Genetic manipulation of sucrose-storing tissue to produce alternative products

[摘要] The main aim of the work presented in this dissertation was to explore the possibilityto genetically manipulate the sucrose storing crops, sugarcane and sweet sorghum, toconvert their sucrose reserves into higher-value alternatives. For the purpose of thisstudy we focussed on fructans as alternative sucrose-based high-value carbohydrates,since these fructose polymers are of significant commercial interest. To investigatethe technical feasibility of transforming sugarcane and sweet sorghum to produce thisnovel carbohydrate, we proposed to transfer the fructosyltransferase genes fromCynara scolymus into these plants by means of particle bombardment.In order to apply this technology to sweet sorghum, an in vitro culture system suitablefor transformation had to be established. For this purpose an extensive screeningprocess with different combinations of variables were conducted. Though therelationships between these variables proved to be complex, it was concluded thatimmature zygotic embryos could be used to initiate a genotype-independent totipotentregeneration system with a 65% callus induction rate, provided that initiation takesplace during summer. Stable transformation and regeneration of these calli werehowever not successful and will have to be optimised to allow future applications.By introducing fructosyltransferase genes into sugarcane, we succeeded intransforming sugarcane into a crop that produces a variety of fructans of the inulintype.Low molecular weight (LMW) inulins were found to accumulate in the matureinternodes of 42% of the transgenic sugarcane plants expressing the sucrose:sucrose1-fructosyltransferase (1-SST) gene, and in 77% of the plants that incorporated both1-SST and fructan:fructan 1-fructosyltransferase (1-FFT), while only 8% of theseplants accumulated high molecular weight (HMW) inulins. Our results demonstratedthat sugarcane could be manipulated to synthesise and accumulate fructans withoutthe induction of phenotypical irregularities.Inulins with a degree of polymerisation up to 60 were found in sugarcane storagetissue. In these HMW inulin-producing plants, up to 78% of the endogenous sucrosein the mature sugarcane culm was converted to inulin. This enabled inulinaccumulation up to 165.3 mg g-1 fresh weight (FW), which is comparable to that found in native plants. These transgenic sugarcane plants, therefore exhibit greatpotential as a future industrial inulin source.Fructan production was detected in all the sugarcane plant tissue tested,predominantly as 1-kestose. In contrast with the fact that fructan accumulation inleaves did not affect the endogenous sucrose concentrations in these organs, thesucrose content of mature internodes that accumulated high levels of 1-kestose wasseverely reduced. However, increases in total sugar content, in some instances up to63% higher than control plants, were observed. This phenomenon was investigatedwith the use of radio-labelled-isotopes. An increase in the allocation of incomingcarbon towards sucrose storage, resulting in higher carbon partitioning into both 1-kestose and sucrose, were detected in the culms of transgenic compared to controllines. This modification therefore established an extra carbohydrate sink in thevacuoles that affected photosynthate partitioning and increased total soluble sugarcontent. The data suggests that sucrose sensing is the main regulatory mechanismresponsible for adapting carbon flow in the cells to maintain sucrose concentration.

[发布机构] Stellenbosch University