COMPONENTS OF SEED AND POLLEN YIELD OF LOBELIA CARDINALIS: VARIATION AND CORRELATIONS

Plants from three Lobelia cardinalis populations were grown under common garden conditions to assess intra- and interplant variation in seed and pollen production. Seed number per flower and mean seed weight varied systematically with floral position on the inflorescence (lowest values were from terminal flowers) but pollen grain number per flower did not vary systematically with floral position. Most of the remaining variance in seed and pollen grain number per flower and mean seed weight was distributed among plants; clones produced very similar amounts of pollen and seed. Seed yield was positively correlated with seed production per flower and with total flower production, but not with mean seed weight; pollen yield was also positively correlated with pollen grain production per flower and total flower production. Seed and pollen yield were simple linear functions of plant size but only pollen yield was a simple linear function of flower production; seed yield was a quadratic function in which the second order term was negative. This quadratic relationship resulted from a negative correlation between seed number per flower and total flower production. This correlation, in addition to the wide variation among plants in pollen number per flower, accounts for the weak correlation of seed and pollen yield. I conclude from these data that it is unlikely that plants in natural L. cardinalis populations transmit genes to the population's seed crop equally through pollen and ovules—emphasizing the importance of measuring both male and female components of reproductive success.     

Identification and functional characterization of the AGO1 ortholog in maize

Eukaryotic Argonaute proteins play primary roles in miRNA and siRNA pathways that are essential for numerous developmental and biological processes. However, the functional roles of the four ZmAGO1 genes have not yet been characterized in maize (Zea mays L.). In the present study, ZmAGO1a was identified from four putative ZmAGO1 genes for further characterization. Complementation of the Arabidopsis ago1‐27 mutant with ZmAGO1a indicated that constitutive overexpression of ZmAGO1a could restore the smaller rosette, serrated leaves, later flowering and maturation, lower seed set, and darker green leaves at late stages of the mutant to the wild‐type phenotype. The expression profiles of ZmAGO1a under five different abiotic stresses indicated that ZmAGO1a shares expression patterns similar to those of Argonaute genes in rice, Arabidopsis, and wheat. Further, variation in ZmAGO1a alleles among diverse maize germplasm that resulted in several amino acid changes revealed genetic diversity at this locus. The present data suggest that ZmAGO1a might be an important AGO1 ortholog in maize. The results presented provide further insight into the function of ZmAGO1a.     

Practical considerations for maintaining germplasm in maize

The main goals of genetic resource management are to acquire, maintain, distribute, characterize, regenerate, preserve, evaluate, and utilize the genetic diversity of crops and their wild relatives. The objectives of this study for ex-situ conservation of maize (Zea mays L.) are to review and describe: (1) practical regeneration methods that are based on population genetic theory; (2) practical problems encountered in choosing core subsets of a maize collection. Whenever possible, regeneration procedures should control the number of pollen parents (male gametes; through controlled hand pollination) and the number of female parent gametes (by harvesting equal numbers of kernels from each seed plant). When the number of pollen and seed parents are controlled during regeneration, the effective population size (N_e) is twice the size of the original population (N). Examples of practical methods for controlling the number of male and female parents are presented. The procedure involves random-paired plant crosses and taking equal numbers of seeds from each maize ear. To form a core subset, accessions of a maize race are subdivided through a stratified sampling procedure. Delineation of a core subset from a Tuxpeño racial collection is described as an example.Part of this study was presented at the International Workshop on Plant Core Collections, Brasilia, Brazil, August 24–28, 1992     

Down‐regulation of OsSPX1 caused semi‐male sterility,resulting in reduction of grain yield in rice

OsSPX1, a rice SPX domain gene, involved in the phosphate (Pi)‐sensing mechanism plays an essential role in the Pi‐signalling network through interaction with OsPHR2. In this study, we focused on the potential function of OsSPX1 during rice reproductive phase. Based on investigation of OsSPX1 antisense and sense transgenic rice lines in the paddy fields, we discovered that the down‐regulation of OsSPX1 caused reduction of seed‐setting rate and filled grain number. Through examination of anthers and pollens of the transgenic and wild‐type plants by microscopy, we found that the antisense of OsSPX1 gene led to semi‐male sterility, with lacking of mature pollen grains and phenotypes with a disordered surface of anthers and pollens. We further conducted rice whole‐genome GeneChip analysis to elucidate the possible molecular mechanism underlying why the down‐regulation of OsSPX1 caused deficiencies in anthers and pollens and lower seed‐setting rate in rice. The down‐regulation of OsSPX1 significantly affected expression of genes involved in carbohydrate metabolism and sugar transport, anther development, cell cycle, etc. These genes may be related to pollen fertility and male gametophyte development. Our study demonstrated that down‐regulation of OsSPX1 disrupted rice normal anther and pollen development by affecting carbohydrate metabolism and sugar transport, leading to semi‐male sterility, and ultimately resulted in low seed‐setting rate and grain yield.     

Stability of deletion,insertion and point mutations at the bronze locus in maize

Summary Phenotypic revertants from several kinds of mutations, including deletions, have been detected by pollen analysis at the wx and Adh loci in maize. Mutations in these genes give phenotypic revertants with median frequencies of 0.7 and 0.5×10^–5, respectively. However, the nature of such revertants can only be analyzed following their recovery from conventional matings. In the current study large seed populations derived from crosses involving several bz (bronze) locus mutations in maize were examined for reversion to a Bz (purple) expression. Deletion, insertion and point mutations were included in the study. Principally, over 2 million gametes of the bz-R mutation, which is shown here to be associated with a 340 base pair deletion within the transcribed region of the gene, have been screened for reversion. No revertants from it or any of the other bz mutations have been recovered, even though a total of almost 5 million gametes from homoallelic crosses have been examined to date. Results from seed analysis are discussed in reference to those from pollen analysis in maize.     

PROXIMATE AND ULTIMATE SOURCES OF WITHIN-INDIVIDUAL VARIATION IN SEED MASS IN PRUNELLA VULGARIS (LAMIACEAE)

Substantial variation in seed mass within individual seed parents is at odds with predictions of models for the evolution of optimum offspring size and with empirical observations of directional selection on seed mass. To elucidate the ultimate causes of this variation, I examined several proximal sources of within-individual variation in seed mass in the perennial herb Prunella vulgaris. Position of inflorescence, position of flower within an inflorescence, date of anthesis, and number of seeds produced per flower all explained some within-individual variation in seed mass. Hand pollination in the field failed to reveal any effect of pollen source (self pollen or outcross pollen) on seed mass. My results, in conjunction with those from studies of selection on seed mass in P. vulgaris, do not support hypotheses that within-individual variation in seed mass is favored by the pattern of natural selection on seed mass. Rather, the results suggest that seed parents are not capable of producing a uniform seed crop in the face of changes in resource availability in the course of a season. The inability to produce a uniform seed crop may persist because of selection for variability in traits correlated with seed mass or because of a true constraint on the evolution of uniform offspring size.     

EFFECT OF MALE POLLEN DONOR AND FEMALE SEED PARENT ON ALLOCATION OF RESOURCES TO DEVELOPING SEEDS AND FRUIT IN CHAMAECRISTA FASCICULATA (LEGUMINOSAE)

In Chamaecrista fasciculata, fruit abortion levels are high and seed mass is highly variable, necessary preconditions for differential resource allocation of the female to seed and fruit sired by different males. This study investigated the relative role of pollen donor and seed parent on the allocation of resouces to developing seed and fruit, and assessed the role of genetic relatedness in contributing to any observed paternal effect in C. fasciculata. In addition, pollen donor effects were contrasted to within-seed parent sources of variation in resource allocation due to pollination date and ovule position in the pod. Plants collected from the field were brought to a greenhouse where single-donor crosses were conducted controlling for pollen donor source and interplant distance, a measure of genetic relatedness. Seed mass, number of seed/fruit, fruit maturation time, and fruit abortion rate were measured as indicators of resource allocation to developing seed and fruit. Variation in resource allocation was largely determined by the seed parent. Pollen donor effects were limited to differences between self vs. non-self pollinations, suggesting that inbreeding depression following mating events between related individuals is the source of any variation among pollen donors on differential resource allocation to developing seed and fruit. Once the effect of inbreeding was removed, however, pollination date and ovule position played larger roles than pollen source. Since there was no detectable variation among male pollen donors in their ability to accrue resources from the female seed parent apart from inbreeding effects, it is concluded that the opportunity for postzygotic mate choice is limited in C. fasciculata.     

Brassinosteroid‐mediated reactive oxygen species are essential for tapetum degradation and pollen fertility in tomato

Phytohormone brassinosteroids (BRs) are essential for plant growth and development, but the mechanisms of BR‐mediated pollen development remain largely unknown. In this study, we show that pollen viability, pollen germination and seed number decreased in the BR‐deficient mutant d^{^im}, which has a lesion in the BR biosynthetic gene DWARF (DWF), and in the bzr1 mutant, which is deficient in BR signaling regulator BRASSINAZOLE RESISTANT 1 (BZR1), compared with those in wild‐type plants, whereas plants overexpressing DWF or BZR1 exhibited the opposite effects. Loss or gain of function in the DWF or BZR1 genes altered the timing of reactive oxygen species (ROS) production and programmed cell death (PCD) in tapetal cells, resulting in delayed or premature tapetal degeneration, respectively. Further analysis revealed that BZR1 could directly bind to the promoter of RESPIRATORY BURST OXIDASE HOMOLOG 1 (RBOH1), and that RBOH1‐mediated ROS promote pollen and seed development by triggering PCD and tapetal cell degradation. In contrast, the suppression of RBOH1 compromised BR signaling‐mediated ROS production and pollen development. These findings provide strong evidence that BZR1‐dependent ROS production plays a critical role in the BR‐mediated regulation of tapetal cell degeneration and pollen development in Solanum lycopersicum (tomato) plants.     

Sources of variation in pollinator contribution within a guild: the effects of plant and pollinator factors

Among plants visited by many pollinator species, the relative contribution of each pollinator to plant reproduction is determined by variation in both pollinator and plant traits. Here we evaluate how pollinator movement among plants, apparent pollen carryover, ovule number, resource limitation of seed set, and pollen output affect variation in contribution of individual pollinator species to seed set in Lithophragma parviflorum (Saxifragaceae), a species visited by a broad spectrum of visitors, including beeflies, bees and a moth species. A previous study demonstrated differences among visitor species in their single-visit pollination efficacy but did not evaluate how differences in visitation patterns and pollen carryover affect pollinator efficacy. Incorporation of differential visitation patterns and pollen carryover effects —commonly cited as potentially important in evaluating pollinator guilds — had minor effects (0–0.6% change) on the estimates of relative contribution based on visit frequency and single-visit efficacy alone. Beeflies visited significantly more flowers per inflorescence than the bees and the moth. Seed set remained virtually constant during the first three visited flowers for beeflies and larger bees, indicating that apparent pollen carryover did not reduce per-visit efficacy of these taxa. In contrast, Greya moth visits showed a decrease in seed set by 55.4% and the smaller bees by 45.4% from first to second flower. The larger carryover effects in smaller bees and Greya were diminished in importance by their small overall contribution to seed set. Three variable plant traits may affect seed set: ovule number, resource limitation on seed maturation, and pollen output. Ovule number per flower declined strongly with later position within inflorescences. Numbers were much higher in first-year greenhouse-grown plants than in field populations, and differences increased during 3 years of study. Mean pollen count by position varied 7-fold among flowers; it paralleled ovule number variation, resulting in a relatively stable pollen:ovule ratio. Resource limitation of seed set increased strongly with later flowering, with seed set in hand-pollinated flowers ranging from 66% in early flowers to 0% in the last two flowers of all plants. Variation in ovule number and resource limitation of seed maturation jointly had a strong effect on the number of seeds per flower. Visitation to early flowers had the potential to cause more seed set than visitation to later flowers. Overall, the most important sources of variation to seed production contribution were differences among pollinators in abundance and absolute efficacy (ovules fertilized on a single visit) and potentially differential phenology among visitor species. These effects are likely to vary among populations and years.     

In vitro pollen germination and transient transformation of<Emphasis Type="Italic">Zea mays</Emphasis> and other plant species

To study pollen-specific gene expression, fast and convenient methods involving in vitro pollen germination and bombardment with promoter deletion constructs are needed. Unfortunately, because of variation of pollen germability and tube growth, conducting these experiments is often unsatisfying for many plant species, including maize, especially when pollen is collected at different times of the day or season. We have overcome these problems by defining a novel medium (PGM) that guarantees germination efficiencies of more than 90% for maize pollen from at least 7 genotypes (A188, AC 3572 C, B73, H99, Hi-II, Q2, Tx232). This medium is also suitable to germinate pollen of other monocot species, such asPennisetum americanum andTradescantia species, and dicot species, such asArabidopsis thaliana, Arachis hypogaea, Columnea oesterdiana, Nicotiana tabacum, Phaseolus vulgaris, Pisum sativum, Solanum lycopersicum, Solanum tuberosum, andVicia faba. On average, reproducible germination rates ranging from 50–100% were observed with all plant species tested. In addition, we report a transient transformation assay using the luciferase (Luc) reporter gene. Biolistic parameters were defined to obtain reproducibleLuc activity measurements after bombarding thick-walled pollen, such as maize pollen. For comparison, samples of germinated maize and tobacco pollen were bombarded with the reporter gene under control of the constitutive ubiquitin-and pollen-specificZmMADS2 maize promoters. The important parameters necessary to apply both in vitro pollen germination and transient transformation for a large range of plant species are discussed. An erratum to this article is available at .     

Assessing the Effects of Bt Maize on the Predatory Mite Neoseiulus cucumeris

The investigation of Neoseiulus cucumeris in the context of the ecological risk assessment of insect resistant transgenic plants is of particular interest as this omnivorous predatory mite species is commercially available and considered important for biological control. In a multitrophic feeding experiment we assessed the impact of Bt maize on the performance of N. cucumeris when offered spider mites (Tetranychus urticae) reared on Bt (Bt11, Syngenta) or non-Bt maize (near isogenic line) and Bt or non-Bt maize pollen as a food source. Various parameters including mortality, development time, oviposition rate were measured. Spider mites were used as a prey for N. cucumeris, since these herbivores are known to contain similar levels of Cry1Ab toxin, when reared on Bt maize, as those found in the transgenic leaf material. In contrast, toxin levels in pollen of this transgenic cultivar are very low. No differences in any of the parameters were found when N. cucumeris was fed with spider mites reared on Bt and non-Bt maize. Pollen was shown to be a less suitable food source for this predator as compared to spider mites. Moreover, subtle effects on female N. cucumeris (9% longer development time and 17% reduced fecundity) were measured when fed with pollen originating from Bt maize as compared to non-Bt maize pollen. Our findings indicate that the predatory mite N. cucumeris is not sensitive to the Cry1Ab toxin as no effects could be detected when offered Bt-containing spider mites, and that the effects found when fed with Bt maize pollen can be assigned to differences in nutritional quality of Bt and non-Bt maize pollen. The significance of these findings is discussed with regard to the ecological relevance for risk assessment of transgenic plants.     

Brassinosteroids promote development of rice pollen grains and seeds by triggering expression of Carbon Starved Anther,a MYB domain protein

Transport of photoassimilates from leaf tissues (source regions) to the sink organs is essential for plant development. Here, we show that a phytohormone, the brassinosteroids (BRs) promotes pollen and seed development in rice by directly promoting expression of Carbon Starved Anther (CSA) which encodes a MYB domain protein. Over‐expression of the BR‐synthesis gene D11 or a BR‐signaling factor OsBZR1 results in higher sugar accumulation in developing anthers and seeds, as well as higher grain yield compared with control non‐transgenic plants. Conversely, knockdown of D11 or OsBZR1 expression causes defective pollen maturation and reduced seed size and weight, with less accumulation of starch in comparison with the control. Mechanically, OsBZR1 directly promotes CSA expression and CSA directly triggers expression of sugar partitioning and metabolic genes during pollen and seed development. These findings provide insight into how BRs enhance plant reproduction and grain yield in an important agricultural crop.     

A single silk- and multiple pollen-expressed PMEs at the Ga1 locus modulate maize unilateral cross-incompatibility

The Gametophyte factor1 (Ga1) locus in maize confers unilateral cross-incompatibility (UCI), and it is controlled by both pollen and silk-specific determinants. Although the Ga1 locus has been reported for more than a century and is widely utilized in maize breeding programs, only the pollen-specific ZmGa1P has been shown to function as a male determinant; thus, the genomic structure of the Ga1 locus and all the determinants that control UCI at this locus have not yet been fully characterized. Here, we used map-based cloning to confirm the determinants of UCI at the Ga1 locus and maize pan-genome sequence data to characterize the genomic structure of the Ga1 locus. The Ga1 locus comprises one silk-expressed pectin methylesterase gene (PME) (ZmGa1F) and eight pollen-expressed PMEs (ZmGa1P and ZmGa1PL1-7). Knockout of ZmGa1F in Ga1/Ga1 lines leads to the complete loss of the female barrier function. The expression of individual ZmGa1PL genes in a ga1/ga1 background endows ga1 pollen with the ability to overcome the female barrier of the Ga1 locus. These findings, combined with genomic data and genetic analyses, indicate that the Ga1 locus is modulated by a single female determinant and multiple male determinants, which are tightly linked. The results of this study provide valuable insights into the genomic structure of the Ga2 and Tcb1 loci and will aid applications of these loci in maize breeding programs.     

APOMIXIS IN THE POA SECUNDA COMPLEX

Observation of cleared ovules of Poa secunda Presl showed that plants produce aposporous embryo sacs, and emasculation studies showed that they do not set seed without pollen. The ratio of sexual to asexual embryo sacs on a given plant does not correlate with percent stainable pollen, nor does pollen stainability correlate with meiotic regularity or with chromosome number. Percent apomictic ovules, percent pollen stainability, and regularity of meiosis all vary within a given population; they also vary if the same plant is moved from one site to another. Although there is morphological variation in the species, none of the reproductive features correlates with morphology. Pollen from any morph will stimulate seed set in any other morph, and hybrids have been produced in low frequency. Plants will also set seed using their own pollen. In its reproductive biology, P. secunda exhibits many similarities with P. pratensis L.     

Monoploids in Zea mays L. following crosses with untreated and X-rayed pollen

Monoploids in Zea mays L. occur spontaneously among individual diploid seedlings. Plants with the gametic chromosome number have also been detected among members of multiple seedlings of maize and numerous other species of angiosperms. Previous reports disclosed that Xirradiation of the pollen successfully stimulated reduced parthenogenesis in some other angiosperms, but the results of X-ray treatment were inconclusive in maize. Therefore, a tester stock of maize homozygous for lg _land gl _l was crossed with pollen from inbred CI3A, carrying the dominant alleles. The pollen was exposed to 0, 1000, 2000, and 4000r units of X rays. chromosome counts were made from root tips of plants exhibiting both recessive phenotypes to establish the frequencies of monoploids in the control and X₁ populations.Monoploids were more abundant among the individual seedlings from crosses with untreated pollen than in the X₁ populations. X irradiation of the pollen is not a feasible method for the induction of monoploids in maize. The X-ray treatments greatly increased the frequency of multiple seedlings, and deficiencies were numerous among them. The members of a set of multiple seedlings were always genetically identical, and no monoploid members occurred. It is concluded that the induced deficiencies caused atypical development resulting in zygotic or embryonic cleavage.Scientific Article No. A2070, Contribution No. 5023 of the Maryland Agricultural Experiment Station, Department of Botany.     

Use of maize pollen by adult Chrysoperla carnea (Neuroptera: Chrysopidae) and fate of Cry proteins in Bt-transgenic varieties

We investigated the use of maize pollen as food by adult Chrysoperla carnea under laboratory and field conditions. Exposure of the insects to insecticidal Cry proteins from Bacillus thuringiensis (Bt) contained in pollen of transgenic maize was also assessed. Female C. carnea were most abundant in a maize field when the majority of plants were flowering and fresh pollen was abundant. Field-collected females contained an average of approximately 5000 maize pollen grains in their gut at the peak of pollen shedding. Comparable numbers were found in females fed ad libitum maize pollen in the laboratory. Maize pollen is readily used by C. carnea adults. When provided with a carbohydrate source, it allowed the insects to reach their full reproductive potential. Maize pollen was digested mainly in the insect's mid- and hindgut. When Bt maize pollen passed though the gut of C. carnea, 61% of Cry1Ab (event Bt176) and 79% of Cry3Bb1 (event MON 88017) was digested. The results demonstrate that maize pollen is a suitable food source for C. carnea. Even though the pollen grains are not fully digested, the insects are exposed to transgenic insecticidal proteins that are contained in the pollen.     

Targeted mutagenesis of a conserved anther‐expressed P450 gene confers male sterility in monocots

Targeted mutagenesis using programmable DNA endonucleases has broad applications for studying gene function in planta and developing approaches to improve crop yields. Recently, a genetic method that eliminates the need to emasculate the female inbred during hybrid seed production, referred to as Seed Production Technology, has been described. The foundation of this genetic system relied on classical methods to identify genes critical to anther and pollen development. One of these genes is a P450 gene which is expressed in the tapetum of anthers. Homozygous recessive mutants in this gene render maize and rice plants male sterile. While this P450 in maize corresponds to the male fertility gene Ms26, male fertility mutants have not been isolated in other monocots such as sorghum and wheat. In this report, a custom designed homing endonuclease, Ems26+, was used to generate in planta mutations in the rice, sorghum and wheat orthologs of maize Ms26. Similar to maize, homozygous mutations in this P450 gene in rice and sorghum prevent pollen formation resulting in male sterile plants and fertility was restored in sorghum using a transformed copy of maize Ms26. In contrast, allohexaploid wheat plants that carry similar homozygous nuclear mutations in only one, but not all three, of their single genomes were male fertile. Targeted mutagenesis and subsequent characterization of male fertility genes in sorghum and wheat is an important step for capturing heterosis and improving crop yields through hybrid seed.