Production Of Glyphosate Resistant Transgenic Rice And Its Unintended Effects Analysis

Rice is the major grain crop in China.Weed is the important influencing factor for rice yield.Glyphosate is a highly effective,broad-spectrum,safe,low toxicity,non-selective herbicide,cultivating glyphosate-resistant transgenic rice is significant.As a weak organizational culture ability,indica is hard to genetic transformate in the process that improves rice qualities by transgenic technology.We used three indica rice varieties-Guan28,Minghui 63 and 9311 as meterials to study the effect of aromatic amino acids and the ratio of hormone on the regeneration ability of indica rice callus,optimized the regeneration culture medium of indica rice callus.2mG2-epsps gene cassette was transformed into indica rice Guan 28 via general transformation and marker-free transformation by gene gun mediating.And the transgenic plants were identified by PCR and glyphosate sensitivity.The genetic and expression stability and agronomic traits variation of T2 generation of 2mG2-epsps transgenic Japonica rice were studied.And metabolite profiling of the leaf of transgenic rice was analysised by liquid high-efficiency chromatographic technology.The technology source of unintended effects was prelininary analysised on the agronomic traits phenotype and metabonomics level according to serious contrast.This research provices experimental data for safety evaluation of glyphosate resistant transgenic rice.The main results are as follows:(1)Appropriate concentration of aromatic amino acids and hormone proportion can significantly improve the ability of differentiation for indica rice callus.When the three kinds of aromatic amino acids-Try,Tyr and Phe was added to regeneration medium with equal proportion,Compared with the constrast,differentiation rate of Indica rice embryogenic callus was increased by 3%～20%,The average number of green seedlings per callus was increased by 0.3～1.5 times.When 0.5mg/L KT and lmg/L IAA was added to regeneration medium,the differentiation rate of indica rice embryogenic callus is increased by5%～28%,The average number of green seedlings per callus were increased by 0.36～1.6 times.When the three kinds of aromatic amino acids and hormone was added by the same time,the differentiation rate of Indica rice embryogenic callus reached 64%～75%,The average number of green seedlings per callus reached 4 to 5.6.Compared with constrat,they wre increased by 5%to 31%and 0.52 to 2.4 times,respectively.Additional compositions for the Optimized regeneration medium of indica rice callusis were 0.3mmol/LTry + 0.3mmol/L Tyr+0.3mmol/LPhe+0.5mg/L KT+lmg/L IAA.(2)Indica rice Guan 28 was transformed 2mG2-epsps gene cassette via general transformation by gene gun mediating.The resistant callus were obtained by 2 mmol/L glyphosate screening.On the optimized regeneration medium,the differentiation rate of resistant callus was 39%,the average number of per callus green seedlings was 2.1.Indica rice Guan 28 was transformed 2mG2-epsps gene cassette by marker-free transformation.On the optimized regeneration medium,the differentiation rate of callus was 64.4%,the average number of per callus green seedlings was 4.4,the transformation frequency was 3.3%;On the contrast regeneration medium,the differentiation rate of callus was 43.3%,the average number of per callus green seedlings was 2.1,the transformation frequency was 0%,.Visiblely,optimized regeneration medium is advantage for genetic transformation of indica rice.T1 generation transgenic plants which was obtained by tow transformation method could tolerate 0.2%glyphosate herbicide,the radio of resistant strains and sensitive strains was 3:1,so showed Mendelian unit locus genetic.(3)T2 generation of 2mG2-epsps transgenic Japonica rice was sprayed with 0.2%,0.5%and 0.8%glyphosate,there were different resistances among the different transgenic strains.The radio of resistant strains and sensitive strains in T2 generation of transgenic plants was 3:1,showed Mendelian unit locus genetic.Investigating 8 kinds of agronomic traits in T2 generation of 2mG2-epsps transgenic plants and the non-transgenic tissue culture seeding lines by going through transformational process,we found that 8 kinds of agronomic traits in T2 generation of transgenic plants appeared different levels of variation.Comparing parent plant,the difference of spikelets of main ear and seed-set reached significant level,and the difference of other 6 kinds of agronomic traits didn’t reach significant level for T2 generation of Nipponbare tissue culture seeding lines;the difference of ear length didn’t reach significant level,and the difference of other 7 kinds of agronomic traits reach significant level for T2 generation of Bi 349 tissue culture seeding strain.The results indicate tissue culture technique in transgenic process isn’t the main fator that lead to agronomic traits chang for T2 generation of Nipponbare transgenic plants,but it’s the main fator that lead to agronomic traits chang for T2 generation of Bi 349 transgenic plants.Investigating agronomic traits of T2 generation of transgenic plants those were sprayed or weren’t sprayed with glyphosate.agronomic traits of T2 generation of different transgenic plants those were sprayed by the same concentration of glyphosate showed different variation.For T2 generation of same transgenic strain,when the concentration of glyphosate increased,every agronomic trait declined.(4)We use T2 generation of transgenic homozygosis or heterozygosis resistance strains(T),heterozygosis sensitive strains(N)and parent plants(P)as material to structured PTN system.In this system,the peak time and retention time of each peak were consistent in metabolic profiling of PTN.The result indicate components of aromatic metabolite didn’t change.components of aromatic metabolite in different srains and different rice varieties also didn’t chang.