ALTHOUGH THE NEW TECHNOLOGICAL ADVANCES IN GENETIC ENGINEERING, SOMATIC HYBRIDIZATION AND TISSUE CULTURE HAVE LONG-TERM POTENTIAL FOR GENERATION OF MAJOR VARIATIONS IN OUR CROP SPECIES, TRADITIONAL BREEDING METHODS, USING THE NORMAL SEXUAL PROCESS, WILL CONTINUE TO BE THE MAIN BASIS FOR MOST PLANT IMPROVEMENT PROGRAMMES. HOWEVER, OUR ABILITY TO CONTROL AND MANIPULATE THE POLLINATION PROCESS, POLLEN-PISTIL INTERACTION, FERTILIZATION AND SEED SET ARE STILL POOR, AND WILL DEPEND ON AN INCREASED KNOWLEDGE OF THE REPRODUCTIVE BIOLOGY SUCH AS DEVELOPED HERE. NEW METHODS COULD THEN BE DEVISED FOR ENGINEERING SOME CROPS THROUGH DIRECT POLLEN TRANSFORMATION, FOR THE ASSESSMENT OF POLLEN FERTILIZATION ABILITY, FOR LONG-TERM STORAGE PROCESS, FOR THE ISOLATION AND IDENTIFICATION OF SPERM CELLS, ALL METHODS WHICH CAN BE CONSIDERED INSTRUMENTAL IN CROP MANAGEMENT AND IMPROVEMENT.
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.
THE PURPOSE OF THE STUDY IS TO ENLARGE KNOWLEDGE OF PLANT REPRODUCTIVE BIOLOGY, FOCUSING ON THE POLLEN GRAIN AND THE MALE GERM UNIT (MGU) IT CARRIES.
IT SHALL SUPPORT THE DEVELOPMENT OF IN VITRO TECHNIQUES FOR PLANT BIOTECHNOLOGISTS BASED ON AN INCREASED UNDERSTANDING OF IN VIVO CONDITIONS.
THREE APPROACHES WILL BE FAVOURED :
- MGU CHARACTERIZATION IN THE IN VIVO CONDITIONS, INCLUDING CYTO-KINETICS OF MICRO-GAMETOGENESIS (SIENA + WAGENINGEN), THREE DIMENSIONAL RECONSTRUCTION OF BICELLED POLLEN GRAINS (SIENA + WAGENINGEN) AND OF TRICELLED POLLEN GRAINS (LYON + SIENA + WAGENINGEN), VIABILITY OF STORED POLLEN GRAINS (LYON + WAGENINGEN);
- IN VITRO MGU CHARACTERIZATION, INCLUDING SPERM CELL ISOLATION FROM BICELLED (SIENA + WAGENINGEN) AND TRICELLED (LYON + WAGENINGEN) POLLEN GRAINS, VIABILITY OF STORED SPERM CELLS (LYON), CYTOLOGICAL (LYON + SIENA + WAGENINGEN) AND BIOCHEMICAL (SIENA + LYON) CHARACTERIZATION OF SPERM CELLS;
- EXPLOITATION OF GERMPLAST FOR BIOTECHNOLOGY, INCLUDING THE IDENTIFICATION OF MALE-FEMALE RECOGNITION PHENOMENA AND IN VITRO FERTILIZATION TECHNIQUES (LYON + SIENA + WAGENINGEN).
Funding SchemeCSC - Cost-sharing contracts