Studies On Genetic Variation Of Potato Protoplast-derived Plants And Stability Of RAPD

Collecting,creating new germplasm resources and enlarging the orign of variation are the important works in crop breeding research . Almost all potato cultivars are homogeneous tetraploid .Its complex genetic segregation and troublesome selecting work which make the conventional breeding very difficult .New breeding technology -protoplast fusion , genetic trasformation and somaclonal variation can overcome these shortcomings in some degree and make a new way for breeding .Protoplast-derived plants often generate a high somaclonal variation ,using the specific traits to get variants and selecting new variety could be a way of potato improvement. At the same time,however, disadvantage of this variation to protoplast fusion and genetic trasformation deserve attention . Therefore studying the genetic variation of protoplast-derived plants has very significance .The morphology, chromosome variation and RAPD polymorphism of 54 plants regenerated from mesophyll cell protoplasts were analyzed ; and also DNA extraction and the stability of RAPD were studied.Total 39 clones derived from the protoplasts of dihaploid 3#and 15 clones derived from the protoplasts of dihaploid 8# were analyzed.The results showed that the morphological variations were universal. Including tuber> height N and leaflets.Except 15 regenerated plants occurred much or no variation, 39 had only little variation . The plants derived from the same callus were similar in morphology.Total 54 clones were examined in chromosomal level ,7 clones had normal chromosome number ( 2n= 24 ) ; 40 clones were tetraploid ( 2n= 48 ) ; a few aneuploid and hexploid ( 2n= 72 ) were found .Endomitosis and the ring -like chromosome were also observed during mitosis .Contrasting morphology results with chromosome results, it was obviously that the chromosomal variation often led to morphological variation .Total 23 and 23 random primers were used to analyze RAPD polymorphism of plants from 3# and 8#. respectively. The results showed that all regenerated plants were different from their parents in the site examined. The genetic variation frequency of 3# was from 5.6% to 18.2%, and 8# was from 4.3%-32.6%. The variation of molecular level was also universal .The molecular variation,however, was not always leading to morphologicalvariation.The method of little amount material DNA extraction was improved in some degree. By optimizing RAPD reaction parameter ( The concentration of DNA templates is from 150 ug/.L to 300ug/.L, The concentration of MgCl2 is from 1.5mmol/L to 4mmol/L, The concentration of dNTP is from 100umol/L to 300umol/L, The concentration of primer is from 0.3umol/L to 3.0umol/L.) , the more stable RAPD result was obtained.The genetic variations were first syntheticly analyzed at three different levels for the protoplast-derived plants in potato, which give us an overall knowledge of these variations. The results could provide the theoretical base for potato breeding using protoplast culture system .In addition,little material DNA extraction method was improved and optimized reaction parameter of RAPD could improve its stability in some degree.