Microspore-derived embryogenesis in pepper (Capsicum annuum L.): subcellular rearrangements through development

Background information. In vitro-cultured microspores, after an appropriate stress treatment, can switch towards an embryogenic pathway. This process, known as microspore embryogenesis, is an important tool in plant breeding. Basic studies on this process in economically interesting crops, especially in recalcitrant plants, are very limited and the sequence of events is poorly understood. In situ studies are very convenient for an appropriate dissection of microspore embryogenesis, a process in which a mixture of different cell populations (induced and non-induced) develop asynchronically.Results. In the present study, the occurrence of defined subcellular rearrangements has been investigated during early microspore embryogenesis in pepper, an horticultural crop of agronomic interest, in relation to proliferation and differentiation events. Haploid plants of Capsicum annuum L. (var. Yolo Wonder B) have been regenerated from in vitro anther cultures by a heat treatment at 35 degrees C for 8 days. Morphogenesis of microspore-derived embryos has been analysed, at both light and electron microscopy levels, using low-temperature-processed, well-preserved specimens. The comparison with the normal gametophytic development revealed changes in cell organization after embryogenesis induction, and permitted the characterization of the time sequence of a set of structural events, not previously defined in pepper, related to the activation of proliferative activity and differentiation. These changes mainly affected the plastids, the vacuolar compartment, the cell wall and the nucleus. Further differentiation processes mimicked that of the zygotic development.Conclusions. The reported changes can be considered as markers of the microspore embryogenesis. They have increased the understanding of the mechanisms controlling the switch and progression of the microspore embryogenesis, which could help to improve its efficiency and to direct strategies, especially in agronomically interesting crops.     

Flower developmental stage and organ sensitivity of bell pepper (Capsicum annuum L.) to elevated temperature

High temperatures adversely affect crop productivity of several plant species including bell pepper (Capsicum annuum L. var. annuum). The objectives of this study were: (1) to determine whether flower ontogeny is adversely affected by high temperature during different phases of development, including pre‐ and post‐pollination events; (2) to determine the duration of high temperature exposure necessary to cause reduction in fruit set; and (3) to determine whether injury to the pistil or stamen during development is responsible for reduced fruit set during high temperature. We determined that flower buds at <2·5 mm in length, corresponding to microspore mother cell meiosis to tetrad dissolution, and flowers that reached anthesis during the high temperature exposure had reduced fruit set when exposed to 33 °C for 48 or 120 h. Flower buds at <2·5 mm in length also had reduced pollen viability when exposed to 33 °C for 120 h. Morphological examination demonstrated that meiocytes initiated tetrad formation, but after dissolution the microspores remained small and clumped without a thick exine. High temperature exposure at a late‐development, pre‐anthesis stage did not affect pistil or stamen viability, but high post‐pollination temperatures inhibited fruit set, suggesting that fertilization is sensitive to high temperature stress.     

Differences on the microsporogenesis and tapetal development of male fertile and cytoplasmic male sterile pepper (Capsicum annuum L.)

To clarify the time and cause of pollen abortion, differences on the microsporogenesis and tapetum development in the anthers of male fertile maintainer line and cytoplasmic male sterile (CMS) line pepper were studied using transmission electron microscopy. The results showed that CMS line anthers appeared to have much greater variability in developmental pattern than male fertile maintainer line ones. The earliest deviation from normal anther development occurred in CMS line anthers at prophase I was cytomixis in some microspore mother cells (MMCs), and vacuolisation in tapetal cells. Then, MMCs in CMS line anthers developed asynchronously and a small part of ones at the different stage degenerated in advance appearing to have typical morphological features of programmed cell death (PCD). Most MMCs could complete the meiosis, but formed non-tetrahedral tetrad microspores with irregular shape and different size and uncertain number of nuclei, and some degenerated ahead of time as well. Tapetal cells in CMS line anther degenerated during meiosis, and were crushed at the tetrad stage, which paralleled the collapse of pollens. Pollen abortion in CMS line anthers happened by PCD themselves, and the premature PCD of tapetal cells were closely associated with male sterility.     

辣椒胞质雄性不育系和保持系内源激素含量的比较

以2个辣椒品系(199807、199803)的胞质雄性不育系和相应保持系为实验材料,采用酶联免疫吸附法(ELISA)测定IAA、(Z ZR)、GA3和ABA等内源激素含量,用气相色谱分析仪测定乙烯(ETH)释放量,对辣椒胞质雄性不育系和相应保持系内源激素含量变化规律进行研究.实验结果表明:在四分小孢子之前,花药中的IAA含量不育系显著高于保持系,在四分小孢子时期花药和花蕾中的IAA含量出现转折,到花粉粒成熟期的花蕾和花药以及开花期叶片中的IAA含量均是不育系显著低于保持系;小孢子各发育时期花药以及花期叶片中GA3含量均是不育系高于保持系,但花粉粒成熟期化蕾中GA3含量为不育系低于保持系;小孢子不同发育时期的花药以及花期叶片中ABA含量始终足不育系显著高于保持系,而花粉粒成熟期花蕾中ABA含量不育系与保持系没有显著差异;花粉粒成熟期的花蕾和花期叶片中ETH释放量表现为不育系显著高于保持系.同时,花粉粒成熟期的花蕾、花药和叶片中IAA/ABA、(Z ZR)/ABA、GA3/ABA、IAA/GA3、(Z ZR)/GA3等5个激素的比值均有不育系低于保持系的趋势.本实验结果说明辣椒的育性表现与花器和叶片等组织中内源激素的含量变化有关,花药和花期叶片中IAA亏缺、GA3和ABA增加以及化蕾和叶片中ETH过度产生,都有可能导致辣椒雄性不育.     

甜椒胞质雄性不育雄配子发育的解剖学和超微结构研究

运用石蜡切片和电子显微镜超薄切片方法观察了甜椒(Capsicum annuum L．)细胞质雄性不育系8A和保持系8B雄配子发育过程。结果表明：不育系和保持系都能正常进行减数分裂,绒毡层细胞无明显差异,形成了正常的四分孢子。在四分体单核居中期后,不育系的绒毡层细胞异常膨大并伸进药室,挤压花粉粒,同时绒毡层细胞提前降解,不育系单核晚期花粉粒开始崩出内含物。致使不育系的雄配子在双核花粉粒形成之前就完全裂解,不能发育成正常的花粉粒。此外,超薄切片还观察到不育系花粉粒在单核早期绒毡层细胞线粒体空泡化,这种变化表明雄性不育的遗传缺陷包括在花药发育早期发生的线粒体结构变化。     

The effect of high temperature and high atmospheric CO2 on carbohydrate changes in bell pepper (Capsicum annuum) pollen in relation to its germination

Pollen viability and germination are known to be sensitive to high temperature (HT). However, the mode by which high temperature impairs pollen functioning is not yet clear. In the present study, we investigated the effect of high temperature on changes occurring in carbohydrate of bell pepper (Capsicum annuum L. cv. Mazurka) pollen in order to find possible relations between these changes and pollen germination under heat stress. When pepper plants were maintained under a moderate HT regime (32/26 degrees C, day/night) for 8 days before flowers have reached anthesis, pollen count at anthesis was similar to that found in plants grown under normal temperatures (NT 28/22 degrees C). However, the in vitro germination, carried out at 25 degrees C, of pollen from HT plants was greatly reduced. This effect matched the marked reduction in the number of seeds per fruit in the HT plants. Maintaining the plants at high air CO2 concentration (800 &mgr;mol mol-1 air) in both temperature treatments did not affect the in vitro germination of pollen from NT plants, but restored germination to near the normal level in pollen from HT plants. Under NT conditions, starch, which was negligible in pollen at meiosis (8 days before anthesis, A-8) started to accumulate at A-4 and continued to accumulate until A-2. From that stage until anthesis, starch was rapidly degraded. On the other hand, sucrose concentration rose from stage A-4 until anthesis. Acid invertase (EC 3.2.1.26) activity rose parallel with the increase of sucrose. In pollen from HT plants, sucrose and starch concentrations were significantly higher at A-1 pollen than in that of NT plants. Under high CO2 conditions, the sucrose concentration in the pollen of HT plants was reduced to levels similar to those in NT pollen. In accordance with the higher sucrose concentration in HT pollen, the acid invertase activity in these pollen grains was lower than in NT pollen. The results suggest that the higher concentrations of sucrose and starch in the pollen grains of HT plants may result from reduction in their metabolism under heat stress. Elevated CO2 concentration, presumably by increasing assimilate availability to the pollen grain, may alleviate the inhibition of sucrose and starch metabolism, thereby increasing their utilization for pollen germination under the HT stress. Acid invertase may have a regulatory role in this system.     

Graft-induced genetic changes and the inheritance of several characteristics in pepper (Capsicum annuum L.)

 The general characteristics of several graft-induced changes in pepper were investigated in a cross experiment. F₁, F₂, and BC₁ progenies derived from crosses of the original stock and scion cultivars ‘Spanish Paprika’ and ‘Yatsubusa’, respectively, as well as their graft-induced variant strain G₅S₂₅ were analyzed for inheritance of the most conspicuous graft-induced variant traits. As part of a research program with the aim of revealing the mechanism of graft induction, the present study was carried out to examine the stability of the phenotypic changes and the characteristics of the graft-induced variants. For the fruit apex, a two-gene system was suggested, with other factors having a modifying influence. One of the two apex genes acted for pointed fruits and the other for inverted-blunt fruits. The inverted-blunt gene, the apex gene of the stock, was unambiguously present in the graft-induced variants, while the pointed gene that acted in the dominant mode in the original scion was inactive and expressed only under certain conditions in a mosaic state. The stable inverted-blunt cultivar used for the stock maintained certain factor(s) for pointed fruit, but the presence of that factor(s) could not be detected in graft-induced variants. The results of pungency analysis suggested a gene for non-pungency that appeared to be introduced in the graft-induced variants. The fruiting habit and fruiting direction that appeared in a mosaic state in graft-induced variants were found to combine factors of the stock with the appropriate characteristics of the scion asymmetrically. The bushy plant type appeared in a transgressive state in the variants, showing a definitely higher number of branches on the main stem and more frequent ramifications on the complete plant than on either the stock and scion cultivars or the progency derived from sexual crosses. A change in mature fruit color from red to yellow occurred in an early generation of graft-induced variants. Our results demonstrate that some of the characteristics of the stock were introduced into the progeny obtained from selfed seeds of the scion and that novel characteristics appeared as a result of graft induction. Received: 30 December 1997 / Accepted: 3 March 1998     

Spectrum of resistance to root-knot nematodes and inheritance of heat-stable resistance in in pepper (Capsicum annuum L.)

Capsicum annuum L. has resistance to root-knot nematodes (RKN) (Meloidogyne spp.), severe polyphagous pests that occur world-wide. Several single dominant genes confer this resistance. Some are highly specific, whereas others are effective against a wide range of species. The spectrum of resistance to eight clonal RKN populations of the major Meloidogyne species, M. arenaria (2 populations), M. incognita (2 populations), M. javanica (1 population), and M. hapla (3 populations) was studied using eight lines of Capsicum annuum. Host susceptibility was determined by counting the egg masses (EM) on the roots. Plants were classified into resistant (R; EM ≤ 5) or susceptible (H; EM >5) classes. The french cultivar Doux Long des Landes was susceptible to all nematodes tested. The other seven pepper lines were highly resistant to M. arenaria, M. javanica and one population of M. hapla. Variability in resistance was observed for the other two populations of M. hapla. Only lines PM687, PM217, Criollo de Morelos 334 and Yolo NR were resistant to M. incognita. To investigate the genetic basis of resistance in the highly resistant line PM687, the resistance of two progenies was tested with the two populations of M. incognita: 118 doubled-haploid (DH) lines obtained by androgenesis from F₁ hybrids of the cross between PM687 and the susceptible cultivar Yolo Wonder, and 163 F₂ progenies. For both nematodes populations, the segregation patterns 69 R / 49 S for DH lines and 163 R / 45 S for F₂ progenies were obtained at 22°C and at high temperatures (32°C and 42°C). The presence of a single dominant gene that totally prevented multiplication of M. incognita was thus confirmed and its stability at high temperature was demonstrated. This study confirmed the value of C. annuum as a source of complete spectrum resistance to the major RKN. Received: 2 July 1998 / Accepted: 11 March 1999     

高温干旱对不同品种辣椒生长及呼吸作用的影响

研究了高温干旱胁迫对不同品种辣椒生长及呼吸作用的影响。结果表明,高温胁迫对正椒13号的生长无影响,对鸡爪×吉林的生长稍有抑制;干旱及高温干旱严重地抑制了辣椒的生长。干旱导致叶片相对电导率的增加高于高温,而高温加剧了干旱伤害程度,并且对鸡爪×吉林的伤害更大。高温处理引起了辣椒总呼吸、细胞色素呼吸和交替呼吸的增加。干旱胁迫抑制了细胞色素呼吸,但诱导了交替呼吸的增强;高温干旱共同胁迫加剧了总呼吸和细胞色素呼吸的下降,交替呼吸只在胁迫第一天被促进,随后立即下降。高温、干旱和高温干旱胁迫下正椒13号表现出了较鸡爪×吉林更强的交替呼吸和总呼吸。说明高温、干旱和高温干旱胁迫下辣椒保持较高的总呼吸和交替呼吸与其抗高温和/或干旱能力相关。     

聚乙二醇、赤霉素、磷酸氢二钾和低温处理种子后的甜椒幼苗生长

聚乙二醇(PEG)、KH2PO4、赤霉素(GA3)和低温处理可以不同程度上提高甜椒二叶一心、四叶一心和六叶一心期的壮苗指数、根系活力、根系吸收面积和叶绿素含量,改善甜椒幼苗的生长;甜椒壮苗指数、根系活力和根系活跃吸收面积与种子活力指数相关性显著,种子活力指数与甜椒苗龄的相关性大小依次为:二叶一心＞六叶一心＞四叶一心期.     

Isolation and characterization ofo-diphenol-O-methyltransferase cDNA clone in hot pepper (Capsicum annuum L.)

A cDNA clone,CaOMTl encoding ano-diphenol-O-methyltransferase (OMT), which is involved in capsaicin biosynthesis, was isolated by screening of a cDNA library prepared from the mRNA of pepper (Capsicum annuum L.) pericarp. Nucleotide sequence analysis ofCaOMTl revealed that it had an open reading frame of 1080 bp which encodes a polypeptide with a predicted molecular weight of 39,430 D, corresponding well with the size of the known OMT’s of tobacco, poplar, aspen, alfalfa, and cabbage. It also had five conserved boxes which appear in all known OMT’s. The nucleotide sequence ofCaOMTl had 89–74% identity with the OMT cDNA’s of tobacco, aspen, alfalfa, and poplar, but a relatively lower identity of 59% with the OMT cDNA of maize. Amino acid sequence analysis also revealed that CaOMT1 has high identity with the known OMT’s which have a substrate ofo-diphenolic compounds, especially 5-hydroxyferulic acid and caffeic acid. It supportsCaOMTl which encodes an OMT. Southern blot analysis suggested thatCaOMTl might exist in the form of multiple copies in the pepper genome.CaOMTl is expressed preferentially in pepper fruit and its expression levels increased during pepper fruit development, but decreased during fruit ripening, suggesting that theCaOMTl gene is fruit development-related.CaOMTl is the first reported cDNA clone for enzymes related to the phenlypropanoid pathway in pepper.     

High-resolution genetic mapping of the pepper (Capsicum annuum L.) resistance loci Me3 and Me4 conferring heat-stable resistance to root-knot nematodes (Meloidogyne spp.)

The PM687 line of Capsicum annuum L. has a single dominant gene, Me ₃ , that confers heat-stable resistance to root-knot nematodes (RKN). Me ₃ was mapped using doubled-haploid (DH) lines and F₂ progeny from a cross between the susceptible cultivar ’Yolo Wonder’ (’YW’) and the highly resistant line ’PM687’. Bulked-segregant analysis with DNA pools, from susceptible or resistant DH lines, was performed to identify RAPD and AFLP markers linked to Me ₃ . There was no polymorphism between bulks of ten DH lines using over 800 RADP primers (4,000 amplified fragments analysed). Using 512 AFLP primers (74,000 amplified fragments analysed), and bulked DNA templates from 20 resistant and 20 susceptible plants, we identified eight repulsion-phase and four coupling-phase markers linked to Me _3. Analysed in 103 DH progeny, they defined a 56.1-cM interval containing the target gene. The nearest were located 0.5, 1.0, 1.5 and 3.0 centimorgans (cM) on both sides of the gene. Analysis of the F₂ progeny (162 plants) with the nearest coupling-phase marker confirmed its close position. Another resistance gene to RKN, present in ’PM687’ (Me ₄ ), was shown to be linked to Me ₃ , 10 cM from it. In order to localize Me ₃ and Me ₄ on our reference intraspecific pepper linkage map, two AFLP markers were mapped. The Me ₃ nearest marker was 10.1cM from a RAPD marker named Q04_0.3 and 2.7cM from a RFLP marker named CT135. We investigated map-position orthologies between Me ₃ and two other nematode resistance genes, the tomato Mi-3 and the potato Gpa ₂ genes, which mapped in the telomeric region of the short arm of the tomato and potato chromosome 12 (or XII for potato). Received: 23 March 2000 / Accepted: 2 January 2001     

Flower and nectar attributes of pepper (Capsicum annuum L.) plants in relation to their attractiveness to honeybees (Apis mellifera L.)

Flower morphology, nectary structure, nectar traits and rates of honeybee foraging on pepper plants were studied. The nectary appears as swellings on the basal part of the ovary. The nectariferous cells are smaller and denser than the neighbouring parenchyma. Stomata are present in the nectary epidermis, but do not appear on the other parts of the ovary epidermis. Seven pepper breeding lines were grown near a bee yard in Rehovot. Five to six fold differences in nectar volume were found between the extreme genotypes. Nectar volumes were higher during noon and afternoon hours, as compared with morning hours. High correlation coefficients between nectar volume and sugar concentration were found. These were significant for the four high nectar yielding genotypes, ranging between r = 0.65 to r = 0.94. Male-fertile flowers produced more nectar and higher sugar concentration than sterile ones. Skewed distribution was observed in nectar volume of F₂ populations, but relatively low heritability values were calculated. Pepper nectar contains fructose and glucose only. The former occupies 52–82 % of the total sugar content. Pepper genotypes varied in frequency of honeybee visits and significant correlation between sugar quantity and number of honeybee visits per flower was evident. Fertile pepper flowers are not very attractive to honeybees and male-sterile flowers are even less so. The considerable variation in nectar characteristics can be exploited to increase attractiveness to honeybees, thus facilitating bee pollination in commercial production of F₁hybrid seeds and improve fruit quality.     

Response of in vitro pollen germination and pollen tube growth of groundnut (Arachis hypogaea L.) genotypes to temperature

Air temperatures of greater than 35 °C are frequently encountered in groundnut‐growing regions, especially in the semi‐arid tropics. Such extreme temperatures are likely to increase in frequency under future predicted climates. High air temperatures result in failure of peg and pod set due to lower pollen viability. The response of pollen germination and pollen tube growth to temperature was quantified in order to identify differences in pollen tolerance to temperature among 21 groundnut genotypes. Plants were grown from sowing to harvest in a poly‐tunnel under an optimum temperature of 28/22 °C (day/night). Pollen was collected at anther dehiscence and was exposed to temperatures from 10° to 47·5 °C at 2·5 °C intervals. The results showed that a modified bilinear model most accurately described the response to temperature of percentage pollen germination and maximum pollen tube length. Genotypes were found to range from most tolerant to most susceptible based on both pollen characters and membrane thermostability. Mean cardinal temperatures (T_min, T_opt and T_max) averaged over 21 genotypes were 14·1, 30·1 and 43·0 °C for percentage pollen germination and 14·6, 34·4 and 43·4 °C for maximum pollen tube length. The genotypes 55‐437, ICG 1236, TMV 2 and ICGS 11 can be grouped as tolerant to high temperature and genotypes Kadiri 3, ICGV 92116 and ICGV 92118 as susceptible genotypes, based on the cardinal temperatures. The principal component analysis identified maximum percentage pollen germination and pollen tube length of the genotypes, and T_max for the two processes as the most important pollen parameters in describing a genotypic tolerance to high temperature. The T_min and T_opt for pollen germination and tube growth, rate of pollen tube growth were less predictive in discriminating genotypes for high temperature tolerance. Genotypic differences in heat tolerance‐based on pollen response were poorly related (R² = 0·334, P = 0·006) to relative injury as determined by membrane thermostability.     

高温胁迫下苋菜的叶绿素荧光特性

为了探明高温胁迫对苋菜(Amaranthus tricolor L.)光合过程的影响,用不同温度(25、30、35、40、45℃)处理苋菜植株1h后,随即测定了其叶绿素荧光动力学参数和快速光响应曲线特征参数的变化.结果表明:40℃以上高温胁迫下,苋菜叶片的光系统Ⅱ(PSⅡ)潜在光化学效率(Fv/Fo)、最大光化学效率(Fv/Fm)下降;最大荧光(Fm)、光合电子传递速率(ETR)、PSⅡ实际光化学效率(Yield)、光化学淬灭系数(qP)也均有所下降;而初始荧光(F.)和非光化学淬灭系数(NPQ)在40℃以上高温胁迫下显著上升.叶绿素荧光快速光响应曲线测定结果表明,初始斜率α、最大相对电子传递速率ETRmax和半饱和光强Ik在40℃以上高温胁迫下有所下降.研究表明,40℃以上高温胁迫对苋菜的光能的吸收、转换、光合电子传递和强光耐受能力等均有一定的影响.     

Bacterial Stem Rot in Greenhouse Pepper (Capsicum annuum L.) in Sardinia (Italy): Occurrence of Erwinia carotovora subsp. carotovora

An unusual bacterial disease was observed in pepper plants during research carried out in greenhouses in central‐north Sardinia. The characteristics were: the presence of lesions and exudates on stems, soft rot of the pith, and a brownish‐black colour in the petioles and leaf‐veins. Only two isolates of 21 were pathogens. One was obtained from exudate present on the stem and the other from pith. Experimental infections revealed that the bacterial isolates were particularly aggressive in the stems and fruit of pepper and tomato. Biochemical, physiological and serological tests in conjunction with fatty acid profile analysis confirmed that they were Erwinia carotovora subsp. carotovora (Jones) Bergey et al. The product of 434 bp polymerase chain reaction (PCR) enabled a preliminary identification of isolates to be made. Restriction fragment length polymorphism (RFLP) analysis of amplification products showed that the isolates DPP 23_ef and DPP 24_m, strain type CFBP 2046 and DPP 28₁, isolated from pepper fruit, belonged to the RFLP group 12, whereas DPP 29, also isolated from pepper fruit, was included in RFLP group 1. Measures to prevent and control this recently introduced disease are suggested in the conclusion of this paper.     

Influence of temperature and salinity on the germination of Lotus creticus (L.) from the arid land of Tunisia

Effects of salinity, temperature and their interactions on the rate and final percentage of germination were evaluated for two populations (Msarref, Oued dkouk) of the invasive glycophyte Lotus creticus Linné, grown under arid environmental conditions of the Tunisia. Seeds that were not treated with NaCl germinated well in a wide range of temperatures. For both populations, maximum germination occurred in distilled water at 25°C and lowest germination for all salinities was at 35°C. Germination was substantially delayed and significantly reduced with an increase in NaCl to levels above 300 mm . Compared to the Oued dkouk population, final germination and germination rate of the Msarref population was completely inhibited at 300 mm NaCl. The interactive effect of temperature and NaCl concentration on final germination and germination rate was significant (P < 0.01), indicating that the germination response to salinity depended on temperature. The inhibition of Oued dkouk population seed germination at high salt concentration was mostly due to osmotic effects while ionic effects were noted at Msarref population. The germination behaviour of the Oued dkouk population would therefore imply adaptive mechanisms to saline environments, while in the Msarref population such mechanisms seem to be absent. Since seed germination is more sensitive to salinity stress than the growth of established plants, the greater tolerance to salinity of Oued dkouk population would be an adaptive feature of this population to saline environment.     

高温高湿条件下意大利黑麦草青贮技术研究

研究以水田冬闲期栽培的意大利黑麦草(Lolium multiflorum)为材料,结合常规的青贮方法进行了小规模控制实验,试验设计了隔水(将青贮过程中产生的渗出液排出)和不隔水,以及不同添加物的青贮处理.青贮结束后,对青贮黑麦草的品质和营养价值进行评定,探讨不同处理对黑麦草青贮的作用效果,以及南方高温高湿条件下青贮黑麦草的合理方法和最佳条件.结果发现,对黑麦草进行隔水添加乳酸的青贮处理从感观评定、pH值和营养成分的分析结果来看,都在一定程度上优于其他处理,由此可以认为隔水添加乳酸的处理相对实现了较稳定和较优质的青贮.