Anna Alwen

In-situ seed production after pollination with in-vitro-matured, isolated pollen

Immature tobacco (Nicotiana tabacum L.) pollen has been isolated from anthers in three distinct stages of development, including the microspore stage. In in-vitro cultures, fully functional, mature pollen was obtained. In a germination medium, this pollen produced pollen tubes. After application to stigmas in situ, the in-vitro-matured pollen fertilized ovules, and seeds were produced. Genetic tests with seedlings obtained from pollinations with in-vitro-matured pollen from a transgenic plant revealed normal Mendelian segregation of two marker genes, the neomycin-phosphotransferase II gene and the nopaline-synthase gene. These results are of interest with respect to the control of self-incompatibility, cytoplasmic male sterility and pollen-allergen formation, and it offers an alternative route for gene transfer in those plants which cannot be regenerated in vitro.

Sporophytes and Male Gametophytes from in Vitro Cultured, Immature Tobacco Pollen

Anther culture is one of the few biotechnologies which has found its way into breeding practice. Still however, many species are recalcitrant and yields are low, indicating that the mechanisms by which immature male gametophytes change their development pathway to form a sporophyte directly and asexually, are not well understood.

Plant endogenous β-glucuronidase activity: how to avoid interference with the use of theE. coli β-glucuronidase as a reporter gene in transgenic plants

We have detected a plant β-glucuronidase activity, present in several tissues and organs of plant species belonging to different families. The fluorimetric β-glucuronidase assay was used to partially characterize this activity in post-ribosomal supernatants of tobacco leaves. The tobacco activity is very stable at low temperatures, but quickly inactivated above 45°C. It is relatively resistant to proteases and insensitive to-SH group reagents and to ionic conditions. It does not require, nor is it inhibited by, divalent cations. Although these properties are shared by theEscherichia coli β-glucuronidase, the two activities can be distinguished by: (i) their different sensitivity to the specific inhibitor saccharic acid-1,4-lactone; (ii) their different thermal stability (iii) their different pH optima (5.0 for the plant activity and close to neutral for the bacterial enzyme). Therefore, under appropriate experimental conditions, it should be possible to assay theE. coli β-glucuronidase in transgenic plants without interference from the endogenous plant activity.

Transformation of Pollen

In most transformation systems today, in vitro regeneration of plants is an essential step and very often the main limiting factor for yield and success. Circumvention of in vitro regeneration would therefore be highly advantageous. The simplest way to do this would be to transfer DNA into the gametes and, after gamete fusion, to exploit seed embryogenesis for plant formation. If feasable, such an approach would be technically simpler and faster than methods based on in vitro regeneration. It should also be universal in its application. In addition, one would avoid the problem of somaclonal variation resulting from in vitro regeneration. Somaclonal variation in transgenic commercial lines of crop plants can spoil the advantage obtained by transferring a transgene. The problem of chimaera that is intrinsic to in vitro regeneration, would also be avoided. Gametes in the strict sense are not easily accessible in plants although progress has been achieved in isolating both sperm and egg cells (1). Both, male as well as female gametes, can be used for gene transfer. The main advantage of the egg cell might be the efficient formation of a whole plant either in situ or after isolation and in vitro culture. The latter remains to be shown, however. The male gamete in higher plants is not a free, mobile cell as in lower plants or in animals, but is part of a larger structure, the pollen. And only the pollen as such is able to perform fertilization in situ, while the isolated sperm cell would have to be fused in vitro with the egg cell. Attempts to use male gametes for gene transfer have therefore concentrated on the transfer through pollen.

Embryogenic cultures of tobacco pollen as a model system to study plant rejuvenation.

Formation of pollen in angiosperms takes place in the anthers, the male sexual organs. During normal male gametophytic development in vivo, each diploid microspore mother cell generates, through meiosis, four haploid microspores; after one or two mitosis and maturation, they will eventually give rise to mature pollen grains. However, in in vitro cultures of isolated anthers or pollen, pollen grains are able to follow a different, sporophytic developmental pathway, leading to the formation of embryos and haploid plants directly and asexually. Recent advances in the technique of isolated pollen culture in the model plant tobacco, allow the strict control of pollen development in both the sporophytic and the gametophytic direction.