EMS-induced lincomycin resistance in red pepper (Capsicum annuum L.)

Summary Ethyl methane sulphonate (EMS) is a potential mutagen to induce lincomycin resistance in Capsicum annuum. Mutagenized cotyledons were cultured on shoot regenerating medium containing lincomycin (100 mgl⁻¹). Approximately 14% of regenerated shoots were chlorophyll deficient and about 4% of regenerated shoots were green from mutaganized cotyledons. The regenerated green plants were resistant to lincomycin but sensitive to chloramphenicol, kanamycin, spectinomycin, and streptomycin. Reciprocal crosses were made between resistant and sensitive plants. Inheritance of lincomycin resistance was transmitted as a non-Mendelian trait. Lincomycin resistance is a first selectable and maternally inherited organelle encoded genetic marker described in chili pepper. Such mutants should be useful in designing biochemical selection schemes to recover somatic hybrids and cybrids.     

Proteome analysis of bell pepper (Capsicum annuum L.) chromoplasts

We report a comprehensive proteome analysis of chromoplasts from bell pepper (Capsicum annuum L.). The combination of a novel strategy for database-independent detection of proteins from tandem mass spectrometry (MS/MS) data with standard database searches allowed us to identify 151 proteins with a high level of confidence. These include several well-known plastid proteins but also novel proteins that were not previously reported from other plastid proteome studies. The majority of the identified proteins are active in plastid carbohydrate and amino acid metabolism. Among the most abundant individual proteins are capsanthin/capsorubin synthase and fibrillin, which are involved in the synthesis and storage of carotenoids that accumulate to high levels in chromoplasts. The relative abundances of the identified chromoplast proteins differ remarkably compared with their abundances in other plastid types, suggesting a chromoplast-specific metabolic network. Our results provide an overview of the major metabolic pathways active in chromoplasts and extend existing knowledge about prevalent metabolic activities of different plastid types.     

Cytomorphology of induced octoploid Chili pepper (Capsicum annuum L.)

Summary Octoploidy was induced in Chili pepper (Capsicum annuum cultivar cerasiformis) through the application of colchicine and the cytomorphological features of two octoploid plants were described. In general, the octoploids did not exhibit gigas characters when compared to the tetraploids; on the contrary they were less vigorous, suggesting that the optimum and desirable ploidy level for Capsicum is probably tetraploid. Chromosome associations such as octovalents and hexavalents, in addition to IVs, IIIs, IIs and Is, were recorded at diakinesis and metaphase I. Meiosis was highly irregular and the pollen and seed fertility was very low. Cytological features of octoploid Chili peppers are compared with octoploids of Physalis and Petunia.     

Exogenous reference RNA for normalization of real-time quantitative PCR

We have utilized an in vitro transcribed 3' mRNA fragment of the plant gene ribulose bisphosphate carboxylase (RuBisCO) as an exogenous standard for normalization of quantitative PCR data. Both K562 cells and primary erythroid CD34+ progenitor cells were treated with sodium butyrate and changes in gamma-globin mRNA levels were assayed using a previously published TaqMan probe and primer set, while RuBisCO levels were assayed by a SYBR Green detection assay. The data presented show that a correction to measured gamma-globin induction was necessary with both cell types. The correction for the CD34+ progenitor cells was a striking 95% increase, while that for the K562 cells was 44%. The use of an exogenous reference such as in vitro transcribed mRNA for the RuBisCO plant gene provides a robust and sample-independent method for the normalization of quantitative PCR data in bacterial and animal cells.     

Cytokinins and leaf development in sweet pepper (Capsicum annuum L.)

Stimulation of leaf expansion by an exogenous cytokinin was studied in isolated leaf discs of sweet pepper with emphasis on the assimilate utilization of the tissue. Leaf discs were floated on solutions containing sucrose and plant growth regulators. Benzyladenine (BA) promoted the area expansion rate of the leaf discs. Sucrose at 100 mM resulted in increased area expansion rate compared with 10 mM sucrose. However, the increased sucrose concentration had no influence on the effect of BA. Over a period of 24 h, treatment with BA did not result in any change of sucrose uptake nor of the partitioning of assimilated carbon in the leaf discs. Neither did BA treatment affect the activity of acid invertase (EC 3.2.1.26) or pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) in the leaf discs. We conclude that the observed promotion of leaf area expansion by exogenous BA is not mediated through the uptake of sucrose or the carbohydrate metabolism of the leaf tissue.Abbreviations BA N⁶-benzyladenine - GA₃ gibberellic acid - PPi-PFK pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) This study was supported by grants from the Danish Research Counsil (SJVF 13-4148 and 13-4547 to P.U. SJVF 13-4146 and 13-4494 to T.H.N.) and from The Research Center for Plant Biotechnology to P.U.     

Androgenesis induction in microspore culture of sweet pepper (Capsicum annuum L.)

Isolated microspore culture experiments were carried out in sweet pepper (Capsicum annuum L.) F₁ hybrid genotypes. In the first experiment, four culture media (W14, B5, MS and NLN) were compared to test their effectiveness in inducing the formation of microspore-derived structures in two genotypes. The experiments revealed the superiority of B5 medium. In the second experiment, the effects of different ratios of 2,4-dichlorophenoxyacetic acid (2,4-D) (0, 0.1, 0.2 and 0.5 mg l⁻¹) and kinetin (0, 0.2 and 0.5 mg l⁻¹) were also investigated in B5 medium with two genotypes. The effect of growth regulators were investigated on the production of microspore-derived calli and embryo-like structures (ELSs), the ratio of the two and plant regeneration (number of regenerated plantlets) in microspore culture. The histological experiments revealed the differences between the microspore-derived ELSs and calli. The most promising results were obtained on the investigated parameters in the presence of 0.1 mg l⁻¹ 2,4-D and 0.2 mg l⁻¹ kinetin producing the highest number of plantlets in both genotypes tested. In the response of 11 genotypes, the androgenesis induction was successful in each sweet pepper genotypes tested using the best basic medium and growth regulators combination. In case of 11 genotypes, the number of ELSs ranged from 20 to 100/Petri dish (an average of 48.1 ELS/Petri dish), while the number of green plantlets varied from 0 to 8 plantlets/Petri dish (an average of 1.5 plantlets/Petri dish) depending on the genotype. The spontaneous rediploidization rate obtained was 25% in isolated microspore.     

内参基因在昆虫实时荧光定量PCR中的研究进展

作为一种高效的定量PCR技术,实时荧光定量PCR(qRT-PCR)因其灵敏度高、特异性强、定量准确等优点,已被广泛运用于昆虫基因表达和转录分析。然而,为了控制样本RNA在质量和逆转录效率上存在差异,必须筛选表达稳定的"看家基因"作为内参基因,对目的基因表达量进行校正和标准化。许多学者研究表明,昆虫种类和实验条件的不同,导致选择的内参基因也不尽相同。因此,本文综述了前人有关昆虫内参基因的研究及其稳定性评价,为其它昆虫内参基因的研究提供理论参考依据。     

Plant regeneration from sweet pepper (Capsicum annuum L.) hypocotyl explants

Morphogenetic potential of hypocotyl and cotyledon explants of the three Polish Capsicum annuum L. cultivars (Kujawianka, Passat and Zorza) was studied to develop a reliable plant regeneration protocol. Out of 8 and 15 combinations of growth regulators used in the first and second series respectively, the best medium contained BAP (5 mg·l⁻¹) and IAA (1 mg·l⁻¹). Media containing thidiazuron (TDZ) and IAA proved to be worse than those with BAP and IAA. Additionally, it was indicated that hypocotyl explants placed upside-down developed more adventious buds. ‘Passat’ was the most responsive variety (approximately 40 % of both types of explants produced buds). In the second part of experiment the aim was to stimulate shoot induction in the conditions most adapted to Agrobacterium transformation. ‘Bryza’ replaced cv ‘Kujawianka’ and proved to be better than ‘Passat’, previously distinguished as a highly responsive cultivar. The experiments confirmed a significant effect of the hypocotyl explant length and induction period on shoot regeneration. Finally, the optimum concentration of carbenicillin and kanamycin was determined.     

Suppression of ethylene levels promotes morphogenesis in pepper (Capsicum annuum L.)

Ethylene and polyamines (PAs) are two phytohormones that play important roles during in vitro morphogenesis of several plant species. The interaction between ethylene and PAs has been of interest because both have S-adenosylmethionine as a precursor. To study the influence of ethylene and PAs on in vitro morphogenesis of an ornamental pepper, we added an ethylene scavenger, PAs, a PA inhibitor, and compounds that affect ethylene biosynthesis and activity to the regeneration medium. Regeneration frequencies increased in response to treatment with ethylene inhibitors (aminoethoxyvinylglycine and silver thiosulfate) and an ethylene scavenger (mercury perchlorate). Treatment with the ethylene precursor 1-aminocyclopropane-1-carboxylic acid reduced the regeneration frequency, increased callus formation, and increased ethylene levels; similar results were obtained in response to treatment with the PA inhibitor methylglyoxal-bis(guanylhydrazone). By contrast, treatment with PAs (particularly spermidine and spermine) decreased ethylene levels, increased the regeneration frequency, and increased shoot bud formation. These results suggest a coordinated regulation of ethylene and polyamines because the suppression of ethylene levels using ethylene inhibitors, polyamines, or mercury perchlorate increased the in vitro regeneration frequency and morphogenic responses of Capsicum annuum L.     

Establishment of a highly efficient transformation system for pepper (Capsicum annuum L.)

Application of modern genetic manipulation has been limited in pepper ( Capsicum annuum L.) due to the lack of an efficient transformation system. Following the development of an efficient protocol for in vitro regeneration of pepper cotyledons, we investigated the key factors affecting transformation and established a highly efficient genetic transformation system using the pepper cotyledon as starting material. In this system, cotyledon explants are preconditioned for 2 days on kanamycin (km)-free DM1 medium [Murashige and Skoog (MS) salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients and a hormone combination of 1.0 mg/l indoleacetic acid (IAA) and 5.0 mg/l 6-benzyladenine (BA) solidified with 0.7% agar, pH 5.8], followed by co-cultivation with Agrobacterium tumefaciens on DM1 for 2 days and delay selection on DM1 with 500 mg/l carbenicillin (carb) for 2 days. The explants are then placed on DM1 containing 10 mg/l AgNO(3), 50 mg/l km-sulfate and 500 mg/l carb. After 4-5 weeks, the explants with buds are transferred to EM1 medium (MS salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients, 10 mg/l AgNO(3) and a hormone combination of 1.0 mg/l IAA, 3.0 mg/l BA and 2.0 mg/l gibberellic acid, solidified with 0.7% agar, pH 5.8) with 50 mg/l kanamycin and 500 mg/l carbenicillin for the elongation of buds. After 3-6 weeks, 1- to 2-cm-long elongated shoots are excised and planted on RM1 medium (MS basal medium supplemented with a hormone combination of 0.2 mg/l NAA and 0.1 mg/l IAA, solidified with 0.8% agar, pH 5.8) with 25 mg/l km and 200 mg/l carb for rooting. We tested four genotypes of pepper, and all presented a high differentiation efficiency (81.3% on average), elongation rate (61.5%) and rooting efficiency (89.5%). Polymerase chain reaction analysis results showed that 40.8% of the regenerated plantlets were transgenic plants.     

Stable reference gene selection for quantitative real-time PCR normalization in passion fruit (Passiflora edulis Sims.)

Molecular Biology Reports - Passiflora edulis is a tropical fruit with high nutrient and medicinal values that is widely planted in southern China. However, the molecular biology of P. edulis has...     

Evaluation of appropriate reference genes for gene expression studies in pepper by quantitative real-time PCR

Quantitative real-time polymerase chain reaction (qRT-PCR) has been extensively used in several plant species as an accurate technique for gene expression analysis. However, the expression level of a target gene may be misconstrued due to unstable expression of the reference genes under different experimental conditions. Therefore, it is necessary to systematically evaluate these reference genes before experiments are conducted. Recently, more and more studies have focused on gene expression in pepper (Capsicum annuum L.). In this study, ten putative reference genes were chosen to identify expression stability by using geNorm and NormFinder statistical algorithms in ten different pepper sample pools, including those from different plant tissues (root, stem, leaf and flower) and from plants treated with hormones (salicylic acid and gibberellic acid) and abiotic stresses (cold, heat, salt and drought). EF1?? and UEP exhibited the most stable expression across all of the tested pepper samples. For abiotic stress or different hormone treatment, the ranking of candidate reference genes was not completely consistent, except for EF1?? which showed a relatively stable expression level. For different tissues, the expression of Actin1 was stable and it was considered an appropriate reference gene. It is concluded that EF1??, UEP and Actin1 are suitable reference genes for reliable qRT-PCR data normalization for the tissues and experimental conditions used in this experiment.     

Molecular characteristics and expression of cytoplasmic genes in cytoplasmic male sterility of pepper (Capsicum annuum L.)

This study compared the sequence variations and expressions of 12 chloroplastic and 8 mitochondrial genes in three pepper (Capsicum annuum L.) cytoplasmic male sterile (CMS) lines, their maintainers and two control cultivars. The results showed that the three CMS lines were highly similar in chloroplastic and mitochondrial fragment sequences, with average similarities of 96.81 and 98.73?%, respectively, and their chloroplastic trnH?CpsbA intergenic spacer, photosystem II 47?kDa protein (psbB) genes, mitochondrial apocytochrome b (cob) and RNAD fragments have 1, 9, 8 and 7 distinct sites from the maintainer lines, respectively, and could be used as informative sites to distinguish CMS lines from the maintainer lines. Meanwhile, the expressions of mitochondrial cob, RNAD and pvs in the reproductive organs (flowers) of CMS lines are different from those of the maintainer lines, but their expressions in the vegetative organs (roots and leaves) are similar. The results indicate that cytoplasmic DNA polymorphisms are rare in CMS lines, and mitochondrial cob, RNAD and pvs genes are closely related to pollen abortion.     

Increased capsaicin content in PFP-resistant cells of chili pepper (Capsicum annuum L.)

Cell suspensions of chili pepper (Capsicum annuum L.) were subjected to a selection process on semisolid medium containing the amino acid analog p-fluorophenylalanine (PFP). Four cell lines with different degrees of resistance were selected and suspension cultures were established from each of them. Resistance was retained even after 75 days of culture in the absence of PFP. PFP-resistant cell lines accumu lated higher levels of capsaicin than sensitive lines even after prolonged culture in PFP-free medium. Capsaicin production in non-selected cells was only 26.8% of that found in one cell line resistant to 500 M PFP. The capsaicin content in the non-selected cell suspension and in one of the resis tant cell lines was 6.7% and 24.9% respectively, that of fruits.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - PFP p-fluorophenylalanine - d. wt. dry weight - f. wt. fresh weight     

Alleviation of low-temperature photoinhibition in gamma-irradiated red pepper (Capsicum annuum L.) plants

We studied the radiation-induced stress resistance in red pepper leaves under conditions of low-temperature photoinhibition or artificially induced photo-oxidative stress. Plants irradiated with 4, 8, or 16-Gy gamma rays were more resistant to both stress factors than were the controls. However, exposure to a low temperature for 12 h with illumination or photo-oxidative treatment for 1 h differentially affected the irradiated leaves, although they had similar stress intensities as defined by their maximal photochemical efficiencies (Fv/Fm). Decreases in Fv/Fm induced by the two stress factors were instead alleviated, dose-dependently, by as much as 22 to 41% (low temperature) or 14 to 29% (photo-oxidation) in the irradiated groups. In contrast, non-photochemical quenching (NPQ) and the de-epoxidation state of xanthophyll cycle pigments could not be correlated with this enhanced stress resistance in the irradiated groups. These results suggest that the adaptive response of plants exposed to gamma radiation is more effective in protecting against low-temperature photoinhibition than against photo-oxidative stress. We also discuss here the involvement of antioxidative defense systems for increased resistance against low-temperature photoinhibition in irradiated red pepper.