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Sexual plant reproduction requires multiple pollen–pistil interactions from the stigma (pollen adhesion, hydration, and germination) to the ovary (fertilization). Understanding the factors that regulate pollen tube growth is critical to understanding the processes essential to sexual reproduction. Many pollen tube growth assays (PTGAs) have shorter and slower pollen tube growth when compared to pollen tube growth through the style. The identification and study of factors that regulate pollen tube growth have been impeded by a lack of an efficient and reproducible PTGA. The objective of this research is to develop a robust assay for Nicotiana tabacum pollen tube growth in an environment that supports sustained and normal growth yet is amenable to testing the effects of specific factors. In this paper, we introduce a novel PTGA, which uses pistils from N. tabacum that lack a mature transmitting tract (TT) due to tissue-specific ablation. The TT-ablated style supports normal pollen tube growth and the hollow structure of the style allows modification of the growth environment by direct injection of test material. This PTGA is robust and allows for rapid and accurate measurement of pollen tube length and pollen tube morphology, supporting pollen tube growth from 20 to 35°C and at pH ranging from 4.8 to 7.6. Use of the ablated style for a PTGA is a novel method for the culture of pollen tubes with sustained growth in vivo while permitting the application of treatments to the growing pollen tubes.

Style morph frequencies (short∶mid∶long) were determined for a total of n = 11 918 plants in 16 Minnesota populations of Lythrum salicaria L. Nine populations were in the establishment phase, with population sizes ranging from n = 56 to n = 2 192. Most of these populations exceeded previously reported population sizes in the native European habitat. A nonparametric statistical test, the chi-square (χ2), can be used to determine if populations are at isoplethic equilibrium (1∶1∶1, short∶mid∶long); a χ2 value >5.99 is significant at the 5% level. Only one established population (White Bear Lake, n = 1991, χ2 = 3.0) fitted the null hypothesis for isoplethy, although all established populations contained all three style morphs. Pooled values for these populations indicated an excess of mids and longs, with shorts being deficient. Colonizing populations had a higher percentage of mids (54%) when compared to established populations (33.7%). Short styles were almost nonexistent (8%) in colonizing populations. Five out of the seven populations lacked at least one style morph. A review of the literature reporting style morph frequencies in tristylous L. salicaria revealed that no statistical analysis for isoplethy has been performed. Darwin originally assumed that all populations would be isoplethic, possessing equal numbers of all three style morphs, but concluded, without statistical analysis, that, instead, populations were anisoplethic. Since tests for statistical deviations from the expected frequencies (1∶1∶1) have not been used, χ2 analysis was performed. Several of these populations were at isoplethic equilibrium (Nadder χsu2 = 1.7, Blelham χ2 = 1.69, Potsdam χ2 = 1.5, Vestfold χ2 = 0.4, Buskerud χ2 = 5.62, Kilchberg χ2 = 0.35, Lausanne χ2 = 3.32, Canberra χ2 = 5.29, Massachusetts χ2 = 3.13), suggesting that the general conclusion of anisoplethy in tristylous L. salicaria is inappropriate.

Discriminating styles (DS), pollen-mediated pseudo-self compatibility (PMPSC), and general pseudo-self compatibility (PSC) phenomena were investigated by re-analyzing data from Petunia hybrida where known S genotypes were used. This demonstrated how female coefficient of crossability (FCC)/male coefficient of crossability (MCC) scatter diagrams and regression analyses aid in identifying and quantifying PSC within an self incompatible (SI) population. One of the female testers was identified by statistics to be SI, not DS, in contrast to what was reported in the original report, where all the plants were assumed to have operating DS. In addition, none of the females expressed PMPSC. Based on regression analysis and chi-square tests, a threshold between 27% and 31% PSC was estimated to be necessary for expression of DS. The presence of DS was also required to test for the existence of PMPSC as reported previously. The upper left-hand quadrant of the FCC/MCC scatter diagram which contains all the deviants from the theoretical SI model, is the location expression of DS has been identified. Placement for PMPSC deviants is not possible, due to the interrelationship with DS. Percent PSC did not directly equate with the different types of PSC phenomena but was useful for identifying and ranking DS in female parents. The compatible tester used in this experiment did not always produce the highest outcross seed set with the females as expected. Therefore, due to the confounding effects of the different types of PSC, it is important to choose the compatible testers with care. Regression analyses of FCC/MCC values indicated that S2.2 and S1.2 male testers did not behave in a similar fashion to S1.1 testers. It is hypothesized that this disparity could be the result of the expression of a general PSC gene, different from the DS or PMPSC genes, which is linked to the S2 allele. Since these general PSC effects associated with the S2 allele are minor in comparison to DS and PMPSC, it was necessary to distinguish the difference using statistical analysis.

The impact of ovule number on seed set calculations for self-incompatible (SI) species was investigated. Diploid Chrysanthemum was chosen for this study because accurate counts of the potential number of ovules could be made. Individuals in populations of C. carinatum, C. coronarium, C. c. subsp. spatiosum, and C. segetum were crossed in complete diallels. All species exhibited similar results. Therefore, only the diallel data from C. coronarium subsp. spatiosum were presented. The seed set data with and without ovule counts were processed by SIGMAS, a computer program designed to analyze SI data. Incorporation of the actual number of ovules into seed set diallels provided the most realistic representation of values for self-incompatibility studies. Data derived from equations excluding ovule counts might lead to inaccurate genetic interpretations. Ovule counts were significant between and within genotypes for self (disc and ray florets), but not cross (ray florets only) pollinations. The disc florets in self-pollinations were found to be responsible for increasing the variability in ovule number. The statistics indicate that the disc and ray florets composed two distinct populations. At the diploid level with a single daisy flower type, the disc floret numbers were variable, whereas ray florets were relatively static. This was not the case with polyploid chrysanthemums, where both ovule populations were dynamic and interactive. The conservative nature of percent pseudo-self-compatibility (%PSC) deems it necessary to obtain an accurate measure of female fertility. Values for this could be obtained using a bulk pollination or a tester with unmatched S alleles.

It is difficult to assess self-incompatibility (SI) as a phenomenon or to distinguish it from other reproductive barriers. Our objective was to demonstrate a method of separating reproductive barriers and other factors affecting SI expression. The analysis consists of four parts: (1) calculating male (MCC) and female (FCC) coefficients of crossability using seed set from a diallel crossing design; (2) determining significant male and female differences using a 1∶1 paired Chi-square test; (3) constructing scatter diagrams of FCC versus MCC; (4) performing linear regression analysis between FCC and MCC. Diallel data from eight genera with various reproductive barriers were used to illustrate and validate the usefulness of this method. SI can be distinguished from other reproductive barriers (incongruity, inbreeding depression, post-fertilization phenomena) using 1∶1 Chisquare and regression analysis. In addition, the incorporation of ovule counts is crucial for demarcating the SI phenomenon in species where ovule numbers vary between self- and outcross pollinations. Male and female steriles can be identified with the MCC/FCC equations and subsequently removed from further analysis.