Acquirement,Bioassay And Safety Evaluation Of Transgenic Poplar Harboring Binary Insect-resistant Genes

Poplar belongs to Salicacea, Populus, which is the most widely-distributed and strong-adapted species in the world. It is not only the most important pulp material but also the perfect bioenergy tree species. But the widely-distributed man-made poplar forest is vulnerable to attacks of insect pests; genetic engineering offers an effective way to cultivate pest-resistant cultivars. China is the first country planting the commercialized Bt-poplar. The transformation of binary or more genes can delay the evolvement of Bt-resistance strain and increase the pest resistance spectrum. In our study, a binary gene construct that contains a chitinase gene encoding insect-specific chitinase from Manduca sexta and the BmkIT gene encoding a scorpion insect toxin from Buthus martensii Karsch were introduced into a poplar cultivar, Zhonglinmeihe by the newly-invented in situ bud transformation approach. The procedure was reported in detail, insect-resistances bioassay was presented, the mechanism of insect resistances was discussed and the feed biosafety of transformed poplar leaves was evaluated. The results were as follows.1. A novel approach for in situ bud transformation of Populus by Agrobacterium with binary pest-resistant genesTransgenic poplar shoots were obtained using Agrobacterium-mediated in situ bud transformation. Buds of Populus cathayana Rehd were co-cultivated with an Agrobacterium strain LBA4404 harboring the binary vector pBI101-Bmk-chi. With the procedure described in the thesis, a transformation efficiency of 1% and 2.24% were achieved in 2006 and 2007, respectively. Stable integration of the transgene sequence was confirmed by PCR and Southern hybridization. The transgenes could be inherited by vegetative propagation. The novel approach circumvented tedious tissue culture procedures and was simple and versatile, which was readily to be integrated into conventional tree breeding, especially to the species or cultivars not responding to tissue culture.2. Insect-resistance bioassays on transgenic poplar with novel binary insect-resistant genesInsect-resistance bioassays on transgenic poplar leaves and stems with chitinase gene and BmkIT gene were conducted with the 1st instar larvae of Hyphantria cunnea and Anoplophom glabripennis. The results showed that transgenic poplar leaves had significant lethal and inhibition effects on Hyphantria cunnea larvae compared with the untransformed control (P< 0.05). Most H. cunnea larvae were not able to develop into pupa and some pupae were not able to eclosion as well as some eclosioned molts were abnormal with imperfect wings which died soon without mating. However, the 8 tested transgenic lines had no significant effect on development of A. glabripennis larvae.3. The effects of transgenic poplar with binary insect-resistant genes on midgut structure and acetylcholinesterase activityLarvae of Hyphantria cunnea were fed on the leaves of poplar plants transformed with chitinase gene and BmkIT gene. The larval midgut structures were observed and the acetylcholinesterase activity in their heads was measured. The results showed that larval midgut structures were incomplete and deformed where cells were disordered and detached. At the same time, transgenic leaves inhibited acetylcholinesterase activity in larval head significantly.4. Effects of transgenic poplar leaves with binary insect-resistance genes used as feed for rabbitsThe aim of the study was to evaluate the feed safety of transgenic poplar (Populus cathayana Rehd) leaves on rabbits. Fifty-four New Zealand White Rabbits (Oryctolagus cuniculus) were fed for 70 consecutive days with fresh poplar leaves containing chitinase-BmkIT gene combination or their untransformed counterparts as control. Rabbit body weight was recorded. Blood, small intestine and muscle samples of the rabbits in the treatment group was subjected to PCR assay for the possible presence of exotic DNA at the end of the trial. Hematological and biochemical data in blood samples were compared for the rabbits; organ histological structures were observed, and the organ weights in the two groups were measured as well. The results of the growth study revealed no significant differences for average body weight, daily weight gain, and feed efficiency between the two groups. Feed-derived foreign DNA was not detected in small intestine, blood, or leg muscle samples, and no obvious pathological change was observed in the small intestine, stomach, spleen, kidney, lung, heart, bladder, pancreas, prostate and ovary. All hematological and blood biochemical data differences revealed in the test were insignificant between the two groups after 70 days of feeding. We conclude that the chitinase-BmkIT poplar leaves had no deleterious or obvious harmful effects on rabbits.