甜椒的离体培养及建立再生体系研究

以荷兰进口甜椒的子叶和下胚轴为外植体,接种到附加不同植物生长调节剂的培养基上,筛选出MS+6-BA 4.0 mg/L+NAA 0.1 mg/L为子叶和下胚轴最佳不定芽分化培养基;MS+IBA 0.2 mg/L和MS+IBA 0.5 mg/L为最佳不定根分化培养基。     

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.     

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.     

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.     

Light-dark regulation of carotenoid biosynthesis in pepper (Capsicum annuum) leaves

The carotenoid content in photosynthetic plant tissue reflects a steady state value resulting from permanent biosynthesis and concurrent photo-oxidation. The contributions of both reactions were determined in illuminated pepper leaves. The amount of carotenoids provided by biosynthesis were quantified by the accumulation of the colourless carotenoid phytoene in the presence of the inhibitor norflurazon. When applied, substantial amounts of this rather photo-stable intermediate were formed in the light. However, carotenoid biosynthesis was completely stalled in darkness. This switch off in the absence of light is related to the presence of very low messenger levels of the phytoene synthase gene, psy and the phytoene desaturase gene, pds. Other carotenogenic genes, such as zds, ptox and Icy-b also were shown to be down-regulated to some extent. By comparison of the carotenoid concentration before and after transfer of plants to increasing light intensities and accounting for the contribution of biosynthesis, the rate of photo-oxidation was estimated for pepper leaves. It could be demonstrated that light-independent degradation or conversion of carotenoids e.g. to abscisic acid is a minor process.     

Carbohydrate metabolism during fruit development in sweet pepper (Capsicum annuum) plants

Growth, accumulation of sugars and starch, and the activity of enzymes involved in sucrose mobilization were determined throughout the development of sweet pepper fruits. Fruit development was roughly divided into three phases: (1) an initial phase with high relative growth rate and hexose accumulation, (2) a phase with declining growth rate and accumulation of sucrose and starch, and (3) a ripening phase with no further fresh weight increase and with accumulation of hexoses, while sucrose and starch were degraded. Acid and neutral invertase (EC 3.2.1.26) were closely correlated to relative growth rate until ripening and inversly correlated to the accumulation of sucrose. Acid invertase specifically increased during ripening, concurrently with the accumulation of hexoses. Sucrose synthase (EC 2.4.1.13) showed little correlation to fruit development, and in periods of rapid growth the activity of sucrose synthase was low compared to the invertases. However, during late fruit growth sucose synthase was more active than the invertases. We conclude that invertase activities determine the accumulation of assimilates in the very young fruits, and a reactivation of acid invertase is responsible for the accumulation of hexoses during ripening. During late fruit growth, before ripening, sucrose synthase is transiently responsible for the sucrose breakdown in the fruit tissue. Results also indicate that pyrophosphate-dependent phosphofructokinase (EC 2.7.1.90) and its activator fructose-2,6-bisphosphate (Fru2,6bisP) are involved in the regulation of the sink metabolism of the fruit tissue.     

根系温度对甜椒生理特性的影响

甜椒苗期的最适根温为２５－３０℃。根温不适宜时,叶片生长明显减慢,光合速率下降,以致干物质积累减少。根温低的甜椒气孔呈前反馈式反应,根温高的气孔则呈反馈式反应,部分关闭和导度降低,胞间ＣＯ２分压减小,净光合速率降低。     

Development and karyology of the tapetal layer of anthers in sweet pepper (Capsicum annuum L.)

The tapetal layer of anthers inCapsicum annuum L. is differentiated from the archesporial complex during the early development stage of the anthers. Further development of tapetum proceeds according to the scheme of the cellular polynuclear type. The high rate of polyploidy is characteristic of the whole layer (from 4n to 8n). Cytokinesis does not follow karyokinesis which conditions the inception of two or three-nuclear cells. The majority of the mononuclear cells show the typical plant endomitosis without the chromosome spiralization, accompanied by the structural nuclear change-over. In other cells chromosome spiralization in the prophase and, after passing through the metaphase, restitution of the cell nuclei were observed. The tapetum development has been studied in 12 cultivars of sweet pepper which did not differ in the course of the karyological processes. The subtle differences during the temporal course of degeneration of the whole layer in the postmeiotic period of development were observed.     

Tapetum Dimorphism and Its Histochemical Study in Sweet Pepper (Capsicum annuum L. Var. grossum)

In sweet pepper, the portion of tapetum toward the interior of the anther comprising large cells is derived from cells of connective of anther whereas the remaining tapetum on the outside of the anther comprising comparatively small cells is derived from the parietal layer. Those ceils of the former prosessing large vacuoles and large nuclei are stained weaker than the cells of the latter by methyl green-pyronin and mercuric-bromophenol blue staining. Large spherical grains which contain acid phosphatase appear in the vacuoles in both kinds of tapetum at sporogenesis stage. During meiosis of pollen mother cells, DNA, RNA and protein sysntheses increase in tapetum. The tapetum derived from connective accumulates more DNA than that derived from parietal layer. The activity of acid phosphatase becomes higher in tapetum when it degenerates. The degeneration of two kinds of tapetum is similar. There are no starch grains in tapetum through its whole course of development.     

In Vivo and In Vitro Formation of Dihydrocapsaicin in Sweet Pepper Fruits,Capsicum annuum L. var. grossum

Dihydrocapsaicin, one of pungent principles in Capsicum fruits, was formed and accumulated in sweet pepper fruits after 6 days’ post-harvest ripening under continuous light in a medium containing vanillylamine and isocapric acid. No capsaicinoids were formed in sweet pepper fruits ripened in the dark even in the presence of both vanillylamine and isocapric acid. The capsaicinoid newly formed during the ripening was almost exclusively dihydrocapsaicin, as much as 92.8% of the total capsaicinoids. Dihydrocapsaicin was also formed by cell-free extracts prepared from the sweet pepper fruits in a reaction mixture containing vanillylamine and isocapric acid. Dihydrocapsaicin formed was quantified by TLC, GLC, GC-MS and MF.     

Effects of aluminum on growth and structure of red pepper (Capsicum annuum L.) leaves

Four-day-old seedlings of red pepper (Capsicum annuum L.) cv. “Trapez” were grown in nutrient solution containing 0, 10, 20 and 40 mg dm^?3 of AlCl₃·6H₂O for 14 days. Observations of leaves were carried out by light microscopy, as well as transmission and scanning electron microscopies. Exposure to aluminum resulted in reduction in the size and thickness of the leaf blades related to a decrease in the size of cells composing the leaves, as well as an increase in the number of stomata in the abaxial epidermis, with a simultaneous reduction of their size. The outer cell wall of the epidermis of the leaves was marked by a significant thickening in the presence of aluminum. The mesophyll cells contained enlarged chloroplasts having a disturbed structure of the lamellar system, filled with large starch grains. Rounded mitochondria were characterised by the electron lighter matrix and the destruction of the mitochondrial cristae. In the vacuoles of the parenchyma cells, as well as in the epidermis of the leaves, dark electron-dense bodies, presumably aluminum deposits, were observed.     

Ferredoxin from sweet pepper (Capsicum annuum L.) intensifying harpinpss-mediated hypersensitive response shows an enhanced production of active oxygen species (AOS)

The hypersensitive response (HR) is a form of cell death associated with plant resistance to pathogen infection. Harpin_pss, an elicitor from the bacterium Pseudomonas syringae pv. syringae, induces a HR in non-host plants. Previously, we reported an amphipathic protein from sweet pepper interfering with harpin_pss-mediated HR. In this report, we isolated and characterized a cDNA clone encoded that amphipathic protein from sweet pepper. This protein is designated as PFLP (plant ferredoxin-like protein) by virtue of its high homology with plant ferredoxin protein containing an N-terminal signal peptide responsible for chloroplast targeting and a putative 2Fe-2S domain responsible for redox activity. Recombinant PFLP obtained from Escherichia coliwas able to significantly increase active oxygen species (AOS) generation when mixed with harpin_pss in tobacco suspension cells. It also showed enhanced HR when co-infiltrated with harpin_pss in tobacco leaves. We used a transgenic tobacco suspension cells system that constitutively expresses the Pflpgene driven by the CaMV 35S promoter to study the function of PFLP in enhancing harpin_pss-mediated hypersensitive cell death in vivo. In response to harpin_pss, suspension cells derived from Pflptransgenic tobacco showed a significant increase both in the generation of AOS and in cell death as compared to the wild type. AOS inhibitors diphenylene iodonium chloride (DPI) and lanthanum chlorate (LaCl₃) were used to study the involvement of AOS in harpin_pss-induced cell death. Our results demonstrate enhanced generation of AOS is necessary to cause enhanced hypersensitive cell death in Pflp transgenic tobacco cells and it is plasma membrane-bound NADPH-oxidase-dependent. Sub-cellular localization studies showed that PFLP is present in the cytoplasm and chloroplast of Pflp transgenic tobacco cells, but only in the chloroplast, not in the cytoplasm, of wild-type tobacco cells. It is possible that PFLP can change the redox state of the cell upon harpin_pss inoculation to increase AOS generation and hypersensitive cell death. Overall, this study will provide a new insight in the functional properties of ferredoxin in hypersensitive cell death.     

DNA divergence as a criterion of a sweet pepper (Capsicum annuum L.) selection for heterosis

The identification of perspective parental lines for the creation of high-yield hybrids is the most labor-consuming stage of selection, because it needs extensive trials of combining ability. Based on evaluation of the genetic divergence of the parental lines, the prediction accuracy of F₁ hybrids performance was investigated. The value of the divergence was calculated using biometric and molecular methods, such as inter simple sequence repeats (ISSR) and random amplified polymorphic DNA (RAPD). Based on estimates of divergence, 10 lines were chosen for cyclic cross (scheme I) and testcross (scheme II). In most crosses, the F₁ hybrids were significantly superior to the parents in the main economically valuable traits. The level of heterosis was higher among hybrids of scheme I. Analysis of the relationship between parental divergence and F₁ performance showed that the hybrid productivity of scheme I was predetermined by ISSR divergence in 86%, and productivity was caused by RAPD divergence in 69%, whereas the F₁ yield of scheme II was not related to the value of genetic distances. Since the values of DNA divergence were closely associated both with midparent level and F₁ performance, we assumed that part of the polymorphic DNA fragments of the parental lines of scheme I is related to heterotic loci (HTL), which may be considered potential key markers for the heterotic selection of the sweet pepper.     

Blossom-end rot in relation to growth rate and calcium content in fruits of sweet pepper (Capsicum annuum L.)

The relative importance of growth rate and calcium concentration in sweet pepper fruits (Capsicum annuum L.) for the induction of blossom-end rot (BER) was investigated in (1) four pollination treatments in one cultivar, (2) four cultivars with the same fruit load and (3) three fruit load treatments in four cultivars. For fruits with the same pollination treatment those eventually developing BER had a higher initial fruit growth rate than those not developing BER. Within the same experiment both the growth rate of the young fruit and BER increased with the number of seeds. The Ca concentration of the pericarp in mature fruits was negatively related to both fruit size and BER incidence. Differences in levels of BER between different pollination experiments could not be explained solely by differences in growth rate of the young fruit, but related to different Ca concentrations in the mature fruits. In the spring, but not in the summer, cultivars more susceptible to BER had a larger final size but lower Ca concentration in the young fruit than the resistant ones. By lowering the fruit load in the summer both the final fruit size and the BER incidence increased, but the Ca concentrations of both proximal and distal pericarp in the young fruit of all cultivars were not consistently affected. Despite a correlation between growth rate and low Ca concentration in the fruit, the incidence of BER may only be predicted from separate effects of fruit growth and of Ca concentration of fruit. The data indicated that at a higher growth rate a higher Ca concentration is required to prevent the induction of BER. The usefulness of the total Ca concentration of the fruit for determining the critical Ca concentration in the induction of BER is discussed.Key words: Capiscum annuum L., sweet pepper, blossom-end rot, calcium, growth rate, pollination, fruit load.      

Development of pepper (Capsicum annuum) seed quality

Changes in seed quality in pepper (Capsicum annuum L.) were monitored during seed development and maturation in two seasons. Seed quality was assessed by a number of different tests, but principally by determining seed storage longevity in laboratory tests and seedling growth in glasshouse tests. Mass maturity (defined as the end of the seed-filling phase) occurred 49–53 days after anthesis (DAA) in 1989 (varying among fruit layers) and 53 DAA in 1990 when seed moisture contents were 51–53%. The onset of both germinability and desiccation tolerance occurred either just before or at mass maturity. Maximum potential longevity (assessed by the value of the seed lot constant K_i) was achieved 63–65 DAA, i.e. not until 10–12 days after mass maturity (DAMM), in both years. Seedling dry weights in the glasshouse growth tests were maximal later still - for seeds harvested 17–21 DAMM in 1989 and 17 DAMM in 1990; the effects on seedling weight arose from differences in times from sowing to emergence (P < 0.005) among different seed harvests, with no significant differences in subsequent relative growth rates (P > 0.25). Seed priming reduced mean germination times for seeds harvested at all stages of development, but had little effect on germination capacity and potential longevity, and did not affect the pattern of changes in potential longevity during seed development and maturation. The results contradict the hypothesis that seed quality is maximal at the end of the seed-filling phase and that viability and vigour begin to decline immediately thereafter.     

Plant regeneration in tissue cultures of pepper (Capsicum annuum L. cv. mathania)

Leaf, stem, hypocotyl, cotyledon, root, shoot tip and embryo explants of Capsicum annuum L. cv. mathania were cultured on Murashige and Skoog (MS) medium supplemented with 6-benzylaminopurine (BAP) or kinetin (Kin) alone or in combination with 3-indoleacetic acid (IAA), 3-indolebutyric acid (IBA), α-naphthaleneacetic acid (NAA) or 2,4-dichlorophenoxyacetic acid (2,4-D). BAP (5.0 mgl⁻¹) in the medium was found to be the best growth regulator for shoot bud differentiation. Shoot buds cultured on 5.0 mgl⁻¹ BAP increased in number but did not elongate. For obtaining complete plantlets, shoot buds were placed on a medium with IBA or NAA (0.1 mgl⁻¹). Histological evidence revealed direct differentiation of buds from cotyledons. Regenerated plants were normal diploids. Unorganized callus could not be induced to differentiate shoot buds.     

A case of early dissolution of the microsporocyte callose wall in male-sterile (CMS) sweet pepper (Capsicum annuum L.)

On squash preparations of anthers from pollen fertile and sterile plants of sweet pepper (Capsicum annuum L. cv. Severka) callose envelopes of microsporocytes, stained specifically with resorcin blue, were investigated microscopically. During normal course of microsporogenesis in fertile plants the envelopes remained intact up to the stage of microspore tetrads. Then callose begins to dissolve, and that from individual microspores towards the envelope periphery. In sterile analogues of the same cultivar the callose breakdown occurred precociously, usually in the course of the second, but sometimes as early as the first meiotic division of PMCs. Having completed meiosis sporadic microsporocytes formed microspore tetrads. Most PMCs contained an undivided four-nucleate protoplast rimmed with a narrow or wider unstained zone of dissolved callose. In certain cases more condensed callose septa pointing to the furrows on the surface of the PMC protoplast were well-observable in this lytic zone, as a residuum of normal mechanism of tetradogenesis.