Non-viable Pollen Grains Research Articles

The Arabidopsis ARID-HMG DNA-BINDING PROTEIN 15 modulates JA signaling by regulating MYC2 during pollen development.

The intricate process of male gametophyte development in flowering plants is regulated by jasmonic acid (JA) signaling. JA signaling initiates with the activation of the basic-helix-loop-helix (bHLH) transcription factor (TF), MYC2, leading to the expression of numerous JA-responsive genes during stamen development and pollen maturation. However, the regulation of JA signaling during different stages of male gametophyte development remains less understood. This study focuses on the characterization of the plant ARID-HMG DNA-BINDING PROTEIN 15 (AtHMGB15), and its role in pollen development in Arabidopsis (Arabidopsis thaliana). Phenotypic characterization of a T-DNA insertion line (athmgb15-4) revealed delayed bolting, shorter siliques, and reduced seed set in mutant plants compared to the wildtype. Additionally, AtHMGB15 deletion resulted in defective pollen morphology, delayed pollen germination, aberrant pollen tube growth, and a higher percentage of non-viable pollen grains. Molecular analysis indicated the down-regulation of JA biosynthesis and signaling genes in the athmgb15-4 mutant. Quantitative analysis demonstrated that jasmonic acid and its derivatives were approximately tenfold lower in athmgb15-4 flowers. Exogenous application of methyl jasmonate could restore pollen morphology and germination, suggesting that the low JA content in athmgb15-4 impaired JA signaling during pollen development. Furthermore, our study revealed that AtHMGB15 physically interacts with MYC2 to form a transcription activation complex. This complex promotes the transcription of key JA signaling genes, the R2R3-MYB TFs MYB21 and MYB24, during stamen and pollen development. Collectively, our findings highlight the role of AtHMGB15 as a positive regulator of the JA pathway, controlling the spatiotemporal expression of key regulators involved in Arabidopsis stamen and pollen development.

Automated monitoring of pollen quality in Douglas fir using digital images: applications for handling and storage

Background: Conservation and breeding programs for forest species require efficient pollen quality monitoring strategies to maximize collection, storage and pollination efforts.
 Question: Can the ImageJ image analysis software be implemented to efficiently monitor pollen viability and germinability for quality testing?
 Studied species: Pseudotsuga menziesii (Mirb.) Franco
 Methods: Tetrazolium staining and germination on 10 % Brewback media was used to test Douglas-fir pollen quality. Two macros for automated counting were developed with ImageJ free software using digitized microscopic images, and were tested to evaluate provenances, germination in different media, dehydration/rehydration and freeze-storage treatments, and finally to compare pollen quality between mature trees and GA-induced strobili from juvenile trees.
 Results: Automated monitoring of pollen quality was feasible because the software suitably distinguishes viable from non-viable pollen grains due to differential tetrazolium staining that translates into pixel density, whereas germinability was assayed based on pollen size and circularity. This tool was used to guide the selection of appropriate pollen handling conditions and compare pollen production in adult and GA-induced juvenile trees. Pollen flash-freezing and storage at -80 °C showed recovery between 74 and 98 % of its initial germinability after rehydration; however, viability detected by tetrazolium is reduced, suggesting that handling and storage affect the reliability of the test.
 Conclusions: The flexibility of the Fiji software allowed the development of an efficient and accurate macro for monitoring P. menziesii pollen quality, reducing invested time and effort involved, which supports future research on its conservation physiology and its successful application for germplasm production.

The effects of heat stress on male reproduction and tillering in Sorghum bicolor

AbstractDue to global climate change, heat stress is increasingly harming the growth, development, overall biomass, and grain yield of numerous crops. Heat stress impairs pollen development and thus reduces seed set in Sorghum bicolor (L.) Moench; however, the effects of heat stress on anther development at specific stages and tiller formation remain incompletely understood. Here we report that exposure to heat stress [42°C/32°C (day/night)] at pollen mother cell (PMC) and booting stages profoundly disrupts tapetum and pollen development, resulting in a significant decrease in grain yield in sorghum. Sorghum plants subjected to 9 days or less of heat stress at the PMC stage exhibited normal pollen viability, but 12 days of heat stress caused almost complete loss of grain yield and the formation of nonviable pollen grains. Similarly, sorghum plants that were heat‐stressed for 3 days at the booting stage produced few seeds. Further analysis revealed that aberrant tapetum and pollen development contributed to the sterility of pollen in these heat‐stressed plants. Notably, in addition to inhibiting plant height, a 12‐day heat stress at the PMC stage promoted the formation of basal tillers, whereas a 3‐day heat stress at the booting stage stimulated the formation of apical tillers, which helped salvage seed yield under heat stress conditions. Moreover, the application of exogenous auxin promoted the formation of apical tillers and leaf numbers per apical tiller. Collectively, our findings suggest that sorghum is susceptible to heat stress during both early and late anther development, and auxin might be involved in governing the formation of apical tillers.

High Temperature Tolerance in a Novel, High-Quality Phaseolus vulgaris Breeding Line Is Due to Maintenance of Pollen Viability and Successful Germination on the Stigma.

The common bean (Phaseolus vulgaris L.) is an important nutritional source globally but is sensitive to high temperatures and thus particularly vulnerable to climate change. Derived from a breeding program at CIAT (Colombia), a heat-tolerant breeding line, named heat-tolerant Andean-type 4 (HTA4), was developed by a series of crosses of parents with a small-bean tepary genotype (Phaseolus acutifolius L.) in their pedigree, which might be the donor of heat stress (HS) tolerance. Importantly, in HTA4, the large, commercially desirable Andean-type beans was restored. To assess underlying tolerance mechanisms, HTA4, together with a heat-sensitive Colombian variety (Calima), was exposed to HS (31 °C/24 °C HS vs. 26 °C/19 °C day/night) under controlled environment conditions. Vegetative growth and photosynthetic performance were not negatively impacted by HS in either genotype, although senescence was delayed in Calima. HS during the reproductive stage caused an increase in pod number in Calima but with few fully developed seeds and many pods aborted and/or abscised. In contrast, HTA4 maintained a similar filled pod number under HS and a higher seed weight per plant. Pollen showed high sterility in Calima, with many non-viable pollen grains (24.9% viability compared to 98.4% in control) with a thicker exine and fewer starch granules under HS. Calima pollen failed to adhere to the stigma and germinate under HS. In HTA4, pollen viability was significantly higher than in Calima (71.1% viability compared to 95.4% under control), and pollen successfully germinated and formed pollen tubes in the style under HS. It is concluded that HTA4 is heat tolerant and maintains a high level of reproductive output due to its ability to produce healthy pollen that is able to adhere to the stigma.

Floral development of one of the oldest dioecious lineages of Arecaceae reveals different stages of dicliny in pistillate and staminate flowers

AbstractArecaceae subtribe Mauritiinae are a lineage of Neotropical dioecious species in subfamily Calamoideae, the earliest diverging and most diverse group of palms regarding sexual systems. Ontogenetic and anatomical studies of reproductive structures contribute to the understanding of evolutionary relationships, but there is a lack of detailed information. We describe the anatomical floral development of Lepidocaryum tenue, Mauritia flexuosa and Mauritiella armata (subtribe Mauritiinae) using standard light microscopy techniques. Three central moments in the development of pistillate and staminate flowers were identified: the initial phase of development of the floral meristem; the phase of determination of the whorls that will compose the pistillate and staminate flowers; and the phase of differentiation (pre-anthesis) of these whorls. Although the pre-anthetic flowers are diclinous, they are initially monoclinous, suggesting a hermaphrodite ancestor for Mauritiinae. The development is asynchronous since, in pre-anthesis, the pistillate flowers have staminodes that produce non-viable pollen grains, whereas the staminate flowers do not even have a pistillode. Staminodes may have a secondary function in attracting floral visitors.

Breeding value of multi-sprout pollinator lines of different inbreeding generations as parental components of sugar beet hybrids on а CMS basis

Aim. Creation of combination-capable lines of multi-sprout pollinators of sugar beets of different levels of homozygosity, establishment of criteria for their selection based on basic productivity, reproductive capacity and hybridization potential at various stages of the selection process for the formation of parental components of hybrids on the cytoplasmic male-sterile (CMS) basis. Methods. Field (experiments, phenological observations), laboratory (to determine sugar content), measuring and weighing (to determine the crop structure), statistical (mathematical processing of research results). The research was carried out at the Tobacco Research Station of the National Scientific Center "Institute of Agriculture of the National Academy of Agrarian Sciences" in the laboratory of sugar beet breeding during the 2015–2021. The starting material was three hybrid multi-sprout populations of sugar beet Um. 17/02-1705, Um. 21/01-33 and Um. 14/05-752. Linear materials were obtained by the inbreeding method. Results. It was established that the lines of multi-sprout pollinators of sugar beets of the second-third generations of inbreeding (I2-I3) are characterized by the highest productivity. The yield of root crops exceeded the standard by 8.1–9.3%, sugar content by 8.6–8.8% and sugar collection by 16.5–17.9%. As the depth of inbreeding increases, the quality of pollen deteriorates sharply. If in the lines of the first generation of inbreeding (I1), the fertility of pollen in plants of of multi-sprout pollinators was 95.1%, then in the lines of the fifth generation (I5) it decreased to 31.0%. The number of non-viable pollen grains in them was 72.2%, seed germination was 29%. Experimental hybrids with the best performance were obtained by using as pollinators lines of the second to third generations of inbreeding. According to the complex characteristic "sugar collection", they exceeded the group standard by 19.7–21.3%. Conclusions. It has been established that for the formation of line materials of sugar beet multi-sprout pollinators, as parent components of hybrids on а CMS basis, the main criterias for the selection of valuable plant genotypes, in addition to the hybridization potential, are high level of their basic productivity and reproductive capacity. The best experimental sugar beet hybrids were obtained by using lines of multi-sprout pollinators of the second to third inbreeding generations.

Tomato POLLEN DEFICIENT 2 encodes a G-type lectin receptor kinase required for viable pollen grain formation.

Pollen development is a crucial biological process indispensable for seed set in flowering plants and for successful crop breeding. However, little is known about the molecular mechanisms regulating pollen development in crop species. This study reports a novel male-sterile tomato mutant, pollen deficient 2 (pod2), characterized by the production of non-viable pollen grains and resulting in the development of small parthenocarpic fruits. A combined strategy of mapping-by-sequencing and RNA interference-mediated gene silencing was used to prove that the pod2 phenotype is caused by the loss of Solanum lycopersicum G-type lectin receptor kinase II.9 (SlG-LecRK-II.9) activity. In situ hybridization of floral buds showed that POD2/SlG-LecRK-II.9 is specifically expressed in tapetal cells and microspores at the late tetrad stage. Accordingly, abnormalities in meiosis and tapetum programmed cell death in pod2 occurred during microsporogenesis, resulting in the formation of four dysfunctional microspores leading to an aberrant microgametogenesis process. RNA-seq analyses supported the existence of alterations at the final stage of microsporogenesis, since we found tomato deregulated genes whose counterparts in Arabidopsis are essential for the normal progression of male meiosis and cytokinesis. Collectively, our results revealed the essential role of POD2/SlG-LecRK-II.9 in regulating tomato pollen development.

Gene silencing, knockout and over-expression of a transcription factor ABORTED MICROSPORES (SlAMS) strongly affects pollen viability in tomato (Solanum lycopersicum)

BackgroundThe tomato (Solanum lycopersicum L.) is an economically valuable crop grown worldwide. Because the use of sterile males reduces the cost of F1 seed production, the innovation of male sterility is of great significance for tomato breeding. The ABORTED MICROSPORES gene (AMS), which encodes for a basic helix-loop-helix (bHLH) transcription factor, has been previously indicated as an essential gene for tapetum development in Arabidopsis and rice. To determine the function of the SlAMS gene (AMS gene from S. lycopersicum) and verify whether it is a potential candidate gene for generating the male sterility in tomato, we used virus-induced gene silencing (VIGS), CRISPR/Cas9-mediated genome editing and over-expression technology to transform tomato via Agrobacterium infection.ResultsHere, the full-length SlAMS gene with 1806 bp from S. lycopersicum (Accession No. MK591950.1) was cloned from pollen cDNA. The results of pollen grains staining showed that, the non-viable pollen proportions of SlAMS-silenced (75%), −knockouted (89%) and -overexpressed plants (60%) were significantly higher than the wild type plants (less than 10%; P < 0.01). In three cases, the morphology of non-viable pollen grains appeared tetragonal, circular, atrophic, shriveled, or otherwise abnormally shaped, while those of wild type appeared oval and plump. Furthermore, the qRT-PCR analysis indicated that SlAMS in anthers of SlAMS-silenced and -knockouted plants had remarkably lower expression than in that of wild type (P < 0.01), and yet it had higher expression in SlAMS-overexpressed plants (P < 0.01).ConclusionIn this paper, Our research suggested alternative approaches to generating male sterility in tomato, among which CRISPR/Cas9-mediated editing of SlAMS implied the best performance. We also demonstrated that the downregulation and upregulation of SlAMS both affected the pollen formation and notably led to reduction of pollen viability, suggesting SlAMS might be essential for regulating pollen development in tomato. These findings may facilitate studies on clarifying the SlAMS-associated molecular regulatory mechanism of pollen development in tomato.

OsCPK29 interacts with MADS68 to regulate pollen development in rice

Pollen development and its germination are obligatory for the reproductive success of flowering plants. Calcium-dependent protein kinases (CPKs, also known as CDPKs) regulate diverse signaling pathways controlling plant growth and development. Here, we report the functional characterization of a novel OsCPK29 from rice, which is mainly expressed during pollen maturation stages of the anther. OsCPK29 exclusively localizes in the nucleus, and its N-terminal variable domain is responsible for retaining it in the nucleus. OsCPK29 knockdown rice plants exhibit reduced fertility, set fewer seeds, and produce collapsed non-viable pollen grains that do not germinate. Cytological analysis of anther semi-thin sections during different developmental stages suggested that pollen abnormalities appear after the vacuolated pollen stage. Detailed microscopic study of pollen grains further revealed that they were lacking the functional intine layer although exine layer was present. Consistent with that, downregulation of known intine development-related rice genes was also observed in OsCPK29 silenced anthers. Furthermore, it has been demonstrated that OsCPK29 interacts in vitro as well as in vivo with the MADS68 transcription factor which is a known regulator of pollen development. Therefore, phenotypic observations and molecular studies suggest that OsCPK29 is an important regulator of pollen development in rice.

Elevated Temperature Disrupts Pollen-Pistil Dynamics and Seed Set in Okra (Abelmoschus esculentus L. Moench)

Elevated temperature can interfere with pollen formation and function in okra (Abelmoschus esculentus). The study was aim to quantify the impact of elevated temperatures on the reproductive stage in okra. In both the stain analysis and pollen tube growth test, pollen viability was decreased at elevated temperatures. The highest number of non-viable pollen grains were observed at 35°C and 40°C. The stigma was nonsignificantly high in receptivity at all temperatures; however, the seed set showed a significant decline under elevated temperatures. The findings offer the potential to look further into approaches, to genetic enhancement of heat-tolerant plants that will secure okra productivity during future climatic variation.

Viability and receptivity of reproductive structures in Lima bean (Phaseolus lunatus L.) for improvement programs

Artificial hybridization in Phaseolus lunatus L. is still little used in genetic improvement programs in Brazil due to the low establishment percentages. This work aimed to evaluate pollen viability and stigma receptivity in different Lima bean genotypes throughout the day. Accessions ‘Roxinha -MT’ and ‘Cana Longa-PB’ were used in the experiment. Five flower buds were collected from each genotype at 6:00 a.m., 10:00 a.m., 2:00 p.m., and 6:00 p.m. The stigma receptivity was determined using 3% hydrogen peroxide, whereas pollen viability was determined with 2% acetocarmine colouration. The genotype and time factors acted independently on the percentage of viable and non-viable pollen grains and stigma receptivity. The genotype ‘Roxinha’ presented 88.54% viable pollen grains, a value 84.27% higher than that of the ‘Cana Longa’ genotype. This percentage varied as a function of time, with the highest means observed at 6:00 a.m. Stigma receptivity was influenced only by the time of the day, undergoing a great reduction in receptivity, from 88% in the early morning to slightly more than 40% around noon. These results indicate that 6:00 a.m. is the most suitable time to perform artificial crosses in Lima bean plants.

Flower stages, germination and viability of pollen grains of Annona squamosa L. in tropical conditions

The success in the application of artificial pollination techniques, aspects of floral biology should receive special attention, especially regarding the viability studies of pollen grains. In this sense, two experiments were carried out, aiming to determine the floral stages: floral opening (anthesis), female, male and senescence stages of A. squamosa L flowers under tropical climate conditions, and to evaluate the germination and viability of pollen grains submitted to different storage conditions. In the first experiment, observations and data collection began when flowers were still closed. Readings were taken every two hours for 46 hours until all the flowers reached the senescence stage. For the second, the experimental design was completely randomized, in a 2 x 7 factorial scheme, with 2 storage conditions (environment at 27 ± 2°C and refrigerated at 5 ± 2°C) and 7 pollen grain conditioning times (0, 4, 8, 12, 16, 20 and 24 hours), with 4 repetitions for the analysis of pollen grain germination and 3 repetitions for the analysis of pollen grain viability, and each repetition consisted of one blade. The variables evaluated were percentage of viable and non-viable pollen grains and percentage of germinated and non-germinated pollen grains. For tropical climatic conditions, the flower anthesis (female stage) of the sugar-apple begins at 00:00 h, extending until 12:00h on the same day. The flowers of the tree reach a functionally staminate stage (male stage) from 6 a.m. the next day. Pollen grains stored at room conditions (27 ± 2°C) remain viable for up to 24 hours after collection, showing that storage in a cold environment at low temperatures (5 ± 2°C) is not necessary. Seed pollen grains have a germination percentage of 51.25% when stored under ambient conditions (27 ± 2°C) for up to 4 hours after collection.

An L-type lectin receptor-like kinase promotes starch accumulation during rice pollen maturation.

Starch accumulation is key for the maturity of rice pollen grains; however, the regulatory mechanism underlying this process remains unknown. Here, we have isolated a male-sterile rice mutant, abnormal pollen 1 (ap1), which produces nonviable pollen grains with defective starch accumulation. Functional analysis revealed that AP1 encodes an active L-type lectin receptor-like kinase (L-LecRLK). AP1 is localized to the plasma membrane and its transcript is highly accumulated in pollen during the starch synthesis phase. RNA-seq and phosphoproteomic analysis revealed that the expression/phosphorylation levels of numerous genes/proteins involved in starch and sucrose metabolism pathway were significantly altered in the mutant pollen, including a known rice UDP-glucose pyrophosphorylase (OsUGP2). We further found that AP1 physically interacts with OsUGP2 to elevate its enzymatic activity, likely through targeted phosphorylation. These findings revealed a novel role of L-LecRLK in controlling pollen maturity via modulating sucrose and starch metabolism.

A rice GDSL esterase/lipase protein (GELP) is required for anther and pollen development

Male reproductive development is vital for crop seed production. However, the mechanism underlying remains largely unknown. Here, we isolated a male sterile mutant osgelp34-1 in rice. Cytological analysis indicated that the mutant exhibited delayed degradation of anther wall layers and abnormal pollen development, which resulted in the production of shrunk and non-viable pollen grains. MutMap and CRISPR/Cas9 analysis confirmed that a point mutation in OsGELP34 is responsible for the male sterile phenotype. OsGELP34 is highly expressed in reproductive tissues and encodes a putative GDSL lipase. OsGELP34 protein is located to the endoplasmic reticulum (ER) and conserved in land plants. Collectively, our findings elaborated that OsGELP34 plays a vital role in rice male reproduction and has potential applications in rice hybrid breeding.

A new image-based tool for the high throughput phenotyping of pollen viability: evaluation of inter- and intra-cultivar diversity in grapevine

BackgroundLow pollen viability may limit grapevine yield under certain conditions, causing relevant economic losses to grape-growers. It is usually evaluated by the quantification of the number of viable and non-viable pollen grains that are present in a sample after an adequate pollen grain staining procedure. Although the manual counting of both types of grains is the simplest and most sensitive approach, it is a laborious and time-demanding process. In this regard, novel image-based approaches can assist in the objective, accurate and cost-effective phenotyping of this trait.ResultsHere, we introduce PollenCounter, an open-source macro implemented as a customizable Fiji tool for the high-throughput phenotyping of pollen viability. This tool splits RGB images of stained pollen grains into its primary channels, retaining red and green color fractionated images (which contain information on total and only viable pollen grains, respectively) for the subsequent isolation and counting of the regions of interest (pollen grains). This framework was successfully used for the analysis of pollen viability of a high number of samples collected in a large collection of grapevine cultivars. Results revealed a great genetic variability, from cultivars having very low pollen viability (like Corinto Bianco; viability: 14.1 ± 1.3%) to others with a very low presence of sterile pollen grains (Cuelga; viability: 98.2 ± 0.5%). A wide range of variability was also observed among several clones of cv. Tempranillo Tinto (from 97.9 ± 0.9 to 60.6 ± 5.9%, in the first season). Interestingly, the evaluation of this trait in a second season revealed differential genotype-specific sensitivity to environment.ConclusionsThe use of PollenCounter is expected to aid in different areas, including genetics research studies, crop improvement and breeding strategies that need of fast, precise and accurate results. Considering its flexibility, it can be used not only in grapevine, but also in other species showing a differential staining of viable and non-viable pollen grains. The wide phenotypic diversity observed at a species level, together with the identification of specific cultivars and clones largely differing in this trait, pave the way of further analyses aimed to understand the physiological and genetic causes driving to male sterility in grapevine.

Cytogenetic evaluation of chili (Capsicum spp., Solanaceae) genotypes cultivated in Valle del Cauca, Colombia

The genus Capsicum comprises a large group of hot and sweet peppers, with different morphological varieties and degree of fruits pungency. Due to its active principles is widely used in cooking and in traditional medicine, being sold in large worldwide. In order to provide cytogenetic information which supporting plant breeding programs, this study aimed to analyze the meiotic behavior, chromosome number and pollen viability of the three species of greater economic importance of Capsicum (C. annuum, C. frutescens and C. chinense). Therefore, fruits and flower buds were collected from the Experimental Center of the Universidad Nacional de Colombia, campus Palmira- CEUNP to the corresponding mitotic, meiosis and pollen viability analysis. Plants presented different meiotic abnormalities, where all can disrupt the process of meiosis cell division, creating unbalanced chromosome numbers and varied pollen fertility. Chromosome number variation, checking 2n=2x=24, 2n=2x=26, 2n=4x=48 to 2n=6x=72 chromosomes also occurred. A low degree of non-viable pollen grains was observed for C. frutescens, so these accession should not considered as a parental for plant breeding programs.

Seed, sugar and acid characteristics of ‘Zaomi’ Ponkan (Citrus reticulata Blanco), a spontaneous mutant of ‘Xinnu’

Abstract Ponkan is one of the two easy-peeling mandarins grown in China, and ‘Xinnu’ Ponkan ( Citrus reticulata Blanco) is a major cultivar in the industry. We selected a spontaneous mutant of ‘Xinnu’ named ‘Zaomi’ Ponkan. Compared to the wild type, ‘Zaomi’ Ponkan exhibited alternating agronomic traits comprising earlier colouring, thinner peel, fewer seeds and higher sugar content. Pollen analysis showed that ‘Zaomi’ produced a greater number of deformed, nonviable pollen grains. Experiments in self- and cross-pollination suggested that self-incompatibility of ‘Zaomi’ contributes to the seedlessness of ‘Zaomi’ × ‘Zaomi’. Fruit quality analysis demonstrated that ‘Zaomi’ was a high-sugar mutant that was not affected by pollinating combination. This study on the fruit characteristics of ‘Zaomi’ indicates that partial male sterility and self-incompatibility contributes to fewer or no seeds; natural pollination promotes the overall fruit quality of ‘Zaomi’ Ponkan. These results provide an explanation of the seed formation of ‘Zaomi’ Ponkan and supply information relevant to its commercial planting.

Development and Evaluation of Diploid and Polyploid Hibiscus moscheutos

Hibiscus moscheutos L. is an herbaceous hibiscus native to eastern North America that has been a popular landscape and container plant exhibiting large and colorful flowers in the summer. However, unsightly fruit develop and remain on the stalks at the end of the blooming season, which greatly decreases the ornamental value. Thus, breeding for sterility was attempted through ploidy level manipulation to reduce formation and growth of seed stalks, and to improve blooming vigor and longevity. Colchicine and oryzalin were used as mitotic inhibitors to induce tetraploid breeding lines that could be used to develop sterile triploids. Germinated seedlings of ‘Luna Red’ were soaked in three concentrations of each doubling agent for three different durations. Exposure to a low concentration of colchicine solution for a long time or to a low concentration of oryzalin for a short period was found to be effective in yielding a high number of tetraploids with a low rate of mortality. Triploids were obtained from the traditional method of crossing tetraploids with diploids. Triploid and tetraploid plants showed a decrease in height with a more compact form. Leaves of tetraploid plants were more ruffled, with an increase in overall leaf thickness, but were not different from leaves of diploids and triploids in regard to leaf mass per area (LMA). Triploid plants bloomed longer but had smaller flowers than diploid plants. Although the whole planting was infected by aerial phytophthora, diploid, tetraploid, and triploid plants were significantly different in their tolerances: all diploid branches were infected, but only a minor infection occurred on one triploid branch, and the transmission remained slow. Flowers of tetraploid plants failed to produce pollen, whereas flowers of triploid plants produced only nonviable pollen grains and fruits aborted after pollination, which led to infertility of induced triploids.

Effects of different temperature regimes on flower development, microsporogenesis and fertility in bolting garlic (Allium sativum).

Garlic (Allium sativum L.) cultivars do not develop fertile flowers and seeds. Therefore, garlic production and improvement depend exclusively on vegetative propagation. Recent advances in garlic research have enabled fertility restoration and the discovery of fertile and male-sterile genotypes; however, the environmental regulation of the reproductive process is still not clear. Garlic seeds are successfully produced in the Mediterrenean region, where the photoperiod is relatively short, whereas spring and summer temperatures are high. We hypothesise that, in bolting garlic, various stages of florogenesis are differentially regulated by temperature and that high temperatures might obstruct pollen production. The effects of eight combinations of controlled growth temperatures on fertile and male-sterile garlic clones were studied. In both genotypes, a gradual temperature increase before and during anthesis favoured intact flower development. Surprisingly, continuous exposure to moderate temperatures during the entire growth period resulted in poor flowering, anther abortion and reduced pollen production. In the male-sterile genotype, no growth regime improved pollen production, which is controlled by genetic mechanisms. In the male-fertile genotype, gradual temperature increase supported pollen production but a sharp transition to high temperatures resulted in rapid flower senescence and pollen abortion, thus supporting our research hypothesis. In both fertile and male-sterile plants, the most vulnerable phase of microsporogenesis is the unicellular microspore stage. Tapetal malformation is the major cause for malnutrition of the microspores, with consequent production of nonviable pollen grains.

Meiotic behavior of Brachiaria decumbens hybrids.

Brachiaria decumbens is a forage grass of inestimable value for livestock in Brazil due to its production of good quality forage even when planted on acid and poor soils, although it is susceptible to pasture spittlebugs. Only one cultivar, cv. Basilisk, has been used as the pollen donor in crosses with Brachiaria ruziziensis since 1988 at Embrapa Gado de Corte Research Center. Breeding within the species only became possible from 2009 when sexual accessions were successfully tetraploidized using colchicine. Three sexual genotypes were obtained and hybridization within B. decumbens was finally achieved. Here, we evaluated microspore tetrads using conventional cytology and found meiotic indexes above 78% for all three female genitors (cD24-2, cD24-27, cD24-45), but a low meiotic index (<22%) in the natural apomictic genitor D62 (cv. Basilisk) and in 49 hybrids. Analysis of the relationship between abnormal tetrad frequency and non-viable pollen grains yielded a highly significant Pearson correlation coefficient. The t-test proved significant for the progeny of cD24-45 x D62, with lower abnormalities and pollen sterility when compared to the other two progenies resulting from cD24-2 and cD24-27 crossed to D62, but these two did not differ. Apomictic hybrids such as S036 and X030 with low pollen sterility have the potential for use in cultivar development, whereas the sexual hybrids T012, X072, and X078 might be of use as female genitors in polycross blocks if they display good agronomic traits.