Novel genetic male sterility developed in (Capsicum annuum x C. chinense) x C. pubescens and induced by HNO2 showing Mendelian inheritance and aborted at telophase of microspore mother cell stage.

A novel genetic male sterile germplasm was developed by successively crossing of (C. annuum x C. chinense) x C. pubescens and by chemical mutagenesis in pepper. The sterile anthers showed morphological abnormalities, but pistils developed normally with fine pollination capability. We investigated fertility segregation through sib-crossing of the same strains and test crossing by male sterile plants with 6 advanced inbred lines. The results showed that male fertility in the pepper was dominant in the F1 generation and segregated at a rate of 3:1 in the F2 generation, suggesting that monogenic male sterility was recessive and conformed to Mendelian inheritance. Cyto-anatomy analysis revealed that microspore abortion of sterile anthers occurred during telophase in the microspore mother cell stage when tapetal cells showed excessive vacuolation, resulting in occupation of the loculi. The microspore mother cells self-destructed and autolyzed with the tapetum so that meiosis in pollen mother cells could not proceed past the tetrad stage.

Effect of ethyl methyl sulfonate concentration and different treatment conditions on germination and seedling growth of the cucumber cultivar Chinese long (9930).

We attempted to create a new germplasm of cucumber cultivar Chinese long (9930) using different doses of ethyl methyl sulfonate (EMS) to induce variability. We tested EMS concentration (0, 0.5, 1.0, 1.5, 2, 3% v/v) with post-treatment (0.1 M Na2S2O3 and water), EMS concentration (0, 0.5, 1.0, 1.5% v/v) over different treatment times (8, 16, 24 h), and EMS concentration (0, 0.5, 1.0, 1.5% v/v) with different treatment temperatures (20 and 28°C). In all experiments with increasing EMS concentration, germination percent, index, and rate were decreased. After addition of stop solution (0.1 M Na2S2O3), post-treatment mutated seeds showed higher germination (84.44%) and rate (37.5%) than seeds treated with water (80 and 34.07%, respectively), while the germination index was high in seeds treated with water. At 20°C, the germination index (4.13) and rate (56.25%) were affected to a greater extent than at 28°C (7.68 and 91.31%, respectively). Treatment times of 16 and 24 h showed similar results for germination percent and rate, while the germination index was decreased over time. There were significant differences in seedling height, fresh true leaf weight, seedling weight, and plant survival with increasing EMS concentration and time. Higher variations in the form of dwarf seedlings were recorded after treatment with 1.5% EMS for 24 h. Based on germination and morphological data, an EMS concentration of 1.5% for 24 h at 20°C and post-treatment with stop solution (0.1 M Na2S2O3) efficiently caused mutation.

Cloning and expression analysis of pepper chlorophyll catabolite reductase gene CaRCCR.

Opening the porphyrin macrocycle of pheophorbide a and forming the primary fluorescent chlorophyll catabolites are key steps in the chlorophyll catabolism pathway. These steps are catalyzed by pheophorbide a oxygenase and red chlorophyll catabolite reductase (RCCR). In this study, a novel RCCR gene, CaRCCR, was isolated from the pepper (Capsicum annuum L.). The full-length CaRCCR complementary DNA is comprised of 1173 bp, contains an open reading frame of 945 bp, and encodes a 314-amino acid protein. This deduced protein belongs to the ferredoxin-dependent bilin reductase family. Amino acid sequence alignment showed that CaRCCR shared a high homology to other higher plant RCCR proteins. CaRCCR expression, as determined by quantitative real-time polymerase chain reaction, was higher in the leaves than the roots, stems, flowers, and immature fruits. CaRCCR expression was almost constant during all phases of leaf development. It was upregulated by abscisic acid, methyl jasmonate, and salicylic acid. Moreover, CaRCCR was induced by high salinity and drought stress treatments; it was also slightly regulated by Phytophthora capsici. Taken together, these results suggest that CaRCCR is involved in defense responses to various stresses.

Kill curve analysis and response of first generation Capsicum annuum L. B12 cultivar to ethyl methane sulfonate.

Pepper seeds (Capsicum annuum L.) var. B12 were mutagenized by four presoaking treatments in ten concentrations of ethyl methane sulfonate (EMS) to determine the sensitivity of the first generation (M1) to mutagens. The spectrum of mutations and induced variability for various quantitative traits, including germination, percent plant height, injury occurrence, survival ratio, first three fruits weight, and number of seeds per first fruit, were observed in the M1 generation. Our results indicated that all of the test parameters decreased with increasing EMS concentration, except for seedling injury. There were significant differences in germination ratio, LD50, plant height, percent injury, and survival ratio among the tested presoaking treatment. The LD50 was 1% EMS in seeds that were not presoaked (T1) and seeds presoaked for 12 h before treating with EMS (T3). In contrast, the LD50 was 0.5% EMS in seeds presoaked for 6 h (T2) and seeds presoaked in water for 6 h then incubated at 28°C for 12 h before EMS treatment (T4). Five dwarf plants were observed in mutagenized seeds without presoaking as compared to control seeds (at the maturity stage of the control plant).

Cloning and expression analysis of heat-shock transcription factor gene CaHsfA2 from pepper (Capsicum annuum L.).

The heat-shock transcription factor (Hsf) gene CaHsfA2 (GenBank accession No. JX402923) was cloned from the Capsicum annuum thermotolerant line R9 by combining the techniques electron cloning and rapid amplification of cDNA ends. The gene, which is 1436 bp in length, had an open reading frame of 1089 bp that encoded 362 amino acids. There was an 831-bp intron between positions 321 and 322 of the cDNA. The deduced amino acid sequence of CaHsfA2 contained the conserved domains of Hsf, including DNA binding domain, adjacent domain with heptad hydrophobic repeats (A/B), activator motifs, nuclear localization signal, and nuclear export signal, and it had the highest E value of hypothesized annotation of HsfA2. CaHsfA2 had the nearest phylogenetic relationship with HsfA2 from Lycopersicon peruvianum and Mimulus guttatus, which was consistent with its botanical classification. After heat-shock treatment at 40°C for 2 h, the expression of CaHsfA2 was observed in different tissues of thermotolerant cultivar R9 and thermosensitive line B6; however, the expression levels of the CaHsfA2 gene were significantly different as follows: expression in B6 leaf > stem > flower > root, and expression in R9 flower > leaf > stem ≈ root.

Biological characteristics and mating type distribution of Phytophthora capsici from China.

Phytophthora capsici from seven provinces of China were investigated for their mating type, hyphal growth, zoospore production, and virulence. All of the morphological characteristics and the results of polymerase chain reaction confirmed that these isolates were indeed Phytophthora capsici. The test of mating type showed that the mating types of 19 representative isolates from China varied. The hyphal growth and the amount of zoospores produced from these isolates differed and there was no evident relationship between them, which indicated the existence of genetic diversity among the isolates in China. Also, the isolates that were more virulent on the pepper cultivars that we checked produced more zoospores than other isolates.

Genetic determinants of the defense response of resistant and susceptible pepper (Capsicum annuum) cultivars infected with Phytophthora capsici (Oomycetes; Pythiaceae).

Based on culture isolation and morphological observation blight-infected pepper plants in Shaanxi Province, China, we identified the pathogen causing pepper phytophthora blight as Phytophthora capsici. Varieties that differed in resistance (CM334, PBC602, and B27) were inoculated with this pathogen. The root activity of resistant CM334 variety was the highest while that of susceptible B27 variety was the lowest. Also, significant differences in the activity of POD, PAL, and ß-1,3-glucanase were found; there was a positive correlation between disease resistance and activity of these three enzymes. We inhibited mycelial growth and sporangia formation of P. capsici using crude ß-1,3-glucanase and PAL enzymes isolated from the resistant variety CM334 after it had been inoculated with P. capsici. These two enzymes had a synergistic effect on inhibition of P. capsici mycelial growth and sporangia formation. Expression of the defensive genes CaPO1, CaBGLU, CaBPR1, and CaRGA in the three varieties was higher in the leaves than in the roots. All three genes were upregulated in infected leaves and roots of the pepper plants, always expressing at higher levels in the resistant cultivar than in the susceptible cultivar, suggesting that the differences in resistance among the pepper genotypes involve differences in the timing and magnitude of the defense response.

Optimization of virus-induced gene silencing in pepper (Capsicum annuum L.).

Virus-induced gene silencing is currently a powerful tool for the study of gene function in plants. Here, we optimized the protocol for virus-induced gene silencing, and investigated factors that affect the efficiency of tobacco rattle virus-induced gene silencing in pepper plants. Consequently, an optimal protocol was obtained by the syringe-infiltration method in the leaves of pepper plants. The protocol involves 2-leaf stage plants, preparing the Agrobacterium inoculum at a final OD600 of 1.0 and then growing the inoculated plants at 22°C. Using this protocol, we achieved high efficiency in silencing CaPDS in different cultivars of pepper plants. We further used the CaPOD gene to illustrate the general reliability of this optimized protocol. Viral symptoms were observed on the leaves of inoculated plants of the Early Calwonder cultivar 25 days post-inoculation, indicating that this protocol can also be used to silence other genes in pepper plants. Real-time polymerase chain reaction analyses revealed that the expression levels of CaPDS and CaPOD were dramatically reduced in inoculated leaves compared to control plants. These results demonstrate that the optimized protocol can be applied to functional genomic studies in pepper to investigate genes involved in a wide range of biological processes.

Maintaining and restoring cytoplasmic male sterility systems in pepper (Capsicum annuum L.).

We studied the efficiency of maintaining and restoring cytoplasmic male sterility (CMS) systems in pepper (Capsicum annuum L.). An Rf-linked molecular marker was employed to analyze the interaction between 6 CMS lines (A), 5 maintainers (B), and 6 restorers (C). Sterility was maintained in the matings of lines 201A x 200B, 203A x 200B, 206A x 200B, 200A x 201B, 206A x 201B, 200A x 202B, 200A x 203B, 200A x 206B, and 201A x 206B. All 6 restorers restored the fertility of lines 200A, 202A, 203A, and 204A, except that 213C could not restore the fertility of lines 200A and 204A. However, the 6 restorers had diverse restoring abilities in individual CMS lines. The Rf-linked molecular marker was amplified by PCR in lines 207C, 208C, and 213C. This DNA marker was only found in the F1 hybrids M39, M14, M19, M25, M13, M20, and M22. We conclude that the restorers 208C and 207C can transmit the Rf gene or the Rf-linked marker to F1 hybrids.

The pollen wall and tapetum are altered in the cytoplasmic male sterile line RC7 of Chinese cabbage (Brassica campestris ssp pekinensis).

Cytoplasmic male sterile line RC(7) of Chinese cabbage produces mature anthers without pollen. To understand the mechanisms involved, we examined the ultrastructural changes during development of the microspores. Development of microspores was not affected at the early tetrad stage. During the ring-vacuolated period, some large vacuoles appeared in the tapetum cells, making them larger, extending to the anther sac center during the monocyte period. At the same time, the tapetum degenerated as the microspores aborted, resulting in pollen-deficient anthers. As a result, the locules collapsed and the anthers shriveled. The callose was degraded in the pollen walls; abnormal deposits of electrodense material gave rise to irregular spike-shaped structures, rather than the characteristic rod-like shape of the B7 bacula. The internal intine wall of RC(7) was thinner than that of the B7 type. At the mitosis I microspore stage, the tapetum cells contained multiple plastids, with numerous small spherical plastoglobuli, and lipid bodies. Based on these observations, we suggest that RC(7) abortion may be due to mutated genes that normally regulate development of the pollen wall and cell walls in the RC(7) line.

Exogenous abscisic acid increases antioxidant enzymes and related gene expression in pepper (Capsicum annuum) leaves subjected to chilling stress.

To elucidate how physiological and biochemical mechanisms of chilling stress are regulated by abscisic acid (ABA) pretreatment, pepper variety (cv. 'P70') seedlings were pretreated with 0.57 mM ABA for 72 h and then subjected to chilling stress at 10°/6°C (day/night). Chilling stress caused severe necrotic lesions on the leaves and increased malondialdehyde and H(2)O(2) levels. Activities of monodehydroascorbate reductase (DHAR), dehydroascorbate reductase, glutathione reductase, guaiacol peroxidase, ascorbate peroxidase, ascorbate, and glutathione increased due to chilling stress during the 72 h, while superoxide dismutase and catalase activities decreased during 24 h, suggesting that chilling stress activates the AsA-GSH cycle under catalase deactivation in pepper leaves. ABA pretreatment induced significant increases in the above-mentioned enzyme activities and progressive decreases in ascorbate and glutathione levels. On the other hand, ABA-pretreated seedlings under chilling stress increased superoxide dismutase and guaiacol peroxidase activities and lowered concentrations of other antioxidants compared with untreated chilling-stressed plants. These seedlings showed concomitant decreases in foliage damage symptoms, and levels of malondialdehyde and H(2)O(2). Induction of Mn-SOD and POD was observed in chilling-stressed plants treated with ABA. The expression of DHAR1 and DHAR2 was altered by chilling stress, but it was higher in the presence than in the absence of ABA at 24 h. Overall, the results indicate that exogenous application of ABA increases tolerance of plants to chilling-induced oxidative damage, mainly by enhancing superoxide dismutase and guaiacol peroxidase activities and related gene expression.

Construction of the intermediate vector pVBG2307 by incorporating vital elements of expression vectors pBI121 and pBI221.

Molecular chaperones of plasmid pBI121 carrying CaMV35S promoter and a nucleotide sequence of plasmid pBI221 were inserted into plasmid pCAMBIA2300 to construct an intermediate vector: pVBG2307. This novel vector pVBG2307 contains a greatly expanded multiple cloning site with an adjacent imported CaMV35S promoter sequence. This vector allows controlled transformation of DNA in both Escherichia coli and Agrobacterium tumefaciens. Cloned PG, orf456, ipt genes and E8, a fruiting promoter, were amplified by PCR of cDNA libraries of Capsicum annum and Lycopersicon esculentum and were then transferred into vector pVBG2307. The viability of this vector was demonstrated, as it regulated PG, orf456, ipt and E8 genes in E. coli and could be transferred into Agrobacterium strain EHA105-4.

Activity and expression of polygalacturonase vary at different fruit ripening stages of sweet pepper cultivars.

Activity and expression of polygalacturonase (PG), a hydrolytic enzyme involved in ultrastructural changes in the pericarp of sweet pepper (Capsicum annaum), were investigated at different ripening stages of the pepper cultivars Mandi and Talanduo. Molecular cloning of CaPG was carried out by constructing a cDNA library from three stages of fruit ripening. Morphological determination, PG assay, RT-PCR, and ultrastructural studies were used to quantify changes in CaPG gene expression in the pericarp from green, color change and fully ripened stages. We found that CaPG gene expression, PG activity and striking changes in the structure of the cell wall occurred with the transition of ripening stages. CaPG gene expression was high (obvious PCR products) in mature and ripened stages of both cultivars; however, the CaPG gene was not expressed in preclimacteric fruits or vegetative tissues. We conclude that developmental regulation of CaPG gene expression is instrumental for sweet pepper fruit ripening; its expression during development leads to dissolution of middle lamella and eventually disruption of the fully ripened cell wall.