Production Of Somatic Hybrid And Observation Of Organelle Morphology During Protoplast Regeneration In Citrus

In order to achieve citrus seedless breeding targets,the fusion between callus protoplast with the CMS features and mesophyll protoplast from seedy varieties was conducted to realize the transfer of CMS character.And the seedless hybrids/cybrids with excellent characteristic from callus and mesophyll parents may be produced.Symmetric fusions were conducted between embryogenic suspension protoplasts of Citrus unshiu and mesophyll protoplasts isolated from seedy Citrus cultivars,in an attempt to obtain the somatic hybrids with sterile cytoplasm from Satsuma mandarin.At the same time,we consecutively observed the organelle morphologic change during the process of protoplast fusion based on cytological techniques.The main results are as follows:1.Symmetric fusions were conducted between embryogenic suspension protoplasts of C.unshiu Marc.cv.‘Guoqing No.1'(G1)and mesophyll protoplasts isolated from the leaves of ‘Nova' tangelo [C.reticulata Blanco ×(C.reticulata Blanco × C.paradisi Macf.)] or ‘Red Anliu' sweet orange(C.sinensis Osbeck cv.‘Red Anliu').Six plants from G1 + ‘Nova' tangelo and eight plants from G1 + ‘Red Anliu'sweet orange were regenerated,respectively.Flow cytometry analysis showed that 6plants regenerated from G1 + ‘Nova' tangelo were tetraploid.Molecular marker analysis showed tetraploid plants had complementary specific bands from both parents and No.1,3 and 6 regenerated plants had chloroplast genome from ‘Nova' tangelo,and that of the other plants was from G1.Four randomly selected plants recovered from G1 + ‘Red Anliu' sweet orange were analyzed by flow cytometry and it showed that three were tetraploids and the remaining one was diploid.Molecular analysis confirmed that all tetraploid plants were true somatic hybrids,with chloroplast and mitochondrial from embryogenic callus parent and the regenerated diploid was identified to be originated from protoplast regeneration of G1,the callus parent.2.Chloroplasts in the fused products significantly reduced after culture for 7 days,and almost completely disappeared 10 days after,which could not be directly transmitted to the daughter cells.This phenomenon may be associated with that mesophyll protoplast cannot regenerate.Structure of endoplasmic reticulum in fused products was restoredafter 7 days of culture and endoplasmic reticulum was concentrated in the cell wall after16 days.Mitochondria from different cells are very different in quantity,morphology and position.In mesophyll protoplast,the number of mitochondria is small and the volume is large,which are located around the chloroplast;the number of mitochondria in calli is large,the volume is small and mitochondria located around the cell membrane and starch grain.Mitochondrial fusion and fission initiated when the cells fuse,and almost 90% of the mitochondria finished the genetic exchange within 16 hours.