Methods currently available for evaluating pollen quality in vitro include, (a) tests of germinability; (b) tests of the stainability of the vegetative cell contents; (c) tests for enzyme activity, and (d) the fluorochromatic procedure (FCR), which tests principally the integrity of the plasmalemma of the vegetative cell. Using germinability in vitro as a standard, a comparison has been made between histochemical methods of classes (b), (c) and (d) in application to various pollens, immature, mature, and treated in ways known to affect viability and membrane state. Predictably, the lowest correlation was obtained with tests of stainability. The highest was given by the FCR, which generally provided an excellent guide to potential germinability. The FCR procedure is subject to various limitations, however, (a) A high correlation between FCR and germinability can only be expected when mature, ripe pollen is used; with immature pollen, the FCR will predict excessively high potential germinability. (b) The FCR may also predict a higher potential level of pollen function than in vitro germinability when the germination medium is sub-optimal. In this situation, however, it will generally give a better guide to fertilising capacity, (c) The FCR is not a test of pollen viability. Like germinability in vitro, it can yield a negative score with pollen which is nevertheless capable of functioning. For example, false negatives will be obtained with some species if the pollen is not properly pre-conditioned by rehydration before testing, an important point in monitoring stored pollen. The paper includes a brief discussion of the rationale of pollen testing.

The evaluation of pollen quality, and a further appraisal of the fluorochromatic (FCR) test procedure.

Membrane state and pollen viability

The pathways of micro- and megagametophyte development in Agave fourcroydes (henequen) and A. angustifolia were studied. We used histology and light microscopy to observe anther ontogeny and ovary differentiation in relation to flower bud size. Both species have the same sexual reproductive strategies and gametophyte development that may be divided into three phases: (1) premeiotic, which includes the establishment of the megaspore mother cell and the pollen mother cell; (2) meiotic, the formation of mature microspores and functional megaspores; (3) postmeiotic, which encompasses the development of mature pollen grains and the formation of the embryo sac. A successive type microsporogenesis was found in both species with formation of T-shaped tetrads and binuclear pollen grains. In vitro germination tests revealed very low pollen fertility. The female gametophyte is formed from two micropylar megaspore cells after the first meiotic division (bisporic type). Male and female gametogenesis occur asynchronously with microsporogenesis finishing before macrosporogenesis. The results so far show that the formation of male and female gametophytes in henequen is affected at different stages and that these alterations might be responsible for the low fertility shown by this species.

https://bsapubs.onlinelibrary.wiley.com/doi/pdf/10.2307/3558424

Reproductive biology of henequén (Agave fourcroydes) and its wild ancestor Agave Angustifolia (Agavaceae). i. Gametophyte development

Phytophthora infestans
 resistant somatic hybrids of
 S. × michoacanum
 (+)
 S. tuberosum
 and autofused 4
 x
 S. × michoacanum
 were obtained. Our material is promising to introgress resistance from
 S. × michoacanum
 into cultivated potato background.
 Solanum × michoacanum (Bitter.) Rydb. (mch) is a wild diploid (2n = 2x = 24) potato species derived from spontaneous cross of S. bulbocastanum and S. pinnatisectum. This hybrid is a 1 EBN (endosperm balance number) species and can cross effectively only with other 1 EBN species. Plants of mch are resistant to Phytophthora infestans (Mont) de Bary. To introgress late blight resistance genes from mch into S. tuberosum (tbr), genepool somatic hybridization between mch and susceptible diploid potato clones (2n = 2x = 24) or potato cultivar Rywal (2n = 4x = 48) was performed. In total 18,775 calli were obtained from postfusion products from which 1,482 formed shoots. The Simple Sequence Repeat (SSR), Cleaved Amplified Polymorphic Sequences (CAPS) and Random Amplified Polymorphic DNA (RAPD) analyses confirmed hybrid nature of 228 plants and 116 autofused 4x
 mch. After evaluation of morphological features, flowering, pollen stainability, tuberization and ploidy level, 118 somatic hybrids and 116 autofused 4x
 mch were tested for late blight resistance using the detached leaf assay. After two seasons of testing three somatic hybrids and 109 4x
 mch were resistant. Resistant forms have adequate pollen stainability for use in crossing programme and are a promising material useful for introgression resistance from mch into the cultivated potato background.

https://link.springer.com/content/pdf/10.1007%2Fs00299-013-1422-5.pdf

Development of somatic hybrids Solanum × michoacanum Bitter. (Rydb.) (+) S. tuberosum L. and autofused 4x S. × michoacanum plants as potential sources of late blight resistance for potato breeding.

Potato pollen stored in liquid nitrogen (−196 C) showed no significant decrease in percentage germination after nine months, whereas pollen stored at −20 C showed a decrease in germination to <1% over the same period.

Advantages of storage of potato pollen in liquid nitrogen

Three staining methods (acetocarmine, fuchsin and oxidation of benzidine) and germination in vitro and in vivo were applied to estimate pollen fertility in Solanum species and dihaploids. Pollen was divided into six classes based on shape and contents of the grains. With acetocarmine, fuchsin, peroxidase and germination in vitro 4, 3, 2 and 1 classes, respectively, are supposed to be included in the percentage of ‘good’ pollen as measured by these methods. This percentage therefore, in more than 96% of the cases studied, shows a decrease in the order indicated. Neither aging of pollen at room conditions nor collecting pollen from flowers on 1–9 days after anthesis does influence the percentage of ‘good’ pollen with acetocarmine and fuchsin, whereas this percentage drops sharply to zero with peroxidase and germination in vitro. The latter two methods apparently measure as ‘good’ pollen only the grains with living cytoplasm. When pollen is collected at three successive dates from the same flowers the percentage of ‘good’ pollen drops sharply with all methods used. There is a relation between quantity of pollen per flower and pollen quality (% ‘good’), low-quantity pollen containing significantly lower percentages of ‘good’ pollen than medium- and high-quantity pollen. The latter two are not significantly different in this respect.

A.W.B. Janssen

Papers

Estimating pollen fertility in Solanum species and haploids