in the greenhouse, all of the randomly selected plantlets had survived and were similar phenotypically to zygotic seedlings. In addition, the effects of irradiance, photoperiod, growth regulators, explant age and cold treatment on SE of root-derived callus were evaluated.

Seven clones containing (CTG)_n/(CAG)_n repeats (n ≥ 4) were isolated by screening Lycopersicon esculentum genomic DNA. Four of the clones contained more than one simple sequence repeat (SSR). The SSRs were analyzed in several L. esculentum cultivars after polymerase chain reaction (PCR) amplification. No length variations were observed, suggesting considerable locus stability. Five clones are from transcribed regions, which might explain the lack of cultivar variations. However the conservation of CTG repeats was limited as differences in some transcribed loci were registered between L. pennellii and other Lycopersicon species. It is noted that in Lycopersicon trinucleotide repeat variation might be used for species identification.

Somatic embryogenesis and plant regeneration were successfully established on Nitsch and Nitsch (NN) medium from immature zygotic embryos of six genotypes of grapevine (Vitis vinifera). The optimum hormone combinations were 1.0 mg dm^-3 2,4-dichlorophenoxyacetic acid (2,4-D) for callus induction and 1.0 mg dm^-3 α-naphthalene acetic acid (NAA) + 0.5 mg dm^-3 6-benzyladenine (BA) for embryos production and 0.03 mg dm^-3 NAA + 0.5 mg dm^-3 BA for embryos conversion and plant regeneration. The frequency of somatic embryogenesis varied from 10.5 to 37.5 % among six genotypes and 15.5-42.1 % of somatic embryos converted into normal plantlets. The analysis of DNA content determined by flow cytometry and chromosome counting of the regenerated plantlets clearly indicated that no ploidy changes were induced during somatic embryogenesis and plant regeneration, the nuclear DNA content and ploidy levels of the regenerated plants were stable and homogeneous to those of the donor plants. RAPD markers were also used to evaluate the genetic fidelity of plants regenerated from somatic embryos. All RAPD profiles from regenerated plants were monomorphic and similar to those of the field grown donor plants. We conclude that somaclonal variation is almost absent in our grapevine plant regeneration system.

To determine whether long-term growth in elevated atmospheric CO₂ concentration [CO₂] and nitrogen fertilization affects woody tissue CO₂ efflux, we measured stem CO₂ efflux as a function of temperature in three different size classes of shoots of Populus nigra L. (clone Jean Pourtet) on two occasions in 2004. Trees were growing in a short rotation coppice in ambient (370 µmol mol^-1) and elevated (550 µmol mol^-1, realised by a Free Air Carbon dioxide Enrichment system) [CO₂], and measurements were performed during the third growing season of the second rotation. Elevated CO₂ did not affect Q₁₀ or specific stem CO₂ efflux (E₁₀) of overall poplar shoots. The lack of any effect of N on stem CO₂ efflux indicated that nutrients were sufficient. Specific stem CO₂ efflux differed significantly between shoot sizes, emphasizing the importance of tree size when scaling-up respiration measurements to the stand level. Variation in stem CO₂ efflux could not be satisfactorily explained by temperature as the only driving variable. We hypothesize that transport of CO₂ with the sapflow might have confounded our results and could explain the high Q₁₀ values reported here. Predicting the respiratory carbon loss in a future elevated [CO₂] world must therefore move beyond the single-factor temperature dependent respiration model and involve multiple factors affecting stem CO₂ efflux rate.

We report on gene silencing of glutathione synthetase (GSHS) that reduces reduced glutathione (GSH) content in somatic embryos of Camellia sinensis L. Using degenerate primers with cDNA of Camellia sinensis, a 457 bp GSHS gene fragment was cloned through polymerase chain reaction. This fragment was used in making ihpRNA. For this it was cloned in sense at AscI and SwaI and in anti-sense at Bam HI and XbaI restriction sites of pFGC5941 that has chalcone synthase (Chs) intron between SwaI and BamHI restriction sites. Resultant RNAi construct was used for C. sinensis somatic embryos transformation through Agrobacterium. After 11, 13 and 15 d of transformation, embryo GSHS transcript levels and GSH content decreased to a great extent which documented the feasibility of RNAi based gene silencing in C. sinensis.

Stomatal closing to abscisic acid (ABA) was studied in leaf epidermal peels of a dexamethasone (Dex)-inducible transgenic line expressing the phospholipase C AtPLC1 antisense in the Columbia genetic background. In the absence of Dex, the Ca²⁺ buffer, ethylene glycol-bis(b-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) and the phopholipase C inhibitor, 1-[6-{[17β-3-methoxyestra-1,3,5(10)-trien-17-yl]amino}hexyl]-1H-pyrrole-2,5-dione (U73122) specifically inhibited the response to 20 µM ABA, whereas the Ca²⁺ buffer, 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) inhibited the response to 20 or 30 µM ABA. Neither EGTA nor BAPTA increased the U73122 effect. Applying 30 µM Dex specifically affected 20 µM ABA-induced stomatal closing through reducing its magnitude as well as suppressing the EGTA, BAPTA and U73122 inhibitory effects. Neither Dex nor U73122 changed the specific inhibitory effects of both the antagonist of cyclic ADP-ribose synthesis, nicotinamide and the GTP-binding protein (G protein) modulators, pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH₂ (GP Ant-2) and mas17 on 30 µM ABA-induced stomatal closing. When tested in combination, substituting nicotinamide for mas17, but not for GP Ant-2, enhanced their inhibitory effect to an extent that BAPTA did not increase. These results supported that AtPLC1 primarily mediates the Ca²⁺-dependent stomatal closing response to 20 µM ABA as much as 30 µM Dex did not affect 20 µM ABA-induced stomatal closing when tested on the wild type Columbia-4 ecotype. Furthermore, the present study suggested that Ca²⁺ mobilization did not involve any dependency between AtPLC1 and a putative G protein-coupled ADP-ribosyl cyclase at the tested ABA concentrations.