Lung Health Care pilot project trims patient pill load and antibiotic prescription in primary health care settings in Kerala, India.

INTRODUCTION: Kerala, the southern Indian state piloted Lung Health Care Project (LHCP) which is a locally adopted version of WHO recommended Practical Approach to Lung health (PAL). The current study assessed the impact of the project on the prescribing practices of doctors and consumption of antibiotics and other drugs. METHODS: This study compared performance of primary health care institutions with regard to drug prescriptions and consumptions before and after the implementation of the project. Chronic respiratory disease (CRD) patients in institutions implemented the project were interviewed in the OPD at exit and their prescriptions were documented at baseline and after six months. Focus group discussions were conducted with doctors to explore the reasons behind changes in drug consumption pattern. RESULTS: In the project implementing institutions, mean number of drugs prescribed for CRDs was 3.88 (SD 1.50) and 2.73 (SD 1.18) at baseline and after six months respectively (p < 0.001). Adjusted odds ratio for prescribing an antibiotic and injection to a CRD patient during impact assessment at institutions implementing project was 0.34 (95% CI 0.15-0.75 p 0.008) and 0.39 (95% CI 0.20-0.74 p 0.004) respectively, as compared to baseline. The factors which helped in reducing antibiotic and injection use as felt by the doctors were presence of a protocol, good quality trainings, supportive supervision and monitoring, availability of alternate drugs and good participation of staff nurses especially in-patient education. CONCLUSION: Strict adherence to diagnostic and management algorithms of Lung health care project in a primary health care setting in India helped in reducing pill burden to patients and prescription of antibiotics and injections.

Refining the Genomic Region Containing a Major Locus Controlling Fruit Maturity in Peach.

Maturity date (MD), defined as the duration between the first calendar day of the year and maturity, and fruit development period (FDP), defined as the duration between full bloom and maturity, are highly variable in peach [Prunus persica (L.) Batsch]. There is a need to discover molecular markers associated with these traits in order to enhance the efficiency and reliability of breeding for extending the harvest season in peach. An association mapping population consisting of 132 peach accessions was phenotypically evaluated for MD and FDP, and genotypically characterized using the genotyping-by-sequencing (GBS) approach. The phenotypic and genotypic data collected were used to conduct a genome-wide association study (GWAS). The GWAS identified three SNPs on chromosome 4 that are significantly associated with both FDP and MD. These three SNPs covered a region of 43,067 bp; we referred to this region as the MD/FDP locus. Seven genes were identified in the MD/FDP locus. One or more of these genes is believed to regulate some aspect of maturity in peach. The data reported here is expected to aid in marker-assisted seedling selection (MASS) targeted towards widening peach germplasm for maturity, particularly early maturity.

TB management by private practitioners - Is it bad everywhere?

INTRODUCTION: Poor prescribing practice is alleged to be one of the major factors fuelling the drug-resistant tuberculosis (DR TB) emergence. A study in Mumbai revealed the extent of inappropriate tuberculosis (TB) management practices of private practitioners and discussed that with the context of high DR TB. Kerala is rated among the well performing States in India as far as TB control is concerned with evidences for a lower level of TB transmission and DR TB. The current study was done in Kerala State to assess the prescribing practices of private sector doctors in the treatment of TB. METHODS: Survey questionnaire to write a standard prescription for treating TB was administered to private practitioners dealing with TB, who attended continuing medical education programme on TB at two major cities in Kerala. RESULTS: Responses from a total of 124 questionnaires were studied. None of them prescribed anti-TB regimen for less than 6 months. Only 7 (5.6%) prescribed a regimen without complete four drugs (H, R, Z, E) in the intensive phase. Out of the 81 doctors who prescribed private anti-TB regimen, 67 (82.7%) had of the opinion that not less than 80% of their patients complete the treatment for the prescribed duration. CONCLUSION: The current study reports a reasonable TB management practice among the private sector doctors from a State with a low prevalence of DR TB and compliments the argument that effective treatment of TB following the principles of standards for TB care can prevent the emergence of DR TB.

Factors associated with sputum culture conversion in multidrug-resistant pulmonary tuberculosis.

INTRODUCTION: Sputum culture conversion in pulmonary multidrug-resistant tuberculosis (MDR-TB) is important to make treatment-related decisions and prevent transmission of disease. OBJECTIVE: To identify factors associated with sputum culture conversion, and to determine time to culture conversion and the impact of culture conversion on successful treatment outcomes in MDR-/rifampicin (RMP) resistant TB. METHOD: Retrospective analysis of data from treatment cards and registers of MDR-/RMP-resistant patients initiated on treatment under India's Revised National TB Control Programme in Delhi, West Bengal and Kerala from January 2009 to December 2011. Proportions were calculated and logistic regression analysis was performed. RESULTS: Of 836 patients, 787 were analysed, 651 (83%) of whom experienced culture conversion: respectively 57%, 73% and 79% culture converted by month 3, 4 and 6 of treatment. The median time to culture conversion was 91.3 days. Patients with body mass index (BMI) 16 kg/m2 (OR 0.403, P = 0.001) and 1618 kg/m2 (OR 0.519, P = 0.039) were less likely to have culture conversion. High rates of culture conversion were observed in patients with successful treatment outcomes compared to those without treatment success (462/469, 99% vs. 183/311, 59%; P 0.0001). CONCLUSION: Low BMI is associated with poor sputum culture conversion in MDR-/RMP-resistant TB patients. Lack of culture conversion can impact successful treatment outcomes.

Directly observed treatment short course for tuberculosis. What happens to them in the long term?

BACKGROUND: Though Directly Observed Treatment Short course (DOTS) is found effective in many controlled trials, few studies have examined its effectiveness under programmatic conditions. DOTS based Revised National TB Control Programme (RNTCP) was initiated in Ernakulam district of Kerala state in June 2000. It now covers all of India. It now seems appropriate to do an evaluation of RNTCP at field level. AIM: This study aims to document impact of DOTS in providing productive life to tuberculosis patients and measure rate of clinical recurrence under program conditions. METHODS: Retrospective cohort study using interview with structured, peer reviewed and validated questionnaire among cohort of new smear positive patients registered in RNTCP from January 2002 to December 2003 and declared cured/Treatment completed. We have contacted 1173 patients (62.2% of the cohort) for the study at their homes by devising a strategy to identify and trace patients from address given in TB registers. RESULTS: Mean age of identified patients is 51.9 years. 82.4% were males. 79% patients report full supervision in the intensive period. After seven years 64.1% are healthy, work and earn; 29.8% report residual respiratory problems; 0.3% of symptomatic patients were diagnosed with smear positive pulmonary tuberculosis. Relapse calculated as worst case scenario for full target population (dead and migrated inclusive) is 9.27%. Age specific mortality is 4-6 times higher than in a comparable general population. CONCLUSIONS: DOTS treatment under program conditions makes a measurable reduction in tuberculosis morbidity. Though high proportion of patients remains productive after DOTS, a significant proportion complains of residual respiratory symptoms. Age specific mortality of Post tuberculosis patients is high compared to general population. Close follow up irrespective of duration of symptoms may help to determine the causes of high residual morbidity and mortality rates.

Social inclusion: An effort to end loss-to-treatment follow-up in tuberculosis.

SITUATION ANALYSIS: Pathanamthitta district is implementing Revised National Tuberculosis Control Program as a pilot district since 1993. The district programme was reporting approximately 5% of their diagnosed smear positive patients as never put on treatment (Initial lost to follow up - ILFU) and 5% of the new smear positive [NSP] Pulmonary TB patients as lost to follow up [LFU] during treatment. Attempts based on reengineering of DOTS were not largely successful in bringing down these proportions. INTERVENTION: A treatment support group [TSG] is a non-statutory body of socially responsible citizens and volunteers to provide social support to each needy TB patient safeguarding his dignity and confidentiality by ensuring access to information, free and quality services and social welfare programs, empowering the patient for making decision to complete treatment successfully. It is a complete fulfilment of social inclusion standards enumerated by Standards for TB Care in India. Pathanamthitta district started implementing this strategy since 2013. OUTCOMES: After intervention, proportion of LFU among NSPTB cases dropped markedly and no LFU were reported among the latest treatment cohorts. Proportion of ILFU keeps similar trend and none were reported among the latest diagnostic cohorts. LESSONS: Social support for TB care is feasible under routine program conditions. Addition of standards for social inclusion in STCI is meaningful. Its meaning is translated well by a society empowered with literacy and political sense.

PpERF3b, a transcriptional repressor from peach, contributes to disease susceptibility and side branching in EAR-dependent and -independent fashions.

KEY MESSAGE: Peach ERF3b is a potent transcriptional repressor for defense-related genes even in the presence of similar levels of transcriptional activators and can interfere with plant development through pathways independent of the EAR motif. Ethylene response factors (ERFs) are a major group of plant transcription factors with either activation or repression capabilities on gene transcription. Repressor-type ERFs are characterised by an intrinsic motif, namely the ERF-associated amphiphilic repression motif (EAR). Here we report the identification of three genes from peach (Prunus persica), PpERF12, PpERF3a and PpERF3b, encoding for ERF repressors. The transcription kinetics of these genes was investigated by qRT-PCR after inoculation of peach leaves with Xanthomonas campestris pv. pruni. All three genes showed higher induction in the susceptible 'BabyGold 5', than in the resistant 'Venture' peach varieties suggesting a negative role for these genes in disease resistance. The functional potency of PpERF3b has been confirmed in vivo by its ability to repress the expression of GUS-reporter gene. To better understand the functional role of PpERF3b, the full-length and the EAR-truncated (PpERF3b∆EAR) genes were overexpressed in tobacco (Nicotiana tabacum). Both transgenic plants (PpERF3b and PpERF3b∆EAR) uniformly exhibited precocious side branching, which suggests the interference of PpERF3b with auxin-mediated dormancy of lateral shoots. Consistent with that the expression of auxin-response factors (Nt-ARF1, Nt-ARF6 and Nt-ARF8) was significantly downregulated in transgenic plants compared to the wild type (WT). Although side branching was independent of EAR motif, the response of transgenic plants to inoculation by Pseudomonas syringae pv. tabaci was EAR dependent. Transgenic plants overexpressing PpERF3b∆EAR showed less disease symptoms than those overexpressing the full-length gene or WT plants. Resistance of PpERF3b∆EAR plants was associated with enhanced induction of pathogenesis-related (PR) genes. Our results indicate that repressor-type ERFs might act through pathways that are dependent or independent of the EAR motif.

Expression of auxin-binding protein1 during plum fruit ontogeny supports the potential role of auxin in initiating and enhancing climacteric ripening.

Auxin-binding protein1 (ABP1) is an active element involved in auxin signaling and plays critical roles in auxin-mediated plant development. Here, we report the isolation and characterization of a putative sequence from Prunus salicina L., designated PslABP1. The expected protein exhibits a similar molecular structure to that of well-characterized maize-ABP1; however, PslABP1 displays more sequence polarity in the active-binding site due to substitution of some crucial amino-acid residues predicted to be involved in auxin-binding. Further, PslABP1 expression was assessed throughout fruit ontogeny to determine its role in fruit development. Comparing the expression data with the physiological aspects that characterize fruit-development stages indicates that PslABP1 up-regulation is usually associated with the signature events that are triggered in an auxin-dependent manner such as floral induction, fruit initiation, embryogenesis, and cell division and elongation. However, the diversity in PslABP1 expression profile during the ripening process of early and late plum cultivars seems to be due to the variability of endogenous auxin levels among the two cultivars, which consequently can change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating PslABP1 was investigated. Our data suggest that auxin is involved in the transition of the mature green fruit into the ripening phase and in enhancing the ripening process in both auxin- and ethylene-dependent manners thereafter.

Differential expression of peach ERF transcriptional activators in response to signaling molecules and inoculation with Xanthomonas campestris pv. pruni.

Ethylene response factors (ERFs) are a large family of transcription factors (TFs) that have diverse functions in plant development and immunity. However, very little is known about the molecular regulation of these TFs in stone fruits during disease incidence. In the present study, we describe the identification of five peach ERFs (Pp-ERFs), aiming to elucidate their potential roles in defense against Xanthomonas campestris pv. pruni (Xcp), the causal agent of bacterial spot disease. The phylogenetic analysis along with sequence comparisons indicated that all Pp-ERFs are transcriptional activators belonging to groups IX and IIV ERFs. The transactivation capacity of these proteins was verified in vivo where they all induced the expression of the GUS reporter gene and in a GCC-dependent manner. The nuclear localization was also confirmed for two of these proteins, Pp-ERF2.b and Pp-ERF2.c, after their transient expression in onion epidermal cells. The induction kinetics of Pp-ERFs after inoculation with Xcp was determined by qRT-PCR. Except for Pp-ERF2.b, transcript levels of Pp-ERFs increased strongly and rapidly in the resistant 'Venture' compared to the susceptible 'BabyGold 5' cultivar after infection with Xcp. In contrast, the expression of Pp-ERF2.b was several-fold higher in the susceptible cultivar after bacterial infection. The expression of Pp-ERFs was also monitored after treating with signaling compounds; salicylic acid (SA) (1 mM), ethephon (1 mM) and methyl jasmonate (MeJA) (50 µM). Although the results generally emphasize the role of ethylene/jasmonic acid (ET/JA) signaling pathways in regulating the expression of Pp-ERFs, there was a coordination of the timing of ET/JA responses, suggesting compensatory rather than synergistic interactions between these pathways during defense against Xcp.

Identification and genetic characterization of a gibberellin 2-oxidase gene that controls tree stature and reproductive growth in plum.

Several dwarf plum genotypes (Prunus salicina L.), due to deficiency of unknown gibberellin (GA) signalling, were identified. A cDNA encoding GA 2-oxidase (PslGA2ox), the major gibberellin catabolic enzyme in plants, was cloned and used to screen the GA-deficient hybrids. This resulted in the identification of a dwarf plum hybrid, designated as DGO24, that exhibits a markedly elevated PslGA2ox signal. Grafting 'Early Golden' (EG), a commercial plum cultivar, on DGO24 (EG/D) enhanced PslGA2ox accumulation in the scion part and generated trees of compact stature. Assessment of active GAs in such trees revealed that DGO24 and EG/D accumulated relatively much lower quantities of main bioactive GAs (GA(1) and GA(4)) than control trees (EG/M). Moreover, the physiological function of PslGA2ox was studied by determining the molecular and developmental consequences due to ectopic expression in Arabidopsis. Among several lines, two groups of homozygous transgenics that exhibited contrasting phenotypes were identified. Group-1 displayed a dwarf growth pattern typical of mutants with a GA deficiency including smaller leaves, shorter stems, and delay in the development of reproductive events. In contrast, Group-2 exhibited a 'GA overdose' phenotype as all the plants showed elongated growth, a typical response to GA application, even under limited GA conditions, potentially due to co-suppression of closely related Arabidopsis homologous. The studies reveal the possibility of utilizing PslGA2ox as a marker for developing size-controlling rootstocks in Prunus.

Cloning and characterization of PR5 gene from Curcuma amada and Zingiber officinale in response to Ralstonia solanacearum infection.

Ginger (Zingiber officinale Roscoe), is an important spice crop that is badly affected by Ralstonia solanacearum wilt. Ginger does not set seed and sexual recombination has never been reported. In spite of extensive search in its habitats, no resistance source to Ralstonia induced bacterial wilt, could be located in ginger. Curcuma amada Roxb. is a potential donor for bacterial wilt resistance to Z. officinale, if the exact mechanism of resistance is understood. Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. Two putative PR5 genes, CaPR5 and ZoPR5, were amplified from C. amada and ginger, which encode precursor proteins of 227 and 224 amino acid residues, respectively, and share high homology with a number of other PR5 genes. The secondary and three-dimensional structure comparison did not reveal any striking differences between these two proteins. The expression of Ca and ZoPR5s under R. solanacearum inoculation was analyzed at different time points using quantitative real-time PCR (qRT-PCR). Our results reveal that CaPR5 is readily induced by the bacterium in C. amada, while ZoPR5 induction was very weak and slow in ginger. These results suggest that the CaPR5 could play a role in the molecular defense response of C. amada to pathogen attack. This is the first report of the isolation of PR5 gene from the C. amada and Z. officinale. Promoter analysis indicates the presence of a silencing element binding factor in ZoPR5-promoter, but not in CaPR5. Prospective promoter elements, such as GT-1 box and TGTCA, implicated as being positive regulatory elements for expression of PR proteins, occur in the 5'-flanking sequences of the CaPR5. Transient GUS expression study confirms its action with a weaker GUS expression in ginger, indicating that the PR5 expression may be controlled in the promoter.

Regulation of two germin-like protein genes during plum fruit development.

Germin-like proteins (GLPs) have several proposed roles in plant development and defence. Two novel genes (Ps-GLP1 and 2) encoding germin-like protein were isolated from plum (Prunus salicina). Their regulation was studied throughout fruit development and during ripening of early and late cultivars. These two genes exhibited similar expression patterns throughout the various stages of fruit development excluding two important stages, pit hardening (S2) and fruit ripening (S4). During fruit development until the ripening phase, the accumulation of both Ps-GLPs is related to the evolution of auxin. However, during the S2 stage only Ps-GLP1 is induced and this could putatively be in a H(2)O(2)-dependent manner. On the other hand, the diversity in the Ps-GLPs accumulation profile during the ripening process seems to be putatively due to the variability of endogenous auxin levels among the two plum cultivars, which consequently change the levels of autocatalytic ethylene available for the fruit to co-ordinate ripening. The effect of auxin on stimulating ethylene production and in regulating Ps-GLPs transcripts was also investigated. These data, supported by their localization in the extracellular matrix, suggest that auxin is somehow involved in the regulation of both transcripts throughout fruit development and ripening.

Molecular characterization of seven genes encoding ethylene-responsive transcriptional factors during plum fruit development and ripening.

Seven ERF cDNAs were cloned from two Japanese plum (Prunus salicina L.) cultivars, 'Early Golden' (EG) and 'Shiro' (SH). Based on the sequence characterization, these Ps-ERFs could be classified into three of the four known ERF families. Their predicted amino acid sequences exhibited similarities to ERFs from other plant species. Functional nuclear localization signal analyses of two Ps-ERF proteins (Ps-ERF1a and -1b) were carried out using confocal microscopy. Expression analyses of Ps-ERF mRNAs were studied in the two plum cultivars in order to determine the role of this gene family in fruit development and ripening. The seven Ps-ERFs displayed differential expression pattern and levels throughout the various stages of flower and fruit development. The diversity in Ps-ERFs accumulation was largely due to the differences in their responses to the levels of ethylene production. However, other plant hormones such as cytokinin and auxin, which accumulate strongly throughout the various developmental stages, also influence the Ps-ERFs expression. The effect of the plant hormones, gibberellin, cytokinin, auxin, and ethylene in regulating the different Ps-ERF transcripts was investigated. A model was proposed in which the role played by the plant hormone auxin is as important as that of ethylene in initiating and determining the date and rate of ripening in Japanese plums.

Expression analysis of a plum pathogenesis related 10 (PR10) protein during brown rot infection.

Plant PR10 is one of the pathogenesis related proteins, induced upon exposure to different stress conditions including fungal infection. PR10 proteins have been implicated in fungal disease resistance in some species; however its transcriptional regulation is not well understood. In the present work we cloned a PR10 gene from European plums (Prunus domestica L.) and monitored the quantitative changes in its transcript levels as a result of fungal infection in two varieties. We also studied the possible involvement of the membrane degrading enzyme phospholipase D-alpha (PLDalpha). In the susceptible variety, 'Veeblue', infection with the brown rot fungus Monilinia fructicola induced PLDalpha and PR10 expression, while in the resistant variety, 'Violette', a constitutive expression of PLDalpha and PR10 transcripts levels were observed. Resistance to M. fructicola also coincides with a sharp decrease in the expression of ABI1, a protein phosphatase and elevated hydrogen peroxide content after infection. Further, inhibition of PLDalpha by hexanal treatment, up-regulated ABI1 and decreased PR10 expression, suggesting a possible relationship between the two. We further confirm these results in Arabidopsis abi1 mutant that shows a higher level of PR10 transcripts.

Differential regulation of four members of the ACC synthase gene family in plum.

The regulation of ACC synthase (ACS) genes was studied in early ('Early Golden') and late ('Shiro') Japanese plum cultivars (Prunus salicina L.) in order to determine the role of this gene family in fruit ripening. Of the four Ps-ACS cDNAs isolated, two (Ps-ACS1 and -3) showed differential expression between the two cultivars. Ps-ACS1 accumulated during fruit ripening of 'Early Golden' ('EG') and 'Shiro' ('SH') in ethylene-dependent and -independent manners, respectively. Ps-ACS3a transcripts accumulated throughout fruit development and during 'EG' fruit ripening. Ps-ACS3b was detected only during ripening of 'SH' fruit. Furthermore, Ps-ACS3a transcript accumulation was negatively regulated by ethylene, whereas Ps-ACS3b was positively induced by the hormone. In both cultivars, the expression of Ps-ACS4 and -5 is under positive and negative feedback control by ethylene, respectively. Genetic analyses of 'EG' and 'SH' cultivars demonstrated that 'EG' is homozygous for Ps-ACS3a whereas 'SH' is heterozygous for Ps-ACS3 (a/b). The role of ethylene-overproducer 1-like in delaying fruit ripening by interacting with Ps-ACS proteins was also studied. The effect of the plant hormones, auxin, gibberellin, and cytokinin, in regulating ethylene production by promoting the induction of the different Ps-ACS mRNAs in plum was investigated. A model is presented in which differences in Ps-ACS alleles and gene expression between early and late plums are critical in determining the ripening behaviour of the cultivars.

Isolation and characterization of four ethylene signal transduction elements in plums (Prunus salicina L.).

Plums are climacteric fruits: their ripening is associated with a burst of ethylene production and respiration rate. Stone fruits, including plum, have a distinct pattern of growth and development, described as a double sigmoid pattern. In order to understand the developmental control of ethylene perception in plum, four ethylene perception and signal transduction components (EPSTCs) were characterized, including two ETR1-like proteins (Ps-ETR1 and Ps-ERS1), a CTR1-like protein, and an ethylene-responsive element-binding factor (ERF). Their regulation was studied throughout fruit development and ripening in early and late cultivars. Analysis of transcript levels revealed that only Ps-ERF1 and Ps-ERS1 accumulated immediately after fertilization. Increases in Ps-ETR1 and Ps-CTR1 transcript levels could not be detected before S3 of fruit development. Marked differences associated with the ripening behaviour of early ('Early Golden') and late ('Shiro') Japanese plum cultivars were observed. The early cultivar showed ripening patterns typical of climacteric fruits accompanied by sharp increases of the four transcript levels in an ethylene-dependent manner. However, the late cultivar exhibited a suppressed-climacteric pattern, with a slight increase in ethylene production related to ripening. The accumulation of the Ps-ETR1 (and not Ps-CTR1) mRNA in the late cultivar was ethylene independent. Ps-ERS1 mRNA was expressed at low, constant levels, while, Ps-ERF1 remained undetectable. The differences between the two plum cultivars in the date and rate of ripening in relation to the differences in the accumulation patterns of the four mRNAs are discussed.

Comparative anatomy and morphology of Vitis vinifera (Vitaceae) somatic embryos from solid- and liquid-culture-derived proembryogenic masses.

Ontogeny of somatic embryos of grapevine (Vitis vinifera) produced from solid- and liquid-culture-derived proembryogenic masses (PEM) was compared using light and scanning electron microscopy. Somatic embryos produced from solid-medium-derived PEM (SPEM) had large cotyledons, little or no visible suspensor structure, and a relatively undeveloped concave shoot apical meristem, whereas those from liquid-medium-derived PEM (LPEM) had smaller cotyledons, a distinct suspensor, and a flat-to-convex shoot apical meristem. The convex shoot apical meristem in LPEM-derived somatic embryos formed as early as the heart stage of development; it was 4-6 cell layers deep and rich in protein. Suspensors persisted in fully developed and mature LPEM-derived somatic embryos. The SPEM-derived somatic embryos exhibited dormancy, as do mature zygotic embryos, which also have a rudimentary suspensor, whereas LPEM-derived embryos were not dormant. We hypothesize that the presence of a persistent suspensor in LPEM-derived somatic embryos modulates development, ultimately resulting in rapid germination and a high plant-regeneration rate.

Expression of a bifunctional green fluorescent protein (GFP) fusion marker under the control of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera).

Activity of three constitutive promoters and enhanced derivatives in transgenic grape (Vitis vinifera L. cv. Thompson Seedless) was characterized using a bifunctional fusion marker containing the enhanced green fluorescent protein (EGFP) and neomycin phosphotransferase (NPTII) genes. Relative differences in transient GFP expression and stable transformation efficiencies were used to compare promoter activity. Expression patterns in transformed somatic embryos revealed that the ACT2 promoter from Arabidopsis thaliana, previously shown to be a strong constitutive promoter in A. thaliana and other species, failed to promote strong expression in grape. In contrast, a promoter isolated from cassava vein mosaic virus (CsVMV) supported high levels of transgene expression equivalent to those achieved using an enhanced double cauliflower mosaic virus (CaMV) 35S promoter. Duplication of the 5'-upstream enhancer region of the CsVMV promoter further enhanced its ability to increase transgene expression. However, the pattern of transgene expression driven by these two viral promoters was significantly different at the whole plant level. The enhanced double CaMV 35S promoter was highly active in most tissues and organs including roots, mature leaves, shoot apices and lateral buds. In contrast, the CsVMV promoter and its double enhancer derivative induced relatively weak expression in these tissues. Our results suggest that activity of the CsVMV promoter, in contrast to the CaMV 35S promoter, was under developmental regulation in transgenic grape plants as compared with the CaMV 35S promoter.

In-vitro selection of Vitis vinifera 'Chardonnay' with Elsinoe ampelina culture filtrate is accompanied by fungal resistance and enhanced secretion of chitinase.

Proembryogenic masses of grapevine (Vitis vinifera L.) 'Chardonnay' (clone 02Ch) were exposed to the culture filtrate of Elsinoe ampelina (deBary) Shear, the causal agent of anthracnose disease. After four or five cycles of recurrent in-vitro selection with medium containing 40% fungal culture filtrate, putative resistant lines RC1 and RC2 respectively, were established. The selected lines inhibited the growth of E. ampelina and Fusarium oxysporium (Schlecht.) (isolated from watermelon) in a dual-culture assay and reduced the growth of mycelium on a conditioned-medium test, thus suggesting the involvement of extracellular compounds in resistance. Sodium dodecyl sulfate-polyacrylamide (SDS-PAGE) gel electrophoresis of extracellular proteins from spent suspension-culture medium showed enhanced secretion of new proteins by selected lines. A 36-kDa protein was immunodetected by a chitinase antiserum. This chitinase continued to express constitutively in differentiated somatic embryos and also in the intercellular fluids of plants regenerated from the selected lines. Somatic embryos from selected lines grew uninhibitedly in a medium containing 40% fungal culture filtrate, whereas non-selected (control) somatic embryos became necrotic and died within a few days. Plants regenerated from selected lines exhibited resistance to infection by E. ampelina in both greenhouse tests and detached leaf bioassays. Results suggest that embryogenic cells can be selected for resistance following in-vitro selection, resulting in resistant plants. Whether or not resistant cells pre-existed in the original embryogenic culture or were induced by the selection pressure could not be determined.