| In the present paper,five kinds of representative fruit crops including citrus,apple,strawberry,pear and kiwi fruit were used to investigate the approaches of in vitro conservation and the genetic variations,The main results were as follows:1 Single-cell sibling lines of 5 citrus genotypes and single-bud sibling lines of 2 apple and 2 strawberry genotypes were established,which provided materials for the studies on somaclonal variation.2 A method to calculate mitosis index and to estimate cell ploidy was put forward and experimentally verified,and then was used to calculate in vivo the mitosis index of different ploid cells in the same citrus callus. The results indicated that quantitatively predominant diploid cells inhibited the mitosis of tetraploid cells in diploid genotype. Later,these tetraploid cells disappeared via programmed cell death under the normal subculture condition. However,this inhibition by the diploid cells was neutralized by low-density culture condition.3 Approach for in vitro conservation of 5 kinds of fruit crops were established and optimized. The average growth mass of material conserved for one year without subculture by slow-growth was just equal to that of 3-week culture under routine subculture condition. The conserved materials survived after being transferred into routine culture condition. With regard to cryopreservation,viability of citrus calli was above 90% whereas shoot-tips of apples and strawberries were 72.7% to 89.3% respectively,as indicated that cryopreservation was more suitable than slow-growth for citrus callus conservation,and slow-growth method was suitable for the conservation of shoot-tips of apple and strawberry.4 Chromosome variation in citrus calli occurred at the early stage of the callus formation was observed. However,the relative genetic stability of chromosome ploidy was maintained during subculture and in vitro conservation,which resulted from the disappearing of variant cells by programmed cell death.5 RAPD markers were adopted to detect the DNA-level variations of citrus callus during subculture and in vitro conservation. As for 8 single-cell sibling lines of Newhall navel,a total of 9 796 bands obtained from the subculture calli with 102 primers. Most of those bands were the same only except 4 bands obtained from the primer OPH-15,as showed a variation frequency of about 0.04%. While no aberration was observed in 9 792 bands obtained from cryopreserved callus. Similarly,8 624 bands without diversity were obtained from 8 Red Marsh single-cell sibling lines conserved in vitro by slow-growth method.6 AFLP markers were also used to detect the DNA-level variations of shoot-tips from single-bud sibling lines of apple and strawberry. After the DNAs of apples were digested by Mse I and EcoR I and amplified with 20 pairs of specific primers,a total of 29 760 bands were observed from Malus pumila var. Gala conserved by slow-growth and 28 480 bands from cryopreserved M. pumila var. M26,no variant band was found. After the DNAs of strawberries were digested by Mse I and Pst I,the latter one sensitive to DNA methylation,and amplified with 16 pairs of specific primers,a total of 20 992 and 21512 bands from Fragaria vesca var Heilongjiang No.l conserved by slow-growth and cryopreserved F. xananassa var. Joho was obtained respectively. As for the former,no difference in band profile was found. But there was a new band scored from every single-bud sibling lines of the latter,which resulted from DNA methylation.7 The transformants of citrus callus,pear and kiwifruit shoot-tips with GUS gene were successfully conserved in vitro by slow-growth and cryopreservation. Chemical stain,RAPD,SSCP marker and Southern blotting confirmed the stable expression and integrity of GUS gene in host genome.8 MSAP markers were used to investigate the DNA methylation status of tested materials. DNA methylation level of Newhall navel callus incapable to regenerate embryoid was higher than that of callus with embryogenesis capacity. Conservation in vitro enh...
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