Optimization Of Potato Anti-PPO Gene Transformation System And Analysis Of PPO Activity And PPO Isozyme Of Transgenic Plants

Many factors such as different kinds of explants, pre-cultivated time, co-cultivated time,culture medium,infected time and so on have influences to the rate of transformation when the Agrobacterium tumefaciens with anti-sense POT32 gene was used to infect different varieties. In this paper,the system of transformation were improved in order to gain more transgenic plants.In addition,the rules about the changes of Polyphenol Oxidase(PPO) activity, physiology biochemical activity and PPO isozyme of transgenic homozygous tetraploid of GD-9-qc were investigated and analysed when they were planted in the field. Research results are as follows: 1.Agrobacterium tumefaciens mediated transformations were conducted for 7 varieties(Atlantic,GANNONGSHU No.1 and No.2,dihaploid line 83-8,NONGSHU No.3,and Shapedy) by using PC3-ASPPO-qc. The transformation lines of the Atlantic , 83-8,NONGSHU No.3 and Shapedy were obtained. The result shows : 1)The best of the callus culture medium and the best regeneration medium were MS +0.5 mg/L NAA +2.0 mg/L 6-BA and MS +5.0 mg/L GA3 +2.25 mg/L 6-BA+ 3.00mg/L Zeatin respectively . 2)Percentage of resistant callus of leaf discsnwas up to 43.74% when pre-cultivated 2 days and co-cultivated 3 days after infected 10 min. Percentage of resistant callus of stem and minituber were up to 50% and 56.25% respectively when pre-cultivated 3 days and co-cultivated 3 days after infected 8 min and 10 min respectively . Various explants would became browning easily then stop growing when pre-culture time,infected time and co-cultivated time were 3 days,10 min and 3 days respectively.As a result, the rate of resistant callus declined.The callus rate reached 89% when infected 8 to 10 min since the contamination were cortroled effectively,as a result,the frequency of tranformation was high correspondingly.3)The　Kanamycin sensitivity experiment showed that suitable concentrations for selection of resistant transformation celles were variable for different explants(25mg/L for leaf discis, 50mg/L for minituber and stem). 4) The frequency of the resistant callus and the rate of transformation were different to various explants. The resistant callus rate and transformation frequence of stem were 23.8% and 3.01%.For mintuber they were 27.56% and 5.4% respectively.In addition,varieties with different genes have different resistant callus rate and transformation frequence. Among all varieties,the resistant callus rate and transformation frequence for 83-8 were 22.5%　and　1.63%　respectively, for NONGSHU No.3,Atlantic　and　Shapody　were　16.3%　and　0.37%,21.5%　and　0.7%,19.5%　and　0.5% respectively. 2.In analysis of PPO activities of transgenic homozygous tetraploid lines of GD-9-qc ,the result showed:1) PPO activity of ground stems was different in different test time remarkably.They were changed from 0.49　to　3.55(U/g.FW). PPO activities of four strains were higher than the comparison . 2) The changes of PPO activitives of different leaves were similar in whole life time.Most of them had 3 PPO activity peaks. 1,2,3 peak were in the cubhood, expanding period and mature period of potato tuber respectively. 3)In the all life time of tuber ,all transgenic lines had similar change laws in PPO activitives,but PPO activity of different varities were different remarkably at the same period. PPO activities at the tuber shapping time and expanding period were higher than other preiods. PPO activities of 9 strains were higher than the comparison .4) Among different tissue (leaf,ground-stem and tuber), PPO activity of leaf was the highest, but for ground-stem it was the lowest and potato tuber took second place. 3.Brown-degree(BD) of potato tuber have positive relationships with PPO activity and the hydroxybenzene content respectively .The correlative coefficient of them were 0.9095 and 0.8158 respectively. The hydroxybenzene content of most transgenic lines were higher than the control line.For example,the hydroxybenzene contents of GD-9-qc-26 and GD-9-qc-44 were 0.5mg/g and 0.4mg/g respectively which were 57.2% and 65.7% higher than the control line. PPO activity of them were 63.74% and 62.56% lower than the control line.BD of GD-9-qc-4 was only 3.01 which was 60.65% lower than the the control line and it,s PPO activity was 4.3(U/g.FW)which 71.6% lower than the control line.4.CAT activities and the soluble-protein content of the transgenic lines changed obviously. The soluble-protein content of the control line was 5.6mg/g but in the transgenic varities it fluctuated from 4.4636 mg/g to 15.65mg/g. CAT activity of the control line was 0.0672(mL/min)but in the transgenic lines it changed between 0.0104(mL/min)and 0.9408(mL/min) 5.According to the analysis of PPO isozyme and POD Isozyme of the transgenic varities,the conclusion is that the anti-sense PPO gene can not only depress the PPO isozyme to some degree,but also have a great influence on the expression of the POD isozyme. The number and the color-degree of the peculiar bands of isozyme can reflect PPO activity diversification.The main band of tuber PPO isozyme of one strain was disappeared and the subordinate bands of 4 strains were reduced. According to the PPO high activity in leaves and low activity in tubers ,2 target transgenic strains (GD-9-qc-28,GD-9-qc-44) were selected.