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Cultivated crops and pollen biotechnology

Research was carried out in the field of pollen biotechnology in order to devise an efficient cellular tool for further genetic manipulations with the germ cells: male germ cells (sperm cells and generative cells) housed in pollen grains and female cells (egg and central cells) housed in the embryo sac. Different approaches have been developed analysis of pollen quality, as a prerequisite for sperm cell isolation;
determination of methods for storing the pollen;
study of sperm cell ontogenesis (male programme, cytological and biochemical techniques);
germ cell isolation and characterization (in vitro techniques, cytological, immunological and physiological studies.

Pollen quality was estimated using multidisciplinary approaches (biophysical, biochemical and cytological tools) in Zea mays. Male germ unit involved isolation of 3-dimensional reconstruction which confirmed the concept of male germ unit (MGU) and sperm dimorphism.
Germ cell ontogenesis was examined on Brassica using cytological and biophysical tools. 3 different stages of protein products were determined: the first corresponded to the uninucleate microspore; the second to the mid binucleate stage; the third (mature pollen) showed a high protein specificity possibly involved in the compatibility or germination processes.
With sperm cell isolation involving Z mays, Brassica, Spinacia, Nicotiana, light and electron microscopic studies demonstrated that sperm cells were intact, viable and appeared to be true protoplasts.
Viable embryo sacs and female germ cell isolation were demonstrated as were in vitro fertilization and gamete manipulation.

Reported by

Università degli Studi di Siena
Via Mattiolo 4
53100 Siena
Italy
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