Screening Capsicum species of different origins for high temperature tolerance by in vitro pollen germination and pollen tube length

Successful fruit set depends on several reproductive processes including pollen germination and tube growth processes. An experiment was conducted to determine the effects of temperature on pollen germination characteristics and to identify species/genotypic differences in Capsicum using the cumulative temperature response index (CTRI) concept. Pollen was collected from plants of seven genotypes from five Capsicum species, adapted to various parts of the world and grown outdoors in large pots. The pollen was subjected to in vitro temperatures ranging from 15 to 50 °C at 5 °C intervals. Pollen germination and tube lengths were recorded for all species after 24 h of incubation at the respective treatments. Species/genotypes differed significantly for in vitro pollen germination percentage and pollen tube length with mean values of 78% and 734 μm, respectively. The mean cardinal temperatures (T_min, T_opt, and T_max) averaged over genotypes, were 15.2, 30.7, and 41.8 °C for pollen germination and 12.2, 31.2, and 40.4 °C for pollen tube growth. The CTRI of each species/genotype calculated as the sum of eight relative individual stress response values, such as maximum pollen germination, maximum pollen tube length; T_min, T_opt, and T_max temperatures of pollen germination, and pollen tube lengths, identified species tolerance to high temperatures. Capsicum annum cv. Mex Serrano from Mexico was identified as tolerant, C. chacoense cv. 1312 and C. spp. cv. Cobanero from Argentina and Guatemala, respectively as intermediate and C. frutescens cv. Early Spring Giant from China, C. annum cv. Long Green from South Korea, C. spp. cv. NM89C130 and C. pubescens cv. 90002 from Guatemala as sensitive to high temperatures. The tolerant species/genotypes can be used in breeding programs to develop new genotypes that can withstand high temperature conditions both in the present climate and particularly in a future warmer climate.     

Pollen biology of ornamental ginger (Hedychium spp. J. Koenig)

An improved in vitro pollen germination assay was developed to assess the viability of stored Hedychium pollen. The effect of polyethylene glycol (PEG) (10, 15, and 20%, w/v) on pollen germination and tube growth was evaluated for Hedychium longicornutum and two commercial Hedychium cultivars, ‘Orange Brush’ and ‘Filigree’. Overall, the inclusion of PEG 4000 in the medium improved both pollen germination and tube growth for the three different genotypes tested and the results varied depending on genotype. In vitro germination was used to assess the viability of Hedychium pollen stored up to two months. Pollen nucleus status was determined for four Hedychium cultivars, ‘Orange Brush’, ‘Anne Bishop’, ‘Filigree’, and ‘Daniel Weeks’. Pollens of ‘Orange Brush’, ‘Anne Bishop’, and ‘Daniel Weeks’ were found to be binucleate but ‘Filigree’ was shown to possess both binucleate and trinucleate pollens. High pollen:ovule ratio values were obtained in several Hedychium taxa. The results obtained on the nuclear pollen status and pollen:ovule ratios will further our understanding of the pollination biology and help clarify the taxonomy and phylogeny of Hedychium species.     

In vitro pollen germination in avocado (Persea americana Mill.): Optimization of the method and effect of temperature

An improved in vitro pollen germination method was developed for avocado (Persea americana Mill.). The effect of different concentrations of sucrose, polyethylene glycol (PEG), Mg and Ca on pollen germination was evaluated in order to determine the optimal pollen germination medium, i.e. that maximizing the percentage of pollen germination and minimizing the percentage of bursted pollen grains. Once the germination medium was optimized we used it to study the effect of temperature on in vitro pollen germination and tube growth in different cultivars from the three botanical varieties of avocado, that differ in their adaptation to environmental conditions. Significant differences in percentage of pollen germination and in pollen tube growth were observed among cultivars. These results could have implications not only for optimizing pollen management in avocado but also to select the best pollinizers for a particular cultivar.     

Pollen germination as affected by pollen age in cherimoya

Cherimoya (Annona cherimola (Mill.)) is a subtropical fruit tree, which is cultivated in a good range of subtropical regions. In most of these areas the crop relies on hand pollination. However, following this practice, erratic fruit set is often produced, which could be related to problems in pollen handling. Indeed, very little is known of the time that the pollen remains viable and on which is the best stage to collect the anthers or pollen from the flower. The aim of this work is to evaluate pollen germinability prior and after anther dehiscence and also how the age of pollen affects pollen vigor, understood as speed of germination. Pollen samples at different times following anther dehiscence were germinated in vitro and in vivo. Pollen up to 90 min following dehiscence performed as well as freshly dehisced pollen. However, the pollen taken 120 min following dehiscence, showed a clear reduction in vigor and germinated much slower in vivo. To overcome this short pollen germinability, pollen was taken from anthers 30 and 5 h prior to natural anther dehiscence and compared with pollen taken at anther dehiscence and 20 h later. However, a reduction in germination rate was obtained in pollen taken prior to anther dehiscence. The narrow stage at which pollen can be collected together to its ephemeral germinability explains erratic results obtained following hand pollination in this crop and these results provide the clues for an adequate pollen handling.     

精胺对梨花粉原位萌发及花粉管生长的影响

为进一步研究活体条件下精胺对梨花粉萌发及花粉管生长的影响,以幸水、新雪、长-二十世纪为材料,采用荧光显微镜观测了不同浓度精胺处理的花粉萌发及花粉管生长情况。试验结果表明:较低浓度的精胺能促进花粉萌发,而超过一定浓度时则表现抑制作用,适宜于花粉萌发的精胺浓度是0-0.025mmol/L。精胺对花粉管生长的促进作用因品种而异,0-0.05mmol/L,精胺促进幸水、新雪花粉管生长的作用主要表现在花柱中上部;而到达基部的花粉管仍较少,与对照间差异不显著。在0-0.025mmol/L的适宜浓度下,精胺可促进长-二十世纪花粉管生长并到达花柱基部。不同浓度的精胺对幸水、新雪坐果影响不大;而适宜浓度的精胺可促进长-二十世纪坐果。     

硼、钙和农药对草莓花粉萌发和花粉管生长的影响

采用花粉液体培养法研究温度、硼、钙及农药对草莓花粉萌发和花粉管生长的影响。结果表明,(1)草莓花粉萌发和花粉管生长适宜的温度为25～30℃。(2)外源硼酸为草莓花粉萌发必需物质,适宜质量浓度为0.3g/L,低质量浓度促进花粉萌发和花粉管生长,而高于0.3g/L则起抑制作用。(3)外源钙为非必需物质,但适量的外源钙离子有利于花粉萌发,适宜质量浓度为0.3g/L,高钙抑制花粉萌发。(4)在培养液中分别加入常规浓度的7种农药,每处理几乎无花粉萌发,加入1/10常规体积分数的7种农药中,只有一遍净、速克灵、奥美特等3种农药处理有少量花粉萌发,说明农药对花粉萌发和花粉管生长有强烈抑制作用。     

Effect of temperature on pollen germination and pollen tube growth of four cultivars of mango (Mangifera indica L.)

Summary

The effect of a constant (10, 15, 20 or 25°C) or a diurnal maximum/minimum (15/5, 20/10, 25/15 or 30/20°C) incubation temperature on in vitro pollen germination and pollen tube growth in the pistils of two poly-embryonic (‘Kensington’ and ‘Nam Dok Mai’), and two mono-embryonic (‘Irwin’ and ‘Sensation’) mango cultivars was studied. In in vitro experiments where pollen was incubated in a liquid germination medium for 24 h in darkness, little difference was found between pollen germination of mono- and poly-embryonic cultivars. Averaged over the four cultivars, 53.9% of pollen germinated at 10°C, this increased to 76.2% when the incubation temperature was increased to 15°C, thereafter up to 25°C the percentage germination remained stable but germination decreased slightly to 68.2% at 30°C. Similarly, there was no difference in percent germination between cultivars when pollen was incubated under diurnal temperature regimes. Mean pollen germination of all four cultivars was 52.3% at 15/5°C and pollen germination increased by 10% when the temperature was raised to 30/20°C. When self-pollinated flowers were incubated for 24 h on a semi-solid agar medium at 10°C, pollen tube growth of the four cultivars was retarded and no pollen tubes reached the ovaries. As the temperature was increased from 15 to 25°C, the mean number of pollen tubes in ovaries increased from 0.04 to 0.25. At 30°C, the mean number of pollen tubes that entered ovaries decreased to 0.04. After incubation under diurnal temperature regimes, the mean number of pollen tubes in ovaries of all four cultivars at 15/5°C was 0.23 and increased to 0.42 when the temperature increased to 30/20°C. At each incubation temperature, there were significant differences in pollen tube growth between cultivars, but there were no differences between the temperature response of pollen from mono- and poly-embryonic cultivars.     

Chitosan specificity for the in vitro seed germination of two Dendrobium orchids (Asparagales: Orchidaceae)

The effects of different types of chitosan on seed germination and protocorm development were determined for two orchid species, Dendrobium bigibbum var. compactum and Dendrobium formosum. Six chitosan types derived from polymer or oligomer chitosan each with 70, 80 or 90% levels of deacetylation (P70, P80, P90, O70, O80 and O90, respectively), were evaluated as direct medium supplements at 0, 10, 20, 40 or 80 mg/L in modified VW medium by following seed germination and protocorm growth for 12 weeks. Chitosan of all six tested types and four concentrations were found to significantly enhance the proportion of D. formosum seeds that germinated, when compared to these germinated without chitosan. In contrast, chitosan caused no enhanced germination rate was noted for D. bigibbum var. compactum with all tested chitosans and doses tested. However, almost all types of chitosan at 10 mg/L, except O90, were able to significantly improve the growth of D. bigibbum var. compactum protocorms, whilst 10 or 20 mg/L of P70 chitosan was the best formula to enhance the growth of D. formosum protocorms. It is concluded that chitosan responses in seed germination and protocorm development were somewhat species and developmental stage dependent. Therefore, the appropriate chitosan application for each plant species should be evaluated first before use.     

Breaking the intergeneric crossing barrier in papaya using sucrose treatment

A breeding programme was undertaken using Carica papaya var. Surya and Vasconcellea cauliflora with a view to raise progenies resistant to ‘papaya ringspot virus’ (PRSV). Earlier studies have clearly demonstrated the cross incompatibility between these two genera. Hence, an attempt was made to break this barrier using sucrose. The pollen of V. cauliflora was collected and pollination was carried out by smearing sucrose solution in the concentrations of 1, 2, 3, 4 and 5% on to the stigmatic surface of the flower. At 5% sucrose concentration, maximum viable seed set (13.73) was obtained. Sucrose at 5% was observed to break the intergeneric barrier by enhancing the pollen germination. There was drastic reduction in the effect of sucrose with the decrease in the concentration levels. The pollen germination studies carried out with and without sucrose clearly demonstrated the efficacy of sucrose in enhancing pollen germination and pollen tube growth. The intervarietal hybridization carried out between the varieties Surya and Pusa Dwarf showed 91.7% set of the fruits with 300 viable seeds per fruit. The hybridity of the progenies was confirmed using ISSR primers by the amplification of DNA from progenies and parents. Four primers UBC 807, 810, 814 and 861 clearly amplified male specific bands, which were present in progenies, but absent in female parent.     

In vitro asymbiotic seed germination and seedling growth of Ansellia africana Lindl.

The effects of sodium hypochlorite treatment and growth medium on in vitro seed germination and seedling growth of the leopard orchid (Ansellia africana Lindl.) were investigated. Forty minutes treatment of seeds with 1.75% sodium hypochlorite stimulated germination (70.6%) and seedling development when measured at Stage 5 (emergence of first leaf) on P668 medium after 8 weeks of culture. The growth medium played a significant role in determining the germination response of A. africana seeds. Dark pretreatment of seed cultures significantly enhanced the germination percentage and the growth of rhizoids on the protocorms. Leaf growth in terms of length was substantially enhanced on P668 medium. It was significantly reduced in modified Knudson C medium after 16 weeks of culture. Seedlings developed on P668 medium showed a significantly better growth performance in relation to leaf length, leaf number, root length, fresh and dry weights per plant in vermiculite after 12 weeks of ex vitro growth in a mist house with 90% relative humidity. Further studies developing a suitable method for in vitro symbiotic germination could assist in reintroduction of this threatened orchid species into the wild.     

Germination and growth of sponge gourd (Luffa cylindrica) pollen tubes and FTIR analysis of the pollen tube wall

Emergence of multiple pollen tubes from single pollen grains occurred both in vitro and in vivo in sponge gourd (Luffa cylindrica (L.) Roem). The frequency with which pollen grains produced multiple pollen tubes in vivo (7.2%) was lower than that under in vitro conditions (14.9%). In pollen grains germinated in vitro, the total length of the multiple pollen tubes was greater than that of single pollen tubes, but individual tubes among the multiple tubes did not reach the same length as single tubes. Moreover, the growth of the single pollen tubes continued for a longer period in vitro than that of the multiple tubes. Fluorescence microscopy showed that callose was present throughout the pollen tube wall except in the apical part of growing pollen tubes, and nuclei moved into the longest of the multiple tubes. Results of Fourier transform infrared microspectroscopy indicated that abnormal cell wall components (peaks at 800–1000 cm⁻¹) were more frequent in multiple pollen tubes lacking nuclei, and the pectin content (1733 cm⁻¹) in multiple pollen tubes was much lower than that in single pollen tubes. These findings suggested that there were significant differences in pollen tube growth and wall composition between single and multiple pollen tubes, and that multiple pollen tubes had much less opportunity than single pollen tubes to reach the embryo sac and achieve double fertilization.     

Characterization of a new fungal antagonist of Phytophthora capsici

The capacity of many microorganisms for antagonism towards pathogens is unknown and previously uncharacterized species may be potentially useful in this respect. This work presents the evaluation of the capacity of a previously uncharacterized Ascomycete, denominated here as UA to protect chili pepper (Capsicum annuum) against Phytophthora capsici, and other soil-borne plant pathogens and establishes the role of UA as a biocontrol agent. Inoculation of UA 2 days before the inoculation of P. capsici led to 77.8% survival of pepper plants. Simultaneous confrontation of both microorganisms in vitro led to 53.1% growth inhibition of P. capsici, while the inoculation of P. capsici 3 days after the inoculation with UA improved growth inhibition up to 73%. Simultaneous confrontation in vitro of UA with Fusarium oxysporum and Fusarium solani led to 41.2 and 50% growth inhibition, respectively, but had no effect on Rhizoctonia solani or a binucleate Rhizoctonia isolate. Moreover, formation of zoosporangia and the germination of zoospores were completely inhibited by exposure to undiluted filter sterilized filtrate. UA produces septate mycelia, but could not be classified in detail due to a lack of spores or reproductive structures. However, sequencing of Internal Transcribed Spacer 1, 2 and the 5.8S genes indicated that this fungus is a member of the Ascomycetes.     

Fructose inhibits pear pollen germination on agar medium without loss of viability

Pollen of Japanese pear (Pyrus pyrifolia Nakai) germinated well on agar medium containing 10% sucrose or glucose, but not on agar containing fructose. The inhibitory effect of fructose was dose-dependent. Sucrose enhanced pollen tube growth much more effectively than glucose. Addition of 5% fructose to 5% or 10% sucrose or glucose media suppressed germination completely. Ungerminated pollen, however, showed similar respiration rate and stainability against acetocarmine dye as germinated pollen. When pollen was transferred onto fructose medium after culturing it on glucose or sucrose medium for 1–2 h, germination was completely impeded. Reversely, pollen transferred to sucrose or glucose medium from fructose medium germinated at almost the same ratio as pollen on sucrose or glucose medium without transfer. Thus, pollen inhibition by fructose is reversible. Compared with uncultured pollen, cultured pollen contained less than half amount of total sugars, even if failed in germination on fructose medium. Germinated pollen on sucrose and glucose media contained sucrose and glucose, but ungerminated pollen on fructose medium contained only trace levels of these sugars, suggests that pollen on fructose medium predominantly uses sucrose and glucose as respiration substrates and cannot maintain the constant levels of these sugars. However, as pollen germination occurred on agar medium without any sugar, fructose may impede a physiological factor that triggers germination, and once the trigger is impeded, many physiological pathways including sugar biosynthesis may be blocked.     

Effects of post-harvest foliar boron and calcium applications on subsequent season's pollen germination and pollen tube growth of pear (Pyrus pyrifolia)

The primary objective of this study was to evaluate the effects of foliar boron and calcium application after harvest on the quantity and activity of pollen in the ‘Housui’ and ‘Wonwhang’ pears on a subsequent year. Pollen grains of the ‘Housui’ pear were cultured on germination medium, to which had been added boric acid (0, 25, 75, 100, 200, 300, 400, and 500 mg L⁻¹) and calcium nitrate (0, 10, 25, 50, 100, 150, 200, 250, 300, 400, and 500 mg L⁻¹). Boric acid, which was added to the germination media, exerted a significant stimulatory effect on both pollen germination and pollen tube growth, although pollen tube growth was inhibited at higher concentrations than 300 mg L⁻¹. Calcium nitrate addition stimulated pollen germination, except at concentration of 500 mg L⁻¹. However, pollen tube growth was significantly inhibited with increasing concentrations of calcium nitrate. In the orchard experiment, boron and calcium were sprayed at concentrations of 0, 100, 200, 500 or 1000 mg L⁻¹ onto leaves after harvest, respectively. Boron and calcium content in the tissues as well as pollen production and growth were determined after these treatments. The foliar application of boron mainly resulted in an increase of boron concentration in buds. It also induced an increase in the weight of the anther and pollen in the following year. On the other hand, the foliar application of calcium resulted in an increase of calcium concentration mainly in the leaves, but pollen weight was decreased at high concentration treatment in the following year. The germination rate and tube growth of collected pollen were highest in the trees which had received boron treatment at a concentration of 200 mg L⁻¹. In contrast, the germination rate and tube growth of collected pollen were decreased by calcium application at concentrations of 500 and 1000 mg L⁻¹ without significant increase at lower concentrations. Consequently, the accumulation of boron in the developed buds of pear trees subjected to post-harvest foliar boron application generated positive effects on both the quantity and quality of pollen in the following year.     

Effects of cadmium and copper on pollen germination and fruit set in pea (Pisum sativum L.)

In this work, pea (Pisum sativum) plants exposed to increasing cadmium and copper concentrations were tested for heavy metals accumulation in flowers and for ‘in vivo’ pollen germination. Based on the Cd and Cu accumulation amounts in the flowers, an evaluation of the same metals effects on ‘in vitro’ pollen germination was achieved. Moreover, the effects of both metals on fruits number and weight and on seed set and yield at individual plant level were examined. While cadmium concentrations did not affect ‘in vivo’ pollen germination, only higher copper concentrations rendered a significant reduction. This is in contrast with the clear negative effect on pollen germination in vitro and might be explained by the different dynamic and bioavailability of both metals. A clear effect of Cd and Cu was observed on two important yield components ie, fruit weight and seed set. Although results obtained herein cannot give a clear cut relationship between the effect of Ca and Cu on reproductive development and its consequences on yields, they represent emerging results on the potential consequences of metals contamination on reproductive development in plants.     

Ameliorative effect of polyamines on the high temperature inhibition of in vitro pollen germination in tomato (Lycopersicon esculentum Mill.)

Effect of polyamines on in vitro pollen germination at high temperatures in tomato (Lycopersicon esculentum Mill.) was investigated. Pollen germination and tube growth were significantly inhibited at 33°C and 35°C compared to those at 25°C. This inhibition was reversed by the addition of spermidine or spermine in the germination medium. Spermidine at 0.5 mM was slightly more effective than spermine at 0.05 mM. Spermidine at 0.05 and 0.5 mM and spermine at 0.05 mM slightly increased pollen germination rates at 25°C. Spermidine at 5 mM and spermine at 0.5 M were inhibitory to pollen germination, regardless of incubation temperatures. Spermidine also promoted germination of pollen grains incubated at 38°C for 1 and 3 h and then at 25°C for the rest of the 20 h incubation period. The effect was higher at 0.5 mM than at 0.05 mM. Treatment of spermidine to intact flowers 1 day before anthesis was also effective in ameliorating the high temperature inhibition of in vitro pollen germination on the polyamine-free medium. Here, the optimum concentration was 5 mM. These results demonstrate that polyamines can counteract the inhibitory effects of high temperature on pollen germination. They also suggest that the endogenous level of polyamines in germinating pollen grains is an important factor for the pollen germinability at high temperature.     

猕猴桃花粉萌发的影响因子

试验以美味猕猴桃4个株系帮增1号、201、35-19N1、35-18N1为材料,测定不同株系花粉的活力和花粉萌发动态,并以蔗糖、硼酸、钙、锰、钼、氯化钠等作为环境因子,看其对花粉萌发和花粉管生长的影响。结果表明:35-19N1萌发和活力最高,萌发率为51.73%;帮增1号萌发最差,萌发率为36.23%。在培养基中硼酸和蔗糖最适宜浓度分别为:150mg/L、10%;培养基中添加Ca(NO3)25mg/L,或Na6Mo7O2480mg/L、MnSO4100mg/L,能明显提高萌发率。不同株系花粉的发芽势高峰有差异。NaCl对花粉萌发和花粉管生长有抑制作用。     

Effect of pistil age and pollen parent on pollen tube growth and fruit production of pistachio

Female pistachio (Pistacia vera) trees were hand pollinated with pollen from three different male genotypes 0,1,2,3 and 4 d after anthesis. Results were measured by mature fruit set and by fluorescence microscopy of pollen germination and tube growth in the pistil. Maximum pollen tube growth and fruit set of split nuts was achieved following pollination within two days of anthesis. Three day old pistils supported pollen tube growth, but fruit set was low. Four day old pistils supported little pollen tube growth or fruit set. Significant differences were also apparent between pollen parents, with low pollen tube growth and fruit set following interspecific pollination with P. atlantica pollen. All pollen tubes were observed to penetrate chalazogamously, and parthenocarpic production of blank fruits occurred in both unpollinated and pollinated treatments. The results indicate that for optimum fruit production of pistachio, pollen transfer must be achieved within 2 d of anthesis and pollination by P. atlantica should be avoided.     

Influence of pistil extract and temperature on in vitro pollen germination and pollen tube growth of olive cultivars

In vitro tests for pollen germination and pollen tube growth of olive cultivars were carried out with the addition of pistil extracts. Low pistil extract concentrations (.0.3–0.6 mg ml^-1 f.w.) enhanced pollen germination and pollen tube growth for the same or different cultivars. Different responses to temperature were observed when pollen and pistil extract belonged to the same or to different cultivars. The results suggest that this in vitro test could be used to assess compatibility groups between olive cultivars.     

Cross-transferable polymorphic SSR loci in Prunus species

This work reports the transferability and polymorphism of previously reported SSRs in 10 Prunus species. The availability of a large number of SSRs in the genus Prunus makes marker choice random, while preventing comparison of results in fingerprinting studies. The availability of SSR markers, polymorphic in a wide sample of Prunus species, would facilitate marker choice, while allowing the comparison of results. In this work, microsatellite markers useful for analyzing 10 different Prunus species (P. persica, P. dulcis, P. armeniaca, P. domestica, P. insititia, P. salicina, P. cerasifera, P. avium, P. cersus and P. mahaleb) were searched through screening SSRs previously reported to be conserved and/or polymorphic in more than one Prunus species. A selected group of 13 SSRs, transferable to the 10 species, was analyzed in terms of their usefulness for analyzing these species. The amplification range, polymorphism and variability detected by these loci are reported. The information provided will be useful for Prunus genetic studies as well as conservation and management of Prunus germplasm resources.