| Studies on the Gcnetic Engineering of TOlerance toLow-temperaturc Swectening of Potato TuberAbstractAll potato (Solanum tuberrosum L.) cuftivars presently used for the production ofpotato chips and fries accumulate excess free reducing sugars when exposed to coldstress (<8C). As raw potatoes are sliced and cooked at high temperatures in oi1,reducing sugars react with the abundant free amino acids in the potato cell, formingunacceptable brown-to black--Pigmellted products via a non-enzymatic Maillard-typereaction. This process of cold-induced sweetening eclipses the benefits of cold storage,i.e. less shrinkage, less disease loss, and decreased sprouting. The enzyme AcInv (acidinvertase, P -fructosidase, EC 3.2.l .26;l) Or synthase (EC 2.4.1 .l3, 2) catalyzes thelast step common to the sweetening process in potato that can catalyses the cleavage ofsucrose to glucose and fructose. lt is idefltified as key factors to raise the contents oftuber reductive sugar. The low-temperature sweetening not only decreases the contentsof starch and dries contents in tubers but also affect the quality of edibility andappearance. Up to now, General breeding methods have getting fewer progresses attuber resistance to low-temperature sweetening because of its technical limits.According to biochemical mechanism of tuber low-temperature sweetening, acidinveftase (AcInv) plays a key role in increasing the contents of reductive sugar. It maybe a short-cut way for potato breeding of low- temperature-resistance varieties byanti-restraining the transcription and expression of AcInv gene.In these studying, potato genergl RNA is extracted from the in vitro of GannongshuNo. 1 Which are stored in low temPerature (l OC) fOr one or two weeks. The first chain ofcDNA is compounded with using of RT-RNase and Oligo (dT)l5. According to knownsequences of AcInv gene, we clone a l.92bp distinctive fragment by PCR usingartificial synthetic special primer and combined it with plasmid pGEM-T Easy Vectorand transform Ecoi DH5 Q with it. Digesting by enzyme and sequence analysisindicated that this fragment is composed by 19l7 bp Oar, which coding a 639 AAfragment Which fits in with reports of AcInv gene well. This recombined p1asmid iscalled as pGEMA. By cuting off the aimed fragment from pGEMA and replacing GUSgene of plasmid pBI121 with it in opposite direction, we construct the plant expressionvector pBIKA of AcInv gene and ifltegrated it illto agrobacterium LBA4404.Analyzing the transformation effects of different recipient material, stem clltting isbetter than others for its high callus induction efficiency, strong ability to differentcapability to stand browning in some degree and easy to obtain. Tuber chip is anotherchoice While 1eaf disc is the most difficult to transform. Microtuber slices were culturedon MS medium with 2%PVP in dark about l0 days to prevent from browning.It was reported that AgNO3 could facilitate potato leaf disc callus induction. In this study, the effect of AgNO3 has also been tested. The result as follows, AgNO3 did not play a distinct part in transformative leaf discs callus induction and bud differentiation, reversal, it intensified browning extent, on the other hand, it improved the induction efficiency of transformative potato slice callus. Transformative potato slices could germinate directly on medium containing 10 mg/L AgNO3 Adding lOmg/L AgNO3 in medium can promote bud differentiation directly from microtuber slices. We have obtained regeneration plants from stem segments, leaf discs and microtuber slices by modulating transformation system.As co-cultivation time is concerned, for stem cutting, leaf discs and potato slices 2 days, 3 days and I day are appropriate respectively. Induction factor acetosyringone (AS) facilitated the transformation of all kind of recipients. And it is found when co-used with I ~iM proline (Pro) together the transformation efficiency was improved. Because of economic factor, the...
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