Reproductive Biology And Techniques Of Chromosome Doubling In Eucalyptus

Eucalyptus is one of the most worldwidely planted hardwood tree species which has a great performance such as vigorous growth, better timber quality and higher disease resistance. However, the process of new variety breeding in this species remained stagnant for a long time due to the weak foundation of reproductive biology and limitations of the crossbreeding technique. Polyploid breeding, which combines the merits of both genome dosage effects and heterosis, will be of great importance in genetic improvement. In this research, three eucalyptus clones (Eucalyptus urophylla ’U51’, E. urophylla × E. tereticornis ’DH101-1’ and E. urophylla × E. grandis ’DH18-16’) were studied on the detail information and relationship between flower development and sporogenesis, and the techniques of chromosome doubling. Major results as follows.1. The challenges in research of techniques on reproductive biology in Eucalyptus has been solved which could efficiently identify the key point of flower development and megasoporogenesis in Eucalyptus. The shedding of outer opercle in Symphyomyrtus which including E. urophylla, E. urophylla × E. tereticornis and E. urophylla x E. grandis is the morphological hallmark of the occurrence of microsporogenesis. A new efficient method has been founded in order to solve the problems of paraffin section in Eucalyptus caused by keratin bud sheath. These results lay a good foundation in further studies in reproductive biology in this tree species.2. Based on the knowledge of the rules of microsporogenesis in E. urophylla × E. tereticornis ’DH101-1’, the process of male meiosis stages could be distinguished by the flower bud diameter growth. Results also showed that, the meiosis in Eucalyptus were normal, cytokinesis were simultaneous, flower development in different position of the branches were asynchronous, the meiosis process in same bud were also asynchronous, the developmental stage of microsporogenesis in the longer stamens was always ahead of the shorter one, a correlation between microsporogenesis and flower bud development has been found, which were occurred a little later then the shedding of outer opercle. Leptotene occured when the diameter of flower bud was bigger than 3.0mm, when the flower bud diameter grew to 3.5mm, the meiosis stages were in prophase Ⅱ to tetrad. These results lay a good foundation in further studies in chromosome doubling of pollen mother cells.3. Based on the knowledge of megasporogenesis and embryo sac development in E. urophylla× E. tereticornis ’DH101-1’, the process of female meiosis stages could be distinguished by micorsporogenesis process between two length of stamen. Observation of massive flower buds sections in E. urophylla × E. tereticornis showed that, a definite proportion of ovules which were bom in the top side of the placenta were dysplasta while the magasporogneisis in the normal ovules were developed normally, the development of embryo sacs belonged to polygonum type. The development of flower buds in different locations on the branch were asynchronous, the diameters of flower buds which were in the bottom of the branch were larger than those in the top sides. There is a sequential relationship between megasporogenesis and microsporogenesis, the meiosis stages of longer and shorter stamens were from metaphase Ⅰ to telophase Ⅰ and interphase while the meiosis stages of megaspore mother cells were in pathytene, the meiosis stages of longer and shorter stamens were from prophase Ⅱ to telophase Ⅱ and from leptotene to pathytene while the meiosis stages of megaspore mother cells were from diplotene to diakinesis, the meiosis stages of longer stamens were in binucleate pollen while the development of embryo sac was mature, the highest corresponding rate of typical stages was up to 84.13%. The meiosis stages of megasporogenesis and embryo sac development could be distinguished by microsporogenesis development which could be observed conveniently. These results lay a good foundation in further studies in chromosome doubling of megaspore mother cells and embryo sacs.4. A method of pollen chromosome doubling by colchicine treatment by sustained-release injection with Ⅳ bag was proposed, and 2n pollen in Eucalyptus was successfully artificially induced for the first time. Based on the knowledge of reproductive biology and the method to distinguish the development stages of sporogenesis, chromosomes doubling techniques were studied in order to verify the most efficient treatment condition. Results showed that, Eucalyptus did not have sensitivity to heat, colchicines treatment brought better results than high temperature treatment, the best way to implement chromosomes doubling with colchicines treatment was by sustained-release injection with IV bag for 6h, the highest rate of effective processing was 28.71%. Eucalyptus 2n pollen was spherical and had a 40% larger volume while normal untreated pollen was tetrahedron. Results of megasoporogenesis and embryo sac chromosomes doubling with colchicines treatment has not been concluded yet before ploidy detection with offspring. The success of the induction of 2n pollen in Eucalyptus indicates a possibility of induction of triploid through chromosomes doubling on megaspore mother cells and embryo sacs, which lays a good foundation in further polyploid breeding works in Eucalyptus.