K. R. Shivanna

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

SummaryMethods 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.

Pollen Biotechnology for Crop Production and Improvement

1. Pollen biology and pollen biotechnology - an introduction K. R. Shivanna and V. K. Sawhney Part A. Pollen Biology: An Overview: 2. Pollen development and pollen-pistil interaction K. R. Shivanna, M. Cresti and F. Ciampolini 3. Gene expression during pollen development D. A. Hamilton and J. P. Mascarenhas 4. Pollination biology and plant breeding systems P. G. Kevan Part B. Pollen Biotechnology and Optimization of Crop Yield: 5. Pollination efficiency of insects A. R. Davis 6. Pollination constraints and management of pollinating insects for crop production R. W. Currie Part C. Pollen Biotechnology and Hybrid Seed Production: 7. Cytoplasmic male sterility P. B. E. McVetty 8. Genic male sterility V. K. Sawhney 9. Self-incompatibility A. McCubbin and H. Dickinson 10. Chemical induction of male sterility J. W. Cross and P. J. Schulz 11. Male sterility through recombinant DNA technology M. E. Williams, J. Leemans and F. Michiels Part D. Pollen Biotechnology and Plant Breeding: 12. Barriers to hybridization K. R. Shivanna 13. Methods for overcoming interspecific crossing barriers J. M. van Tuyl and M. J. de Jeu 14. Storage of pollen B. Barnabas and G. Kovacs 15. Mentor effects in pistil-mediated pollen-pollen interactions M. Villar and M. Gaget-Faurobert 16. Pollen tube growth and pollen selection M. Sari-Gorla and C. Frova 17. Isolation and manipulation of sperm cells D. D. Cass 18. Isolation and micromanipulation of embryo sac and egg cell in maize E. Matthys-Rochon et al. 19. In vitro fertilization with single isolated gametes E. Kranz 20. Pollen embryos C. E. Palmer and W. A. Keller 21. Use of pollen in gene transfer H. Morikawa and M. Nishihara Index.

Pollen viability and pollen vigor

SummaryInvestigations were carried out to correlate pollen viability, assessed on the basis of a fluorochromatic reaction (FCR) test, with pollen vigor, assessed on the basis of the time taken for in vitro germination in pollen grains subjected to high humidity (>95% RH) and temperature (38 °C) or storage stress of Nicotiana tabacum, Agave sp., Tradescantia virginiana, and Iris sp. Both high RH and temperature, as well as storage stresses, affected pollen vigor before affecting pollen viability. The results are discussed in the light of available data on the viability and vigor of stressed pollen and of aged seeds. The need for consideration of pollen vigor, particularly in stored pollen, the inadequacy of the methods presently used, and some of the methods suitable to assess pollen vigor are elaborated.

Responses of tobacco pollen to high humidity and heat stress: viability and germinability in vitro and in vivo

SummaryResponses of pollen grains of Nicotiana tabacum to high humidity (95% RH, 4 h) and temperature (38°/45° C, 4 h) stresses were investigated. Pollen grains were subjected to only RH or only temperature, or to both of these stresses. Their viability was assessed on the basis of the fluorochromatic reaction (FCR) test, and vigour was assessed on the basis of the time taken for in vitro germination as well as on the emergence of pollen tubes through the cut end of semi-vivo implanted styles. None of the stress conditions affected pollen viability and high RH or high temperature stress did not individually affect pollen vigour. However, pollen vigour was markedly affected when both the stresses were given together. Pollen grains subjected to high RH at 38° C took a longer time to germinate in vitro and the pollen tubes emerged later from the cut end of the semi-vivo styles; division of the generative cell was also delayed. Pollen grains subjected to high RH at 45° C failed to germinate in vitro, but did germinate on the stigma. Many pollen tubes subjected to this treatment showed abnormalities, and the growth of pollen tubes in the pistil was much slower than that observed in other treatments. Pollen samples subjected to all of the stress conditions were able to induce fruit and seed set. The implications of these results on the relationship between the FCR test and viability, and between viability and vigour, especially in stressed pollen, are discussed.

Intergeneric hybridization between Diplotaxis siifolia, a wild species and crop brassicas

SummaryAttempts were made to obtain intergeneric hybrids between Diplotaxis siifolia, a wild species, and cultivars of Brassica (B. campestris, B. juncea, and B. napus). The crosses showed unilateral incompatibility. When the wild species was used as female parent, pollen germination and pollen tube growth were normal, but hybrid seeds aborted due to post-fertilization barriers. Reciprocal crosses (cultivars as female parent) showed strong pre-fertilization barriers; although pollen grains showed germination, pollen tubes failed to enter the stigma. Hybrids were realized in two of the crosses, D. siifolia x B. juncea and D. siifolia x B. napus, through ovary culture. The hybrids were multiplied in vitro by multiplication of axillary shoots, or somatic embryogenesis. Detailed studies were carried out on the hybrid D. siifolia x B. juncea. F1 hybrids had shrivelled anthers and were pollen sterile. Amphiploids of this hybrid showed 60% pollen fertility and produced seeds upon self-pollination as well as backcross pollination with the pollen of B. juncea.

Intergeneric hybrids between Enarthrocarpus lyratus, a wild species, and crop brassicas

SummaryAttempts were made to produce intergeneric hybrids between Enarthrocarpus lyratus, a wild species, and several species of crop brassicas: B. campestris, B. nigra, B. oleracea, B. juncea, B. napus and B. Carinata. Hybrids using E. lyratus as female parent were realized by means of embryo rescue in four combinations — E. lyratus x B. campestris, E. lyratus x B. oleracea, E. lyratus x B. napus and E. lyratus x B. carinata. Reciprocal crosses showed strong pre-fertilization barriers and yielded no hybrids except in one combination — B. Juncea x E. Lyratus — in which a single hybrid could be realized. All of the hybrids were multiplied in vitro through the multiplication of axillary shoots. Morphological and cytological studies confirmed hybridity. All hybrids were completely pollen sterile except for E. lyratus x B. carinata, which showed 2% pollen fertility. Attempts to double the chromosome number through the in vitro application of colchicine to axillary meristems of F1 hybrids were successful in only one hybrid, E. lyratus x B. oleracea. Cytological studies of the hybrids indicated the presence of a partial homology between the genomes of E. lyratus and crop brassicas. Backcross progenies were raised from all of the five F1 hybrids to develop malesterile alloplasmic lines.

Production and characterization of interspecific hybrids between Brassica maurorum and crop brassicas

Abstractu2002Seven interspecific hybrids were produced between Brassica maurorum (♀), a wild species resistant to Alternaria blight and white rust, and all the monogenomic (B. campestris, B. nigra and B. oleracea) and digenomic (B. juncea, B. napus and B. oleracea) crop brassicas (♂) through embryo rescue. The hybrids were confirmed by means of morphological and cytological studies. All the hybrids were pollen-sterile. Amphidiploids were induced in three of the hybrids: B. maurorum×B. napus, B. maurorum×B. carinata, B. maurorum×B. nigra. The hybrids were also confirmed through DNA analyses for nuclear and organelle genomes using RAPD and RFLP techniques.

Pollen-Pistil Interaction and Control of Fertilization

Fertilization in flowering plants is a unique phenomenon and involves two fusion processes — syngamy between one of the male gametes and egg, and triple fusion between the other male gamete and fusion nucleus. Successful fertilization leads to fruit and seed development. The processes leading to fertilization are initiated on the stigma. Soon after pollination the crucial decision of recognition followed by subsequent acceptance or rejection of the male partner is made by the pistil. A chain of sequential, integrated processes in the pistil following pollen recognition regulate the post-pollination behaviour of the pollen. If the pollination is compatible, the pollen grain germinates on the stigma, and the pollen tube grows through the style and eventually reaches the embryo sac where it discharges the male gametes. If the pollination is incompatible, the pistil will effectively prevent fertilization, by inhibiting either pollen germination, entry of the pollen tube into the stigma, or growth of pollen tube in the style. Pollen-stigma interaction, therefore, is of paramount importance in the biology of sexual reproduction, because the vital function of selection of the male gametes in flowering plants is performed not by the egg, but by the pistillate tissue (see Shivanna 1979). For a rational understanding of the pollen-pistil interaction and fertilization it is important to understand the structure of the pollen grain and the pistil in relation to their function of recognition and rejection.

Pollen selection for Alternaria resistance in oilseed brassicas : responses of pollen grains and leaves to a toxin of A. brassicae

SummaryThe effects of destruxin B, a host-specific toxin of Alternaria brassicae that causes black spot disease in oilseed brassicas, were studied on in vitro pollen germination and pollen-tube growth of Brassica campestris var “brown sarson”, B. juncea, B. napus cvs “Westar” and “Cresor”, B. nigra and Sinapis alba. Pollen grains of B. nigra, B. juncea and B. campestris were the most sensitive and those of S. alba the least sensitive to the toxin. Effects of the toxin were also studied on the leaves of these species, and the degree of sensitivity of leaves of different species was comparable to that of their pollen grains. The results on the responses of pollen grains as well as leaves to the toxin are in agreement with the degree of susceptibility/resistance of these species to A. brassicae reported in the literature, indicating that the genes imparting susceptibility/restistance are expressed in the pollen, a prerequisite for pollen selection. Results are also presented which show that the toxin fed to the cut end of isolated inflorescence axis is readily taken up by the developing pollen and results in the inhibition of germination of susceptible pollen. This technique offers a simple and effective method for application of selection pressure to eliminate pollen grains susceptible to the toxin from effecting fertilization.

The role of the pistil in screening compatible pollen

SummaryResults of in vitro studies on pollen germination and tube growth in the presence of leachates from bisected pistils in Crotalaria retusa provide evidence for the operation of selection pressure during pollen-pistil interaction — a process which stimulates growth of a limited number of pollen tubes giving them an advantage over others in effecting fertilization.