Inviable Pollen Research Articles

Rice transcriptional repressor OsTIE1 controls anther dehiscence and male sterility by regulating JA biosynthesis.

Proper anther dehiscence is essential for successful pollination and reproduction in angiosperms, and jasmonic acid (JA) is crucial for the process. However, the mechanisms underlying the tight regulation of JA biosynthesis during anther development remain largely unknown. Here, we demonstrate that the rice (Oryza sativa L.) ethylene-response factor-associated amphiphilic repression (EAR) motif-containing protein TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTORS (TCP) INTERACTOR CONTAINING EAR MOTIF PROTEIN1 (OsTIE1) tightly regulates JA biosynthesis by repressing TCP transcription factor OsTCP1/PCF5 during anther development. The loss of OsTIE1 function in Ostie1 mutants causes male sterility. The Ostie1 mutants display inviable pollen, early stamen filament elongation, and precocious anther dehiscence. In addition, JA biosynthesis is activated earlier and JA abundance is precociously increased in Ostie1 anthers. OsTIE1 is expressed during anther development, and OsTIE1 is localized in nuclei and has transcriptional repression activity. OsTIE1 directly interacts with OsTCP1, and overexpression of OsTCP1 caused early anther dehiscence resembling that of Ostie1. JA biosynthesis genes including rice LIPOXYGENASE are regulated by the OsTIE1-OsTCP1 complex. Our findings reveal that the OsTIE1-OsTCP1 module plays a critical role in anther development by finely tuning JA biosynthesis and provide a foundation for the generation of male sterile plants for hybrid seed production.

Knockout of floral and meiosis genes using CRISPR/Cas9 produces male-sterility in Eucalyptus without impacts on vegetative growth.

Eucalyptus spp. are widely cultivated for the production of pulp, energy, essential oils, and as ornamentals. However, their dispersal from plantings, especially when grown as an exotic, can cause ecological disruptions. To provide new tools for prevention of sexual dispersal by pollen as well as to induce male-sterility for hybrid breeding, we studied the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-mediated knockout of three floral genes in both FT-expressing (early-flowering) and non-FT genotypes. We report male-sterile phenotypes resulting from knockout of the homologs of all three genes, including one involved in meiosis and two regulating early stages of pollen development. The targeted genes were Eucalyptus homologs of REC8 (EREC8), TAPETAL DEVELOPMENT AND FUNCTION 1 (ETDF1), and HECATE3 (EHEC3-like). The erec8 knockouts yielded abnormal pollen grains and a predominance of inviable pollen, whereas the etdf1 and ehec3-like knockouts produced virtually no pollen. In addition to male-sterility, both erec8 and ehec3-like knockouts may provide complete sterility because the failure of erec8 to undergo meiosis is expected to be independent of sex, and ehec3-like knockouts produce flowers with shortened styles and no visible stigmas. When comparing knockouts to controls in wild-type (non-early-flowering) backgrounds, we did not find visible morphological or statistical differences in vegetative traits, including average single-leaf mass, stem volume, density of oil glands, or chlorophyll in leaves. Loss-of-function mutations in any of these three genes show promise as a means of inducing male- or complete sterility without impacting vegetative development.

Identification of Heat-Induced Proteomes in Tomato Microspores Using LCM- Proteomics Analysis

Pollen development is highly susceptible to heat stress (HS) and the production of inviable pollen causes reduction in seed- and fruit-set in plants. This study was carried out to identify HS-induced pollen proteins and the associated biological processes in tomato (Solanum lycopersicum). Tomato ‘Micro-Tom’ plants were incubated under 32°C//22°C (day/night, 12/12 h) for two weeks for heat treatment, and the non-treated control plants were incubated for the same time period at 25°C /22°C. Flower buds of 5 mm in length were confirmed to contain the heat sensitive uninucleate microspores. Pollen cells were harvested using laser capture microdissection (LCM) and protein was extracted using a one-step method under high pressure and vacuum. Approximately 60,000 LCMharvested microspore cells yielded about 18-20 μg proteins. The tandem mass tags (TMT) proteomics analysis identified a total of 6018 proteins, 4784 proteins were quantified, 37 proteins were identified as HS up-regulated significantly changed proteins (SCPs), and 83 proteins as HS down (dn)-regulated SCPs. Further analysis using the plant MetGenMap system showed that the HS up-regulated SCPs were enriched in the heat acclimation, pollen wall formation, protein folding/refolding gene ontology (GO) biological processes, and the HS dn-regulated SCPs were placed in the carbohydrate catabolism and de-novo protein biosynthesis GO terms. Biological processes such as mitosis, resistance to oxidative stresses, and carbohydrate and lipid metabolic processes contain both the HS up-, and dn-regulated SCPs. These results indicate that the LCM-TMT proteomics workflow is highly efficient in the identification of HS-induced pollen proteomes. These HS induced SCPs will be used for exploring heat tolerance of tomato pollens. The proteomics data are available via ProteomeXchange with identifier PXD010218.

The Role of Non-Coding RNAs in Cytoplasmic Male Sterility in Flowering Plants.

The interactions between mitochondria and nucleus substantially influence plant development, stress response and morphological features. The prominent example of a mitochondrial-nuclear interaction is cytoplasmic male sterility (CMS), when plants produce aborted anthers or inviable pollen. The genes responsible for CMS are located in mitochondrial genome, but their expression is controlled by nuclear genes, called fertility restorers. Recent explosion of high-throughput sequencing methods enabled to study transcriptomic alterations in the level of non-coding RNAs under CMS biogenesis. We summarize current knowledge of the role of nucleus encoded regulatory non-coding RNAs (long non-coding RNA, microRNA as well as small interfering RNA) in CMS. We also focus on the emerging data of non-coding RNAs encoded by mitochondrial genome and their possible involvement in mitochondrial-nuclear interactions and CMS development.

Ionizing Radiation from Chernobyl and the Fraction of Viable Pollen

Pollen viability is an important component of reproductive success, with inviable pollen causing failure of reproduction. Pollen grains have evolved mechanisms to avoid negative impacts of adverse environmental conditions on viability, including the ability to sustain ionizing radiation and repair DNA. We assessed the viability of 109,000 pollen grains representing 675 pollen samples from 111 species of plants in Chernobyl across radiation gradients that spanned three orders of magnitude. We found a statistically significant but small and negative main effect of radiation on pollen viability rates across species (Pearson’s r = 0.20). Ploidy level and the number of nucleate cells (two vs. three) were the only variables that influenced the strength of the effect of radiation on pollen viability, as reflected by significant interactions between these two variables and background radiation, while there were no significant effects of genome size, pollen aperture type, life cycle duration, or pollination agent ...

Genetic characterization of hybridization between native and invasive bittersweet vines (Celastrus spp.)

Hybridization associated with species introductions can accelerate the decline of native species. The main objective of this study was to determine if the decline of a North American liana (American bittersweet, Celastrus scandens) in the eastern portion of its range is related to hybridization with an introduced congener (oriental bittersweet, C. orbiculatus). We used newly characterized microsatellite loci, a maternally-inherited chloroplast DNA marker, and field observation to survey individuals across the USA to determine the prevalence of hybrids, their importance in the invasion of C. orbiculatus, and the predominant direction of hybridization. We found that only 8.4 % of non-native genotypes were hybrids (20 of 239), and these hybrids were geographically widespread. Hybrids showed reduced seed set (decline of >98 %) and small, likely inviable pollen. Genetic analysis of a maternally inherited chloroplast marker showed that all 20 identified hybrids came from C. scandens seed parents. The strong asymmetry in pollen flow that favors fecundity in introduced males has the potential to greatly accelerate the decline of native species by wasting limited female reproductive effort.

Pollen Insights into Apomictic and SexualMiconia(Miconieae, Melastomataceae)

The genus Miconia, Melastomataceae, has been highlighted for its high proportion of apomictic species, in which the occurrence of low pollen grain viability is commonly recorded. In this study, we compared aspects of pollen development and viability and ploidy level in a sexual species, Miconia pepericarpa, and an apomictic species, Miconia fallax, and investigated the possible causes of pollen grain sterility in apomictic species. Abnormal meiosis was observed only in apomictic M. fallax and leads to the formation of inviable pollen grains, often with little or no cytoplasmic content. Furthermore, the apomictic species is polyploid, and therefore, chromosomal imbalance would be expected. Symmetric mitotic division was found only in M. fallax, also resulting in inviable pollen grains, usually with cytoplasm content but without generative cell differentiation. At the end of development, the apomictic species also displays another difference when compared with the sexual species: the generative cell does not become fusiform but, rather, maintains a spherical shape. It is unknown whether the generative cell shape may interfere with its function.

Image Analysis Protocol for Detecting and Counting Viable and Inviable Pollen Grains

Estimating total pollen number and viability is labor intensive and time consuming. Nevertheless the information is crucial for a range of plant biologists from plant breeders to evolutionary biologists. Viability is determined by dye staining and counting colored (viable) and transparent (inviable) grains under a compound microscope. Existing protocols have been standardized to speed up total pollen counts but their success in determining viability is rather limited because they do not incorporate staining techniques. Some of the published protocols that determine viability do so based on statistical methods but focus on just one parameter such as pollen diameter requiring manual standardization to generate two distinct size distributions for viable and inviable pollen. We demonstrate a digital image processing protocol that can count viable and inviable pollen by distinguishing colored and transparent objects in the image space, saving valuable labor and time. Using pollen grains from two plants, Collinsia heterophylla and Brassica napus, we show that differences between viable and inviable pollen are best described by a complex measure, the grain shape. By measuring several parameters such as area, length, width, circularity and elongation, while retaining all the advantages of traditional staining process, our procedure increases the accuracy of viability estimates. The only drawback of our protocol is that it uses the NIS elements a software specific to Nikon instruments.

Silencing of meiosis-critical genes for engineering male sterility in plants

The potential for pollen-mediated transgene flow into wild or closely related species has provoked unease in terms of transgenic modification of agricultural plant species. One approach to remedy this situation in species whose seeds and fruits are not of particular value is to engineer male sterility into the transgenic lines. In this study, three meiosis-critical genes, namely AHP2, AtRAD51C and SWITCH1 (SWI), were chosen as silencing targets to test the feasibility of incorporating sterility into plants using an RNAi-based approach. Our results indicated that the silencing of each of these genes via hairpin RNA (termed AHPi, RAD51Ci and SWIi lines) in Arabidopsis thaliana yielded a proportion of transgenic plants exhibiting a similar 'partially sterile' phenotype in which less than 50% of pollen was viable. In addition, a 'sterile' phenotype was also evident in a minority of RAD51Ci and SWIi, but not AHPi, lines in which plants yielded no seeds and either produced inviable pollen (RAD51Ci lines) or displayed a complete absence of pollen (SWIi lines). This suggests that AtRAD51C and SWI may function at distinct stages of meiosis. Further analyses of SWIi lines demonstrated that the 'sterile' phenotype was associated with a substantial reduction in the level of targeted gene transcript in floral tissues and resulted from sterility of the male, but not female gametes. This work demonstrates that generating male sterility through the silencing of key genes involved in the regulation of meiosis is feasible, and its advantages and potential applications for transgene containment are discussed.

Arabidopsis Basic Leucine-Zipper Transcription Factors TGA9 and TGA10 Interact with Floral Glutaredoxins ROXY1 and ROXY2 and Are Redundantly Required for Anther Development

ROXY1 and ROXY2 are CC-type floral glutaredoxins with redundant functions in Arabidopsis (Arabidopsis thaliana) anther development. We show here that plants lacking the basic leucine-zipper transcription factors TGA9 and TGA10 have defects in male gametogenesis that are strikingly similar to those in roxy1 roxy2 mutants. In tga9 tga10 mutants, adaxial and abaxial anther lobe development is differentially affected, with early steps in anther development blocked in adaxial lobes and later steps affected in abaxial lobes. Distinct from roxy1 roxy2, microspore development in abaxial anther lobes proceeds to a later stage with the production of inviable pollen grains contained within nondehiscent anthers. Histological analysis shows multiple defects in the anther dehiscence program, including abnormal stability and lignification of the middle layer and defects in septum and stomium function. Compatible with these defects, TGA9 and TGA10 are expressed throughout early anther primordia but resolve to the middle and tapetum layers during meiosis of pollen mother cells. Several lines of evidence suggest that ROXY promotion of anther development is mediated in part by TGA9 and TGA10. First, TGA9 and TGA10 expression overlaps with ROXY1/2 during anther development. Second, TGA9/10 and ROXY1/2 operate downstream of SPOROCYTELESS/NOZZLE, where they positively regulate a common set of genes that contribute to tapetal development. Third, TGA9 and TGA10 directly interact with ROXY proteins in yeast and in plant cell nuclei. These findings suggest that activation of TGA9/10 transcription factors by ROXY-mediated modification of cysteine residues promotes anther development, thus broadening our understanding of how redox-regulated TGA factors function in plants.

Dihaploid Stocks of Switchgrass Isolated by a Screening Approach

Manipulation of ploidy in switchgrass has potential to accelerate inbred production and to provide insight about genome structure through either sequencing or cytogenetic approaches. We have identified two dihaploid individuals isolated from among the progeny of a controlled cross between two individuals of the cultivars Alamo and Kanlow. The dihaploid lines were initially distinguished from the parental lines by their reduced heterozygosity and were subsequently confirmed through estimation of C values by flow cytometry and chromosome counts of metaphase root tip squash preparations. These plants are functionally sterile, with floral bracts that remain closed and inviable pollen. They can be easily distinguished from tetraploid individuals by their reduced stature, smaller epidermal cell size, and lower number of chloroplasts per guard cell. Aberrant meiosis in these individuals is evidenced by a lack of regular pairing at diakinesis and metaphase I and suggests that the non-homologous genomes are distinct from one another. The reduced genome size of these dihaploids will facilitate basic genome studies and genetic analyses that are impossible or problematic in polyploid accessions.

Transcript profiling of male-fertile and male-sterile sorghum indicates extensive alterations in gene expression during microgametogenesis

Male-sterile sorghum carrying the IS1112C cytoplasm represents an unusual example of aberrant microgametogenesis wherein microspores develop into inviable pollen that remain physically intact until anther exsertion. These inviable pollen grains do not deposit starch, yet fluoresce with the vital stain fluoroscein diacetate. We sought to elucidate the extent of differential gene expression in this subtle example of defective microgametogenesis through cDNA-AFLP transcript profiling of near-isogenic male-fertile and male-sterile plants at an early stage representing early-mid microspores to early pollen, 7–11 days prior to anthesis, and a late stage representing young to nearly mature pollen, spanning the terminal 96 h of pollen development. The transition from early to late stages is characterized by changes in abundance of nearly 33% of shared transcripts, and early- or late-specific expression of about 10% of transcripts. Male-sterile plants exhibit extensive changes in regulatory patterns characteristic of fertile plants, including premature expression of late-specific, and prolonged expression of early-specific, transcripts. Genome-wide transcriptome patterns indicate the expression of an estimated 12,000 genes in early-mid microspores, and the abundance of at least 15% of these transcripts is altered in male-sterile plants. A near-isogenic line restored to male fertility is characterized by apparent normalized expression of most of these transcripts. The development of the microgametophyte is thus characterized by dynamic programmed changes in gene expression, and the expression of male sterility compounds these changes in a complex manner.

Chromosomal and morphological studies of diploid and polyploid cytotypes of Stevia rebaudiana (Bertoni) Bertoni (Eupatorieae, Asteraceae)

In this study, we examined the chromosome number and some morphological features of strains of Stevia rebaudiana. The chromosomes were analyzed during mitosis and diakinesis, and the tetrad normality and pollen viability were also assessed. In addition, stomata and pollen were measured and some plant features were studied morphometrically. All of the strains had 2n = 22, except for two, which had 2n = 33 and 2n = 44. Pairing at diakinesis was n = 11II for all of the diploid strains, whereas the triploid and tetraploid strains had n = 11III and n = 11IV, respectively. Triploid and tetraploid plants had a lower tetrad normality rate than the diploids. All of the strains had inviable pollen. Thus, the higher the ploidy number, the greater the size of the pollen and the stomata, and the lower their number per unit area. The triploid strain produced the shortest plants and the lowest number of inflorescences, whereas the tetraploid strain had the largest leaves. Analysis of variance revealed highly significant differences among the strains, with a positive correlation between the level of ploidy and all of the morphological features examined.

Antisense inhibition of a nuclear gene, BrDAD1, in Brassica causes male sterility that is restorable with jasmonic acid treatment.

Male sterility is widely used for the production of hybrid seeds, but the use of genic male sterility is rather limited because of difficulty in maintaining homozygous male sterile plants. Recently, the DEFECTIVE IN ANTHER DEHISCENCE 1 (DAD1) gene, which encodes a phospholipase A1 involved in the first step of the jasmonic acid (JA) biosynthesis pathway, was isolated from a male sterile Arabidopsis mutant. To utilize this gene in Brassica crops, we characterized the BrDAD1 gene, the putative ortholog of DAD1 in Brassica rapa. Out of 25 plants transformed with an antisense gene constructed from the BrDAD1, 3 plants showed a defect of anther dehiscence at the flower bud opening stage and produced inviable pollen. One of the three showed male sterility only, but the other two showed a delay or a lack of flower opening in addition to male sterility. The male sterile and flower-opening phenotypes were rescued by the application of JA as well as linolenic acid. Furthermore, all these characteristics were inherited to the next generation. The present results demonstrate a novel control system for hybrid seed production by the use of nuclear genes.

Gene-expression analysis of sucrose-starch metabolism during pollen maturation in cytoplasmic male-sterile and fertile lines of sorghum

During pollen maturation, single-celled microspores undergo a rapid phase of starch biosynthesis, presumably from sucrose stored in the vacuoles. Very little is known about the genes and the intracellular controls that regulate the sucrose → starch pathway in such microspores. We show here RNA profiles in sorghum microspores during the transition from vacuolated microspore to immature pollen at two specific stages of pollen development: early, showing little or no detectable starch, and late, when pollen are active in starch-filling. We have also examined two near-isogenic cytoplasmic male sterile lines that retain fully turgid immature pollen until the starch-filling stage, but remain starch-deficient, and ultimately form inviable pollen. Using maize cDNA clones, both temporal and genotypic differences were observed in the expression profiles of four metabolic genes (soluble or vacuolar invertase, Ivr2; sucrose synthase, Sus1; phosphoglucomutase, Pgm; and a subunit of ADPG pyrophosphorylase, Bt2), two similar metabolic regulatory genes (Grf1 and Grf2, encoding 14-3-3 proteins) and a transcription factor, ZmMADS1. Western blot analyses were conducted to visualize sucrose synthase, SS2; AGPase, BT2 and the GRF (14-3-3) proteins. Temporal differences of both a qualitative and quantitative nature were observed in all three proteins in both male-fertile and male-sterile lines. We suggest that these changes in gene expression may result from increased and decreased sink strengths and associated changes in carbohydrate metabolism in starch-filled (fertile) and starch-deficient (sterile) pollen, respectively.

Decreased NADH glutamate synthase activity in nodules and flowers of alfalfa (Medicago sativa L.) transformed with an antisense glutamate synthase transgene

Legumes obtain a substantial portion of their nitrogen (N) from symbiotic N2 fixation in root nodules. The glutamine synthetase (GS, EC 6.3.1.2)/glutamate synthase (GOGAT) cycle is responsible for the initial N assimilation. This report describes the analysis of a transgenic alfalfa (Medicago sativa L.) line containing an antisense NADH-GOGAT (EC 1.4.1.14) under the control of the nodule-enhanced aspartate amino-transferase (AAT-2) promoter. In one transgenic line, NADH-GOGAT enzyme activity was reduced to approximately 50%, with a corresponding reduction in protein and mRNA. The transcript abundance for cytosolic GS, ferredoxin-dependent GOGAT (EC 1.4.7.1), AAT-2 (EC 2.6.1.1), asparagine synthase (EC 6.3.5.4), and phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.31) were unaffected, as were enzyme activities for AAT, PEPC and GS. Antisense NADH-GOGAT plants grown under symbiotic conditions were moderately chlorotic and reduced in growth and N content, even though symbiotic N2 fixation was not significantly reduced. The addition of nitrate relieved the chlorosis and restored growth and N content. Surprisingly, the antisense NADH-GOGAT plants were male sterile resulting from inviable pollen. A reduction in NADH-GOGAT enzyme activity and transcript abundance in the antisense plants was measured during the early stages of flower development. Inheritance of the transgene was stable and resulted in progeny with a range of NADH-GOGAT activity. These data indicate that NADH-GOGAT plays a critical role in the assimilation of symbiotically fixed N and during pollen development.

BUMBLE BEE SELECTION OFMIMULUS GUTTATUSFLOWERS: THE EFFECTS OF POLLEN QUALITY AND REWARD DEPLETION

The ability of flower visitors to monitor returns when collecting pollen from flowers has been seldom studied despite the importance of pollen as a food resource, particularly for bees. Californian populations of Mimulus guttatus are polymorphic for pollen quality: many plants produce a high proportion of cytoplasmless pollen grains that render the grains incapable of fertilizing ovules or of supporting bees nutritionally. We found that different genotypes maintained a consistent proportion of inviable pollen within a genotype and over time. The number of pollen grains per flower was also consistent within a plant at each date but declined over time. We studied the ability of British bumble bees (Bombus spp.) to discriminate among plants of Mimulus guttatus on the basis of pollen quality and quantity at three scales: indoors with choices of two genotypes, in outdoor plots of several genotypes that varied in pollen quality, and outdoors at a whole-patch scale where two patches of plants differed in quality. We found that bees could discriminate among plants on the basis of pollen quality provided that flowers still retained most of the pollen. In the two-genotype trials, bees chose genotypes primarily on the quantity of viable pollen, and nectar was much less important. Similarly, where patches of low- and high-pollen quality plants were established, bees responded by visiting the high-quality patch more often and by visiting more flowers within the patch. However, the results from the outdoor plots that contained genotypes of varying phenotypes were inconsistent. A meta-analysis of a large number of separate plots showed that the overall correlation between visitation rate and pollen quality was significant, but variation among plots was also significant. A possible explanation for this inconsistency was suggested in a greenhouse trial in which we showed that, when foraging density was high, depletion of the standing crop of pollen happened quickly, and this reduced the ability of the foragers to choose the higher-quality genotypes. The results have implications for the evolution of pollen production in Mimulus guttatus and reward production in other plants.

Gametophytic expression of GUS activity controlled by the potato Lhca3.St.1 promoter in tobacco pollen

A β-glucuronidase (GUS) gene fusion with the promoter from the potato Lhca3.St.1 gene, encoding a light-harvesting complex protein, shows expression in pollen of transgenic tobacco plants. This was established by histochemical and fluorometric assays, and confirmed by RT-PCR, Western analysis and GUS activity following gel electrophoresis. The activity is minimal in early pollen development and high in mature pollen at anthesis. It shows gametophytic segregation in pollen grains. Pollen from tobacco plants hemizygous for a single Lhca3.St.1-GUS locus exhibited 1 : 1 segregation ratios, whereas dihybrids segregated in the expected 3 : 1 ratio. A sequential combination of histochemical staining for GUS activity and Alexander's stain for pollen viability, offers a convenient system to eliminate artefacts in the histochemical staining, identify inviable pollen and accurately score pollen segregation. The dual staining approach also confirmed gametophytic segregation in tobacco plants hemizygous for the GUS gene under transcriptional control of a doubled CaMV 35S promoter. Gametophytic segregation establishes that the GUS activity is determined by the genetic status of the pollen rather than of the parent plant, and indicates de novo GUS gene expression and translation during pollen maturation. The unanticipated activity of the Lhca3.St. 1 promoter in pollen has implications for the biosafety assessment of transgenic plants intended for agricultural use. Even when a promoter is not generally considered to be active in pollen, its expression in pollen should be evaluated if effects on pollinating insects or food safety concerns for products such as honey can be anticipated.

Reproductive Responses of Capsicum annuum to High Temperatures

Fruit yield reduction due to high temperatures has been widely observed in Solanaceous crops. Our past experiments have demonstrated that Capsicum annuum cultivars Ace and Bell Boy completely fail to produce fruit when grown at constant 33 °C. However, flowers are produced, continually. To determine which stages of flower development are sensitive to high temperatures, pepper buds, ranging in size from 1 mm to anthesis, were exposed to high temperatures for 6 hr, 48 hr, 5 days, or for the duration of the experiment. Fruit set for each bud size was determined. Exposure to high temperatures at anthesis and at the 2-mm size stage for 2 or more days significantly reduced fruit production. To determine whether inhibition of pollination, inhibition of fertilization, and/or injury to the female or male structures prevents fruit production at high temperatures, flowers from pepper cultivars Ace and Bell Boy were grown until flowers on the 8th or 9th node were 11 mm in length. Plants were divided between 25 °C and 33 °C constant growth chambers for 2 to 4 days until anthesis. At anthesis, flowers from both treatments were cross-pollinated in all combination, and crosses were equally divided between 33 or 25 °C growth chambers until fruit set or flowers abscised. All flower crosses resulted in 80% to 100% fruit set when post-pollination temperatures were 25 °C. However, post-pollination temperatures of 33 °C significantly reduced fruit production. Reduced fruit set by flowers exposed to high temperatures during anthesis and pollination is not a result of inviable pollen or ovule, but an inhibition of fertilization or initial fruit development.

A Simple Method to Test Genetic Allelism in Nearly Sterile Interspecific Plant Hybrids

The comparison of electrophoretic isozyme patterns of multimeric enzymes in leaf and pollen extracts from the same individual has been used to distinguish allozymes and isozymes. This is possible because in genetic heterozygotes leaf tissue exhibits both homomers and heteromers whereas pollen shows only homomers. I show that pollen of nearly sterile interspecific plant hybrids contains isozymes that can be examined in the same way. This permits a test to determine whether genes encoding particular isozymes in the parental species have remained allelic; i.e., located at corresponding positions on homologous chromosomes. Such a test is illustrated by examining cytosolic PGI in two classic hybrid combinations in Clarkia. The evidence shows that the coding PGI genes of the parental species are located on different chromosomes (or at different sites on the same chromosome), possibly resulting from reciprocal translocation as proposed by Lewis and Raven. The ability to determine whether genes in different species segregate as alleles or not will likely be useful in studying the extent of gene linkage conservation and as a supplement to RFLP and other DNA-directed maps. Formal genetic analysis is generally restricted to crosses between individuals of a single species because fertile hybrids are necessary to produce an F2 or other segregating progeny. However, for genes encoding multimeric enzymes expressed in pollen and detectable by electrophoretic assays, I show here that tests of allelism can be carried out between species even when their hybrids have very low fertility. This new test is an extention of previous results in which the electrophoretic isozyme pattern of extracts from leaves or other somatic tissues is compared with that of pollen from the same individual (Weeden and Gottlieb 1979). The test depends on the fact that following meiosis the haploid cells that develop into pollen receive one or the other allele at every locus, but not both. Thus if a particular locus is heterozygous for alleles encoding allozymes having different electrophoretic mobilities, leaf tissue exhibits both homomers and heteromers, but only homomers are synthesized in the pollen because any single grain receives only a single allele. For a dimeric enzyme in heterozygous individuals, extracts from a mass of pollen exhibit two homodimers but not the electrophoretically intermediate heterodimer. Its absence is apparent when patterns from the pollen and leaves are compared. In contrast, if the two homodimers are encoded by two different homozygous loci, then both leaf tissue and pollen show the same three-banded pattern. The pollen-leaf comparison has proved useful to test allelism of electrophoretic variants of numerous enzymes within plant species in which formal breeding studies cannot be readily carried out (Crawford 1990). The allelism of variants in different species can also be tested by the pollen-leaf comparison because inviable pollen often characteristic of interspecific hybrids contains many functional enzymes detectable by electrophoresis. In this report, I demonstrate the use of the pollen-leaf test to assess allelism of electrophoretic variants in two classical interspecific hybrids in Clarkia: C. franciscana x amoena and C. amoena x rubicunda (Lewis and Raven 1958a, 1958b). MATERIALS AND METHODS Plants. Interspecific hybrids were produced by crossing Clarkia amoena subsp. huntiana (Jepson) Lewis & Lewis x C. rubicunda (Lindley) Lewis & Lewis and x C. franciscana Lewis & Raven. Clarkia amoena subsp. huntiana was collected in Lake Co., near Manning Creek Bridge on Rt. 175, west of the junction with Rt. 29 (LDG 9025), and in Marin Co., on Lucas Valley Rd., 5.8 miles east of Nicasio Schoolhouse (LDG 714). Clarkia rubicunda was collected in Marin Co., at Ft. Barry (LDG BAR). Clarkia franciscana was represented by two populations, the type locality at The Presidio in San Francisco Co., and a newly discovered population in the foothills above