Nghiên cứu biểu hiện gen mã hoá nhân tố phiên mã ZmDREB2A nhằm tăng cường tính chịu hạn ở một số dòng ngô việt nam tt tieng anh

MINISTRY OF EDUCATION AND TRAINING MINISTRY OF AGRICULTURE AND RURAL DEVELOPMENT VIETNAM ACADEMY OF AGRICULTURAL SCIENCES  DOAN THI BICH THAO DEVELOPING DROUGHT TOLERANT MAIZE BY OVEREXPRESSION OF THE TRANSCRIPTION FACTOR ZmDREB2A IN VIETNAMESE LINES MAJOR: Biotechnology Code: 94202001 SUMMARY OF DOCTORAL THESIS OF AGRICULTURE Hanoi - 2020 i The work has been completed at: Vietnam Academy of Agricultural Sciences Supervisors: Prof Dr Nong Van Hai Dr Bui Manh Cuong Reviewer 1: Reviewer 2: Reviewer 3: This thesis is presented at the Academy-level doctoral thesis Assessment Council Location: Vietnam Academy of Agricultural Science Time: date month 20 This thesis can be found at: The National Library of Vietnam The Library of Vietnam Academy of Agricultural Sciences The Library of Maize Research Institute of Vietnam ii GENERAL INTRODUCTION The essence of the thesis Global climate change, extended drought and uneven distribution of rainfall are seriously difficult for agricultural production in many countries in Asia, Africa, and South America Besides direct impacts on crop cultivation, climate change also reduces the area of agricultural production In Vietnam, about 75% of the area is mountainous thus often facing water shortage, leading to difficulty in farming for many crops Drought is considered as a major global limiting factor of crop productivity, including maize especially in rainfed areas There are several solutions to improve maize production under drought such as cultivation techniques and breeding Breeding drought-tolerant maize varieties can be considered to be a feasible solution There are three main directions in developing drought-tolerant crops, including maize: 1) evaluating and selecting directly in the field; 2) conventional breeding approaches in combination with molecular markers; 3) transferring drought-tolerance related genes In Vietnam, studies on transferring drought-tolerance related genes into maize has been of more interest for the recent years However, studies on the expression of drought-tolerant genes in transgenic plants are limited Therefore, this thesis "Developing drought tolerant maize by overexpression of the transcription factor ZmDREB2A in Vietnamese lines" was carried out Research objectives (1) Developing ZmDREB2A transgenic maize lines; (2) Identifying the presence, stability, and expression of ZmDREB2A gene through generations; (3) Testing ZmDREB2A transgenic maize lines for tolerance to drought under artificial drought stress Novel contributions (1) The thesis is a systematic study including transforming a vector containing ZmDREB2A gene into three maize lines K1, K3, and K7 and then evaluating the expression of ZmDREB2A gene, which is related to drought tolerance (2) Analysis by PCR, RT-PCR, Southern blot, tests under artificial drought stress, and quantification of physiology and biochemistry parameters to evaluate the presence, the stability and the effects of ZmDREB2A on phenotype of transgenic plants; (3) The results of the thesis can possess both scientific and practical impact on the development of drought tolerant maize lines by plant genetic engineering Research impact 4.1 Scientific impact (1) The scientific basis for applying transgenic techniques in improving drought tolerance in crops by enhanced ZmDREB2A recombinant proteins and the expression of their biological functions on maize was confirmed (2) The results of the development of transgenic maize opened the research trend in using transgenic techniques in improving drought tolerance of maize in Vietnam 4.2 Practical impact (3) The development of transgenic maize lines with higher drought tolerance compared to their wild-types contributes to the research trend in applying gene transfer techniques in different crops for enhancing their drought tolerance (4) Drought tolerant transgenic maize lines are materials for breeding drought tolerant transgenic maize varieties in Vietnam (5) Outline of the thesis The thesis contains 179 pages: General introduction (4 pages); Literature review (44 pages); Materials and methods (22 pages); Results and discussion (77 pages), and Conclusion and suggestion (2 page); Thesis related research works (1 page); References (16 pages), including 193 documents with 23 ones in Vietnamese and 170 others in English; Appendix (13 pages) 33 tables and 59 pictures and graphs CHAPTER LITERATURE REVIEW 1.1 THE IMPACTS OF DROUGHT ON GROWTH AND DEVELOPMENT OF MAIZE Under drought stress, maize plants response to water deficit as followings: i) slow germination and low germination percentage; ii) reduction in plant size, abnormal development of organs; iii) closed stomata, reduced photosynthesis, even stopped due to the destruction of the enzyme system; iv) reduction in assimilation, extended anthesis silking interval, yield loss; v) under mild drought stress, an increase in root/shoot ratio; in severe drought, root system gets underdeveloped, leading to difficulties in nutrient uptaking, stagnancy in nutrient transportation in stem, reduction in matter accumulation in kernels 1.6 RESEARCH ON TRANSCRIPTION FACTORS DREB2A IN RESPONSE TO DROUGHT IN PLANTS Studies on the activity of genes controlling transcription factors DREB2 confirmed that the mechanism for enhancing their expression only occurs under stresses Analyzing DREB2A protein structure revealed a negative regulatory region that inhibited the activity of DREB2A If it is removed, DREB2A-CA will be formed and become proactive as a transcription factor [138] Studies in Arabidopsis showed that DREB2A-CA plays a role in enhanced drought tolerance, but not cold; it can even lead to the phenomenon of stunted plants Using RD29A as a stress induced promoter for the expression of DREB2A-CA improves abiotic tolerance in plants, but plants can still be stunted Analyzing the two groups of transcription factors DREB1A and DREB2A showed that differences in the sequences of regions that are associated with DNA leads to various responses to drought and differences in the expression of downstream genes In addition to drought and salinity stresses, the expression of DREB2A-CA in Arabidopsis plays a role heat shock resistance controlled by heat shock protein group (HSP)C It suggests that the expression of DREB2A is involved in enhanced tolerance to various stresses in plants Therefore, DREB2A is of interest to improve tolerance to abiotic stresses such as drought and high temperature Microarray analysis showed that HSP70 and HSFA3 genes, which are related to high temperature tolerance, are regulated by DREB2A The transcription factor DREB2A can bind to the DRE sequence on the promoter of AtHSFA3 gene, to activate its expression In maize, Qiu et al (2007) isolated ZmDREB2A cDNA from maize ZmDREB2A encoding a protein molecule, that consists of 318 amino acids, contains a region associated with ADNERP/AP2 that is specific to DREB There are two forms of ZmDREB2A transcripts: ZmDREB2A-L and ZmDREB2A-S The first one, ZmDREB2A-L is 1336 bp in length, encoding a peptide that includes 89 amino acids As this peptide has no ERF/AP2 domain for DNA binding, it is not a functional form ZmDREB2A-S is 1283 bp in length, encoding a functional peptide that has 318 amino acids with typical ERF/AP2 domains of DREB The S form is 53 bp shorter than the L form (figure 3.2) Under abiotic stresses, post transcriptional processing in plants transforms the ZmDREB2A-L form into the functional ZmDREB2A-S form However, in normal conditions, the majority of ZmDREB2A transcripts are ZmDREB2A-L form [128] Studies in Arabidopsis showed that the ZmDREB2A protein may increase plant tolerance to drought 1.7.3 RESEARCH ON GENETICALLY MODIFIED MAIZE IN VIETNAM It has been more interested in research and application of genetically modified crops in Vietnam for the recent years In inspite of different opinions, Vietnamese government has highly appreciated the role and importance of genetically modified plants in improving crop productivity, thereby built a plan for the development of genetic engineering up to 2020 as well as issued Circulars of policies on genetically modified plants forwards encouraging the research, development and application of genetically modified plants in Vietnam These are the biosafety regulations for genetically modified organisms, genetic specimens and products of genetically modified organisms; for the certification of genetically modified products eligible for use as food, feed, as well as regulations on testing and assessing risks to biodiversity and environment of genetically modified plants Based on that, studies on gene isolation and selection, vector construction are currently in progress However, these researches are only at its dawn and most of the products are at the stage of evaluation, analysis and maintenance at laboratory or green houses So far Vietnam has not developed any commercial genetically modified plant varieties A number of studies in the field of gene transfer have been conducted only in laboratories or green houses at research institutes Currently, a number of genes of drought tolerance, cold tolerance, pest resistance has also been isolated and some vectors designed and then effectively transformed into plants by different methods Chapter MATERIALS, ACTIVITIES AND METHODOLOGY 2.1 MATERIALS 2.1.1 Maize germplasm Three maize lines selected by NMRI: V64, K2, K5, C502N, C88N, M67, C433, K10, K1, K3, K7, 952-TG1, 2224-TG1, N3-TG1, K4, K8, K9, K17, 1257-TG1, N18-TG13 used as materials to evaluate tree regeneration, into K1, K3, K7 used as donor These lines had high regenerative ability with good agricultural trait 2.1.2 Microbial strains Microbial strains were provided by Institute of Genome Research – VAST: DH5α – Ecoli and LBA4404 - A tumefaciens 2.1.3 Vector and oligonucleotide Cloning vector pUC57 carried ZmDREAB2A were synthesized by Genscript Vector pRTRA7/3 were provided by The Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany Vector pBY were received by University of Michigan, USA.Vector pCAMBIA1300 Oligonucleotide (Table 2.1) were designed using GeneBank Database by Invitrogen (USA) and Sigma (USA) 2.2 Research activities Activity Evaluation of regeneration rate and agro-physiological parameters of maize lines Activity Design and transformation of the expression vector containing the gene ZmDREB2A into Agrobacterium Activity Evaluation of transgene stability in T1-T3 generations of maize lines overexpressing ZmDREB2A Activity Evaluation of chlorophyll, proline, carbohydrate contents and drought tolerance of transgenic maize lines 2.3 RESEARCH METHODS 2.3.1 Evaluation of the regeneration rate using a method by Armstrong (1985) 2.3.2 Methods to evaluate agro-physiological parameters, and to select lines as transgenic materials The parameters in the experiments was selected according to QCVN 01-56: 2011/BNNPTNT 2.3.3 Methods to design the plant expression vector containing ZmDREB2A by Sambrook (2001) 2.3.4 Transformation by Agrobacterium-mediated method according to Frame et.al (2002, 2006, 2011) and Ishida Yuji et al (2003, 2007) 2.3.5 Methods to analyze transgenic maize line using PCR by Sambrook (2001) 2.3.6 Methods to analyze the expression of transgene using RT-PCR, Western hybridization by Joyce, (2002) and Sambrook (2001) 2.3.7 Methods to determine chlorophyll (Porra et al., 2006), proline (Bates et al., 1973); non-structural cacbonhydrate (Zheng et al., 2010) contents 2.3.8 Method to evaluate drought tolerance by Camacho (1994) and Zaidi (2002) Chapter RESULTS AND DISCUSSION 3.1 EVALUATION OF REGENERATION RATE AND AGROPHYSIOLOGICAL PARAMETERS OF MAIZE LINES 3.1.1 Evaluation of the regeneration rate in maize germplasms To evaluate the regeneration rate, 20 maize germplasm were planted and harrvested after 12 days of pollination After that, the ratios of callus and regenerated plants were determined Table 3.1 shows the ratios of callus and regenerated plants in various genotypes The K5 line had the highest ratio of creating callus (73.5%), followed by K8 (68.7%); the lowest ratio was observed in the C88N line (3.2%) Others had ability of creating callus ranging from 13.3% to 63.2% Regarding the ability of plant regeneration, K3 was the highest (48.7%), higher than K2 (29.5%) but its ability to develop to fully plant was lower than K2 (68.5%) The other lines with high ability of plant regeneration were K3 (48,7%) with ability of fully plant formation (45.9%), K7 (35.8%; 45.7%), K1 (36.8%; 52.8%), 1257 –TG1 (45.7%; 44.8%) The lowest ratio of creating callus was obeserved in the C88N line The C433 line had a high ratio of plant regeneration (21%), but the ratio of fully plant was low (5.3%) Table 3.1 Number of regenerated plants in different maize germplasms V64 1800 23,1 Number of regenerated plant (%) 12,4 K2 2550 63,2 29,5 68,5 K5 3700 73,5 22,4 33,3 C502N 1800 23,4 12,1 13,3 C88N 2500 3,2 - - M67 1300 24,4 31,3 13,2 C433 690 29,8 21,0 5,3 K10 1000 13,3 21,4 15,4 K1 600 76,4 36,8 52,8 10 K3 600 75,9 48,7 45,9 11 952-TG1 300 68,9 34.1 37.3 TT Maize gerplasms Number of embryos Ratios of creating callus (%) Number of fully plant formation (%) 21,5 12 2224-TG1 300 61,9 33.6 44.8 13 N3-TG1 300 60,8 28.7 34.8 14 K4 600 48,9 38,9 44,8 15 K7 600 77,5 35,8 45,7 16 K8 600 68,7 31,5 40,4 17 K9 600 34,6 12,8 24,6 18 K17 600 32,9 23,9 25,8 19 1257-TG1 300 55,8 45,7 44,8 20 N18-TG1 300 56,9 41,5 38,5 In conclusion, 12 maize lines with high regenerative ability were determined: K2, K5, K1, K3, 952-TG1, 2224-TG1, N3-TG1, K4, K7, K8, 1257-TG1, N18TG1 The agricultural biology, ability to be donors in genetic transformation experiments were evaluated in these lines 3.1.2 Agricultural biology of maize germplasm Results of growth duration, biological characteristic, resilience and yield can be seen in Table 3.6 Most of the maize lines had growth duration ranging from short to medium growth duration They were resistant to pests and diseases at a certain degree and had stable yield Table 3.6 Cob morphology, yield and yield components of maize line in 2014 T T Line K1 13,3 14 26,5 25,7 235,5 227,8 69,7 71,2 25,5 24,8 K2 12,0 12,0 25,4 25,4 241,3 234,6 67,5 68,5 25,3 23,6 K3 10,0 10,6 28,7 27,2 242,5 241,5 68,5 68,1 27,2 25,4 K4 12,7 12,0 28,3 26,4 235,4 238,4 65,5 66,6 27,1 25,3 12,0 12,0 27,6 26,5 225,6 231,4 66,5 67,4 25,6 22,3 12,0 12,4 24,5 25,4 235,5 241,3 67,8 66,9 25,4 23,4 952TG1 2224TG1 No of row/cob S F No of seed/row S F Weight of 1000 seed (gram) S F Ratio of seed/cob (%) S F Yield (quintal/ha) S F K7 12,0 12,0 27,5 26,5 241,5 237,6 71,2 70,5 27,5 26,1 K8 14,7 14 26,3 27,3 224,6 231,5 69,5 70,1 26,5 24,5 N3TG1 10,0 10,8 26,4 26,5 234,2 242,3 65,4 67,8 26,1 23,4 K5 12,7 12 27,1 26,7 243,5 245,2 68,7 70,2 25,2 22,5 13,3 14 26,3 25,6 237,6 241,4 66,6 68,7 24,1 21,7 12,0 12,7 25,6 26,3 234,5 236,5 67,2 66,5 22,8 21,5 7,32 6,58 10 11 12 1257TG1 N18TG1 CV% LSD 0.05 3,55 4,05 S: Spring season; F: Autumn season The number of row/cob in the Spring season ranged from 10.0 to 14.7 rows; in the Autumn season it ranged from 10.6 to 14 rows K1, K8 and 1257-TG had high number of row/cob in both the Spring and Autumn season (13.3 – 14.7 row/cob) In general, maize lines with long cob had a high number of seed/row, but this character depends on seed shape The number of seed/row ranged from 24.5 to 28.7 seed in the Spring season, higher than in the Autumn season (25.4 – 27.3 seed) (Table 3.6) Weight of 1000 seed in the Spring season ranged from 224.6 – 243.5g; in the Autumn season, it ranged from 227.8 to 245.2g The highest value can be seen in the K5 line (243,5 – 245,2 g) The ratio of seed/cob ranged from 65.4 to 71.2% in the Spring season; it was 66.6 – 71.2% in the Autumn (table 3.6) Yield is always considered the most important criterion when assessing materials, to predict the yield potential of the line The yield traits are regulated by multiple genes and are greatly affected by environmental factors and farming conditions In particular, yield is significantly affected by unfavorable factors such as drought, flood or pests Table 3.6 showed that in the Spring season, there was a variation in yield of different lines (CV = 7.32%), which is normal in field experiments Yield of maize lines in the Spring and Autumn seasons were 22.8 – 27.2 quintal/ha and 21.5 – 26.1 quintal/ha, respectively The yield of the lines in the Autumn season tended to be lower than that of the Spring season, which can be explained by the cold weather and the lack of light at the end of the Autumn season, affecting the photosynthesis process and the nutrient transport into the seed formation In addition to characteristics such as pest resistance and yield stability, these 12 pure maize lines have been maintained and bred over many years at the Maize Research Institute They have generally been shown good combining ability, being the parent of a number of promising hybrid combinations Based on the results of regeneration ability of 20 maize lines and the results of field experiments of 12 lines in the Spring and Autumn seasons in 2014, and the evaluation of combining ability of the lines, we selected the K1, K3, and K7 lines to be transgenic materials 3.2 DESIGN AND TRANSFORMATION OF THE EXPRESSION VECTOR CONTAINING THE GENE ZMDREB2A INTO AGROBACTERIUM 3.2.1 Design of the expression vector containing the gene ZmDREB2A In maize, there are two forms of ZmDREB2A transcripts: ZmDREB2A-L and ZmDREB2A-S The first one, ZmDREB2A-L is 1336 bp in length, encoding a peptide that includes 89 amino acids As this peptide has no ERF/AP2 domain for DNA binding, it is not a functional form ZmDREB2A-S is 1283 bp in length, encoding a functional peptide that has 318 amino acids with typical ERF/AP2 domains of DREB The S form is 53 bp shorter than the L form (figure 3.2) Under abiotic stresses, post transcriptional processing in plants transforms the ZmDREB2A-L form into the functional ZmDREB2A-S form However, in normal conditions, the majority of ZmDREB2A transcripts are ZmDREB2A-L form [128] The ZmDREB2A-S form, that is only produced under drought stress, plays an important role in drought tolerance of maize Nevertheless, in natural conditions, the production and maintenance of ZmDREB2A-S transcripts occur in a short period of time, leading to difficulties in cloning this sequence from drought samples Artificial synthesis of ZmDREB2A-S sequences can be a solution Therefore, we introduced the ZmDREB2A-S sequences, that were artificially synthesized by Genscript, into the cloning vector pUC57 The ZmDREB2A-S sequences were attached to a construction including an ubiquitin promoter and a 35S terminator inside T-DNA of the vector pCAMBIA1300; this vector has hygromycin B resistance gene for plant selection 3.2.2 Transformation of the expression vector containing the gene ZmDREB2A into Vietnamese maize lines via Agrobacterium-mediated gene transfer Plant materials for transformation were three maize lines K1, K3, and K7 They were all elite lines, with good agronomic traits, especially a high regeneration rate, compared to other lines which were tested before In this experiment, the immature embryos (12 days after pollination) were isolated to be cultured with Agrobacterium bacteria containing the expression vector Table 3.8 Plant regeneration of K1, K3, and K7 lines Co-cultivation media (CCM) Regeneration (TS1) No of samples % Regenerated plants Percentage (%) 23,67 291 3,45 690 25,84 64 2,40 675 12,71 92 1,73 Regeneration (TS2) S N o N Line K1 8430 2400 28,47 1995 K3 2670 810 30,34 K7 5310 735 13,84 No of embryos No of sample % The formation of callus after two selections was good However, regenerated shoots were found in only a small percentage The regeneration rate of shoot was from 12,71% to 23,67% (Table 3.8) The highest number of regenerated shoots and the regeneration rate were observed in the K3 line (the average rate was 25.84%), followed by the K1 line (23.67%) The K7 line has the lowest regeneration rate, which was 12.71% Regarding the ratio of plants that survived after being transferred to soil, the table 3.9 showed that the K1 line had the highest number of survival plants (152 A B C Figure 3.22 Mutated maize plants A Abnormal tassels; B Seeds in the tassels; C Plants without corn ear These fertile T0 plants were kept in the greenhouse to collect the seeds for evaluation of the T1 generation 3.3 EVALUATION OF THE STABILITY OF THE TRANSGENE ZmDREB2A IN T1, T2, T3 GENETATIONS 3.3.1 Evaluation of the presence of ZmDREB2A gene in T1-generation transgenic maize lines by PCR The results of PCR testing for the presence of ZmDREB2A in T1-generation transgenic plants from 10 T0 plants of K1, K3 and K7 were shown in Table 3.12 In particular, K1 line had 71 plants containing ZmDREB2A sequences That number in the K3 and K7 lines were 123 and 128 plants, respectively The size of the ZmDREB2A sequences in transgenic plants did not change, compared to the original design (948 bp, Figure 3.23) It indicated that there were no insertion or deletion mutations during the transformation process Table 3.12 PCR results in T1 maize lines Number Number Percentage Chi Dissociation of plant of plant of plants square T1 line rate of the with with carrying value transgene (χ2) PCR (+) PCR (-) genes K1 lines K1-1 16 1:4 20 K1-2 18 10 K1-3 15 1:3 25 K1-4 14 1:2 30 K1-5 12 2:3 40 K1-6 12 2:3 40 K1-7 10 10 1:1 50 0.0 K1-8 11 1:1 55 0.20 K1-9 11 1:1 45 0.20 10 K1-10 12 1:1 40 0.80 Total 71 129 35.5 K3 lines 11 10 10 K3-1 K3-2 K3-3 K3-4 K3-5 K3-6 K3-7 K3-8 K3-9 K3-10 Total K7 lines K7-1 K7-2 K7-3 K7-4 K7-5 K7-6 K7-7 K7-8 K7-9 K7-10 Total 10 12 15 16 18 10 11 13 123 10 11 10 11 77 1:1 3:2 3:1 3:1 15:1 1:1 1:1 1:1 1:1 3:1 50 60 75 80 90 45 50 55 45 65 61.5 0.00 0.00 0.26 0.054 0.20 0.00 0.20 0.20 1.06 12 15 16 13 18 14 15 13 11 11 3:2 1:3 3:1 3:1 3:1 1:1 15:1 3:1 1:1 3:1 60 35 75 80 65 45 90 70 45 0.00 0.26 1.06 0.20 0.054 0.26 0.2 0.00 128 72 75 64 Figure 3.23 PCR resuls of ZmDREB2A (948bp) in T1 generation of K3 line (electrophoresis in agarose gel 1%) L: Ladder 1kb, (+): positive control, (-): negative control; wt: wild-type, 1- 74: T1 transgenic plants 3.3.2 Evaluation of the presence of ZmDREB2A in T2, T3 generations of transgenic maize lines by PCR In the T2 genetation, K3 and K7 lines were evaluated, in which plants separate according to the ratio of 3:1 (K3-3.1, K3-4.18, K7-5.5 and K7-8.10) 12 K7-4.12 had 100% T2 plant that carried the transgene (table 3.13) These T2 plants were selected for selfing for the next (T3) generation Table 3.13 PCR results in T2 generation maize lines T2 lines 3 Number of plant with PCR (-) Segregation ratio Ratio of plants carrying transgene(s) Chi square value (χ2) 65 27 3:1 70,7 0.93 75 31 3:1 82 14 - 222 103 92 14 - 108 100 81 21 3:1 67 21 3:1 348 56 Number of plant with PCR (+) K3 Lines K3-3.1 K34.18 K310.10 Total K7 Lines K7-3.3 K74.12 K7-5.5 K78.10 Total 70,8 1,11 85,4 68,3 86,8 100,0 79,4 76,1 86,1 0,00 1,05 0,06 The segregation ratio in K3-3-1.96 was 3:1 (χ2 value was equal to 0.21), that was followed Mendel's law of independent segregation with a single copy of the transgene K3-4.18 had 100% plants with the transgene in T3; it could be assumed that the K3-4.18.32 had homozygous genotype Souhthern hybridization experiment should be performed to confirm K3-3.1.96 and K3-4.18.32 separated according to Mendel’s laws in T1, T2 and T3 generation It could be explained that these two lines had a single copy of the ZmDREB2A These two lines of K3 line were selected to perform Southern hybridization, physiological, biochemical assessment and drought tolerance evaluation Three cobs of K7 in T2 generation were selected for T3 generation K74.12.25 had dissociation rate of the transgene, which was 100% in T3 This results showed that the plants had homozygous transgene However, further experiments are required to get accurate results K7-5.5.19 had a 3: 13 dissociation ratio, according to Mendel's law with a single copy of the transgene; this is the third generation K7-5 line that dissociated at a 3: ratio K7-8.10.65 had a 9: dissociation ratio, which did not follow any segregation rules, although in the two previous generations, the K7-8 series had dissociated according to the Mendel’s law This may be due to transgene was unstable in the transgenic maize genome, because the transgene location appears to be crossexchanging, resulting in a change in the rate of gene transfer across subsequent generations, or the weak inheritance of these genes PCR results and results of evaluation of segregation ratio across generations showed that K3 (K3-3 and K3-4) and K7 (K7-4 and K7-5) lines contained the ZmDREB2A sequences and this transgene was segregated according to Mendel's law during generations The transgene size through generations did not change, compared to the original design Plants with transgenes from these lines were selected for next experiments (Table 3.15) Table 3.15 PCR results of maize lines in T3 generation No 2 T3 Lines Number of plant with PCR (+) K3 lines K3-3.1.96 77 K399 4.18.32 Total 176 K7 lines K7100 4.12.25 K7-5.5.19 71 K792 8.10.65 Total 263 Number of plant with PCR (-) Dissociation rate of the transgene Percentage of plants carrying genes Chi square value (χ2) 22 3:1 100 77,8 100 0.21 0.00 100 100 0.00 29 3:1 9:1 71,0 92,0 0.05 - 22 37 3.3.5 Identification of the target sequences (ZmDREB2A) via Southern hybridization The figure 3.37c showed the results of Southern hybridization in T3 plants There was only one bold band in the lane of the positive control (the plasmid containing ZmDREB2A sequences) There were plants that showed positive signals (one signal band) in the Southern hybridization experiment (2 plants from the K3 line: K3-3.1.96, K3-4.18.32; plants from the K7 line: K7-4.12.25, K7-5.5.19, and K7-8.10.65) In Southern hybridization, the number of signal 14 bands indicates the number of copies of the gene of interest in the genome Hence, there was a copy of ZmDREB2A that was integrated into the genome of plans from the K3 and K7 lines Before being allowed to appear in the market, genetically modified plants need to be tested to make sure that number of exogenous genes is minimal [93] It is difficult to evaluate individuals that have a lot of copies of the exogenous gene That is a reason why Agrobacterium-mediated transformation is now preferred than gene gun Figure 3.37a PCR Figure 3.37b Total Figure 3.37c Southern result for identification DNA after digestion hybridization using plasmid specific of ZmDREB2A (+): probes for containing sequences ZmDREB2A sequences M: marker 1Kb;(-): ZmDREB2A (+): plasmid containing wild-type; (+): plasmid sequences; ZmDREB2A sequences; containing ZmDREB2A 1,5: plants from the 1,5: plants from the K3 line sequences; 1-2: plants K3 line (2 plants: K3- (2 plants: K3-3.1.96, K3from the K3 line (2 3.1.96, K3-4.18.32) 4.18.32) plants: K3-3.1.96, K3- 2,3,4: plants from the 2,3,4: plants from the K7 4.18.32); 3-5: plants K7 line (3 plants: K7- line (3 plants: K7-4.12.25, from the K7 line (3 4.12.25, K7-5.5.19, K7-5.5.19, K7-8.10.65) plants: K7-4.12.25, K7- K7-8.10.65) 5.5.19, K7-8.10.65) The Southern hybridization results confirmed the successful transformation in K3 and K7 plants (K3-3.1.96, K3-4.18.32, K7-4.12.25, K7-5.5.19, K78.10.65) It is the basis for selection, maintenance and development of materials that possess the ZmDREB2A sequences in breeding 3.3.6 Identification the expression of ZmDREB2A in genetically modified plants by RT-PCR In the figure 3.37A, ZmDREB2A cDNA in M1, M4, M5, M6, and M7 samples from K3-4.18.32 (well 5, 11, 13, 15, and 17) were amplified These products had the same size, compared to the positive control (well 1: PCR product from the expression vector) There were plants from the K3 line which possessed the ZmDREB2A sequences and the sequences were expressed 15 Figure 3.37B showed results of M9, M10, M12, M14, M15, and M16 samples from K7-4.12.25 (well 5, 7, 11,15, 17, 19) The product size was similar to the positive control (well 1) It means that ZmDREB2A was expressed in plants from the K7 line Figure 3.37A Expression of ZmDREB2A in K3-4.18.32 (RT-PCR) M: Maker 1kb; Well 1: Positive control (plasmid containing target gene); well 2: negative control (PCR without DNA template); well 3: wild-type; well 4, 6, 8, 10, 12, 14, 16, 18: PCR products from RNA templates; well 5, 7, 9, 11, 13, 17, 19: PCR products from cDNA of K34.18.32 (Well 4,5: sample M1; 6,7: sample M2; 8,9: sample M3; 10, 11: sample M4; 12, 13: sample M5; 14, 15: sample M6; 16, 17: sample M7; 18, 19: sample M8) Figure 3.37B Expression of ZmDREB2A in K7-4.12.25 (RTPCR) M: Maker 1kb; Well 1: Positive control (plasmid containing target gene); well 2: negative control (PCR without DNA template); well 3: wildtype; well 4, 6, 8, 10, 12, 14, 16, 18: PCR products from RNA templates; well 5, 7, 9, 11, 13, 17, 19: PCR products from cDNA of K7-4.12.25 (Well 4,5: sample M9; 6,7: sample M10; 8,9: sample M11; 10, 11: sample M12; 12,13: sample M13; 14, 15: sample M14; 16, 17: sample M15; 18, 19: sample M16) Other wells were PCR products from RNA templates in 1% agarose gel There were no products, indicating that DNA was removed totally during the RNA extraction process There were no PCR products from RNA and cDNA templates (6-7, 8-9, and 18-19) from K3-4.18.32 (Figure 3.37A) as well as other pairs (8-9, 12-13) from K7-4.12.25 (Figure 3.37B) It indicated that the target gene was not expressed in these plants 3.3.7 Detection of ZmDREB2A protein by Western blot Results of the Western blot experiment (Figure 3.38) showed that maize plants (well 3, and 4) from the K3 line and plants from the K7 line (well 6, and 7) had a protein which was approximately 37kDa It indicated that all plants had an exogenous protein that reacted in the specific antibody-substrate reaction There were no color reactions from wild-type or plants from the transformation experiment but without target gene (well 1, 2, and 5) The product size of ZmDREB2A protein in this experiment was similar to other previous studies 16 [128][95] This result showed that ZmDREB2A was translated into protein in K3 and K7 plants Figure 3.38 Western blot in T3 plants containing the ZmDREB2A gene (M): marker; (1): wild-type; (2): K3 plant from the transformation experiment without target gene; (3,4): K3 plants with ZmDRB2A gene in T3; (5): K7 plant from the transformation experiment without target gene; (6,7) K7 plants with ZmDRB2A gene in T3 3.4 CHLOROPHYLL, PROLINE, CARBOHYDRATE CONTENTS AND DROUGHT TOLENRANCE OF TRANSGENIC MAIZE PLANTS 3.4.1 Chlorophyll content of transgenic plants Figure 3.39 The chlorophyll a content in normal and drought conditions Figure 3.40 The chlorophyll n content in normal and drought conditions Figure 3.41 The chlorophyll a/chlorophyll b ratio in normal and drought conditions The chlorophyll a/b content and the chlorophyll a/b ratio (Chl a/Chl b) can be seen in the figure 3.41 The Chl a/Chl b ratio in drought stress was higher, compared to normal condition For example, the Chl a/Chl b ratio of K3, and K3-CG samples were around in normal condition, but it increased up to 5.4 in drought condition Similarly, the Chl a/Chl b ratio of K7, and K7-CG samples were aprroximately 3.4 in normal condition but 5.3 and 5.9 in drought stress, respectively It means that the chlorophyll b level decreased more than chlorophyll a in drought condition These results were similar to other studies about physiology of plants in drought stress A decrease in the total chlorophyll content and an increase in the Chl a/Chl b ratio were observed in maize when plants were treated with artificial drought stress in vitro by sorbitol [83] 3.4.2 Proline and carbohydrate content of transgenic plants 17 The proline content of transgenic plants were showed in the figure 3.42 In normal condition, the proline content in all groups was approximately 0.2 μg/mg However, the proline content in drought stress increased 2.5-4.5 times, compared to normal condition For instance, the proline content of K3 and K3CG samples in drought stress increased up to 2.5 and times, respectively, compared to the control The transgenic plants in drought stress had a 37% increase, compared to the wild-type Similarly, K7 and K7-CG samples in drought stress had higher proline contents, 3.55 and 4.5 times higher than that in normal condition The transgenic plants had a 20% increase in the proline content, compared to the wild-type in drought condition Figure 3.43 showed the non-structural carbohydrate (NSC) content in maize plants In normal condition, there was no differences in the NSC content between transgenic plants and wild-type plants; it was around 3.6 μg/mg In drought stress, the NSC content of K3 and K3-CG samples were 5.27 μg/mg and 6.76 μg/mg respectively The NSC content in drought condition of K7 and K7CG were 5.6 μg/mg 7.55 μg/mg, respectively Compared to the wild-type, K3 and K7 transgenic plants had an increase in the NSC content that was equal to 28.3% and 34.8%, respectively μg/m g 10 NSC content K7-CG… K7 Hạn K7 K7-CG K3-CG… K3 Hạn K3 K3-CG μg/m Proline content g Figure 3.42 Proline content Figure 3.43 Non-structural carbohydrate content Analysis of other physiological and biochemical characteristics also showed that transgenic plants with ZmDREB2A sequences can be more tolerant to drought stress, compared to the control Between the two experimental maize lines, the K7 line showed a better tolerance to drought than the K3 line In addition to morphology, these physiological and biochemical characteristics can provide useful information for selection of elite maize lines in breeding 3.4.4 Drought tolerance of T3 transgenic plants under aritificial drought 3.4.4.1 Drought tolerance of ZmDREB2A transgenic maize lines at the vegetative stage In drought treatments, it was not irrigated during 14 days for testing drought tolerance while plants under optimal condition (control) were watered normally At the ninth days after drought treatment, leaves rolled at different degrees between transgenic plants and wild-types In addition, the growth of plants under drought became slower, compared to optimal condition At the 14th day, the difference in leaf rolling was obvious Both transgenic and wild-type plants were 18 scored in leaf rolling; leaves in the lower parts of plants and tip of leaves started to be dead (Figure 3.45) Figure 3.45 Plants before the recovery period (A): K3: None- transgenic maize line K3; K3-CG: ZmDREB2A transgenic maize line K3 (B): K7: None- transgenic maize line K7; K7-CG: ZmDREB2A transgenic maize line K7 Notice: in each pot: individual plants on the left are transgenic miaze lines; others on the right are their none-transgenic inbreds respectively; CT1: wellwatering treatments; CT2: artificial drought treatsments After 14 days under artificial drought stress, water and fertilizers were applied for the recovery of maize plants from drought stress At the 7th day after rewatering, the survival rate of plants was recorded: it was 86.6% in the transgenic maize line K7-ZmDREB2A, equivalent to 13/15 survival individuals, higher than that of the wildtype K7 (33.3%), equivalent to 5/15 The survival rate of K3ZmDREB2A reached 66.6%, equivalent to 10/15 while the K3 line was 26.6%, equivalent to 4/15 In well-watering treatments, there were no differences between transgenic maize lines and wild-types Regarding drought treatments, ZmDREB2A lines can recover better than wild-types after drought stress Its survival rate was higher than that of the wildtype plants However, under drought stress, plants grew slowly, compared to well-watering treatments (Figure 3.46) Figure 3.46 Plants after recovery period (A): K3: None- transgenic maize line K3; K3-CG: ZmDREB2A transgenic maize line K3 (B): K7: None- transgenic maize line K7; K7-CG: ZmDREB2A transgenic maize line K7 Notice: in each pot: individual plants on the left are transgenic miaze lines; others on the right are their none-transgenic inbreds respectively; CT1: well-watering treatments; CT2: artificial drought treatsments 19 Besides, there were other criteria recorded at this stage including leaf and stem length, root length, root volume, stem fresh weight, root fresh weight, stem dry weight, root dry weight (Table 3.20) Leaf and stem length, root length, root volume: When drought occurs in the vegetative stage, the elongation of individual maize internodes is affected The fact that the decrease in plant height under drought has been reported in previous studies In contrast, under drought stress, an increase in the length of the roots to absorb more water was recognized but a reduction in the development of branch-roots was also observed if there is a severe drought Our results showed that, in optimal contitions, leaf/stem length, root length, and root volumn of the transgenic maize lines were not statistically significant different, compared to wild-types Table 3.20 Criteria of leaves and roots of maize lines tested in pots Materia ls Treat ment Leaf lengt h (cm) Root lengt h (cm) Root volum n (mm3) Stem fresh weigh t (g) Root fresh weigh t (g) Stem dry weigh t (g) Root dry weigh t (g) Total dry matter (g) K7ZmDRE B2A CT2 63.2 23.6 2.09 7.359 0.888 1.72 0.22 1.94 CT1 78.8 26.2 4.65 11.96 1.963 5.779 0.598 6.377 Line K7 CT2 56.7 19.5 1.13 2.06 0.344 0.74 0.14 0.88 CT1 77.8 26.26 4.636 11.97 1.964 5.762 0.589 6.351 K3ZmDRE B2A CT2 56.5 20.6 1.91 7.038 0.842 1.53 0.16 1.69 CT1 73.5 23.2 4.534 11.86 1.943 5.629 0.548 6.177 Line K3 CT2 51.7 16.08 1.01 1.968 0.324 0.588 0.091 0.68 CT1 72.8 4,2 1.99 23.2 5.7 0.92 4.518 0.9 0.013 11.87 2.8 0.168 1.944 5.622 0.537 2.2 0.8 4.7 0.02 0.019 0.0124 CV% LSD CT1 CT2 CT2 CT1 CT1 CT2 CT2 CT1 Figure 3.47 Roots of maize lines after 7th day recovery period A: Maize line K3; B: Maize line K7 Notice: CT1: well-watering treatments; CT2: artificial drought treatsments 20 6.169 0.0279 Under drought stress, leaf/stem length, root length, and root volumn of ZmDREB2A transgenic maize lines were significantly different, compared to wild-type plants The leaf and stem length of K7-ZmDREB2A reached 63.2 cm while it was 56.7 cm in K7 The root length of K7-ZmDREB2A and K7 lines were 23.6 cm and 19.5 cm, respectively (Figure 3.47, table 3.20) The root volume of K7-ZmDREB2A was 2.09 mm3 but it was 1.13 mm3 in K7 Similarly, the leaf and stem length of K3-ZmDREB2A and K3 was 56.5 cm and 51.7 cm, respectively The root length of K3-ZmDREB2A was 20.6 cm and 16.08 cm in K3 (Figure 3.47, Table 3.20) The root volume of K3-ZmDREB2A was 1.91 mm3 and while it was 1.01mm3 in K3 Thus, it was confirmed that a well-developed root system helped maize plants absorb more water under drought stress This result is consistent with previous conclusions [46], [13] that the ratio of root/leaf will be increased under mind drought Stem fresh weight, root fresh weight: Under severe drought, maize cells can be seriously damaged and not restored as normal But for drought-tolerant germplasms, an increase in several cell activities that helps them restore their functions Maize cells in drought stress are in dehydration leading to a decrease in the turgor pressure of cells The experimental results showed that stem fresh weight and root fresh weight of maize lines were significantly increased, compared to wild-types (Table 3.20) In optimal conditions, the stem fresh weight and root fresh weight of maize lines K7-ZmDREB2A and K3-ZmDREB2A were not statistically significantly different from K7 and K3 In contrast, under drought stress, these traits were statistically significant different between ZmDREB2A transgenic maize lines and the wildtype plants The reasons can be seen that the restoration of the controls was weaker or the rate of survival individual plants lowers than that of these transgenic maize lines Experimental treatments under drought, the stem fresh weight of K7-ZmDREB2A and K7 lines were 7.395 and 2.06 gram, respectively, while the root fresh weight was 0.888 gram in K7-ZmDREB2A and 0.344 gram in K7 Similarly, K3-ZmDREB2A reached 7,308 gram in stem fresh weight but only 1.968 gram for K3, the root fresh weight of K3-ZmDREB2A was 0.842 gram and of K3 was only 0.342 gram Stem dry weight, root dry weight and total dry matter: Dry weight can be considered as a parameter expressing maize growth and development activities A better growth and photosynthesis activity will lead to a higher amount of dry matter The data of stem dry weight, root dry weight and total dry matter of experimental maize lines are shown in Table 3.20 Table 3.20 showed that for experimental treatments under well-watering, stem dry weight, root dry weight and total dry matter of ZmDREB2A transgenic 21 maize lines were not statistically significantly different from controls However, in drought stress, these traits of ZmDREB2A transgenic maize lines were statistically significantly different from wild-type plants The stem dry weight of K7-ZmDREB2A (1.72 gram) was higher than of K7 (0.74 gram), the root dry weight of K7-ZmDREB2A was 0.22 gram while in K7 it was only 0.14 gram Similarly, K3-ZmDREB2A reached 1.53 gram in stem dry weight that was 0.942 gram higher than that of K3 (0.588 gram) K3-ZmDREB2A was 0.069 gram higher than that of K3 (0.091 gram) in the root dry weight Total dry matter: Total dry matter would be considered to be the most important indicator in evaluating the growth of maize plants In favorable conditions, they grow well with maximum photosynthesis process leading more accumulation of dry matter compared with stress conditions (drought, cold, pests, diseases, etc) Regarding artificial drought stress, the total dry biomass of K7-ZmDREB2A and K3ZmDREB2A was statistically significantly different from K7 and K3, respectively The total dry matter of K7-ZmDREB2A (1.94 gram) was 1.06 gram higher than that of K7, equivalent to 16.6% of the total dry matter of K7ZmDREB2A under well-watering condition The total dry matter of K3ZmDREB2A (1.69 gram) was 1.01 gram higher than that of their wild-types (0.68 gram), equivalent to 16.3% of their total dry matter in normal irrigation Studies of drought tolerance of maize at the vegetative stage in Vietnam [13], [14] and in the world [46], [59], [190] showed that the root system plays an important role in drought stress That means if a plant has a well-growing root system in drought, they will be able to take advantage of deeper groundwater level and naturally have better tolerance to drought That is the reason why drought takes place at the vegetative stage, ZmDREB2A transgenic maize lines were more tolerant to drought, compared to wild-type plants 3.4.4.2 Drought tolerance of the transgenic maize lines and their wildtype plants at other growth stages The experiment was conducted with treatments: treatment (wellwaterring) and treatment (under drought stress at maize growth stages) Four stages were I - Artificial drought during milk stage to physiological maturity (harvesting), II - From tassel stage to physiological maturity (harvesting), III - days before tassel emergence (whorl stage to the 10th day after pollination ends), IV - During 10 leaf stage (V10 stage) to the 10th day after pollination ends [189] Grain yield and yield components of maize lines Grain yield components including ear length, ear diameter, kernel row number, kernel number per row, shelling percentage, kernel weight Productivity can be determined by grain yield components on aggregate If they are high in yield components, these maize lines are often of high potential yield 22 Table 3.26 Comparison of individual productivity of ZmDREB2A transgenic maize lines at different stress timings in net-house Lines K7-ZmDREB2A K7 K3-ZmDREB2A K3 CV% LSD0,05 CT TN CT2 CT1 CT2 CT1 CT2 CT1 CT2 CT1 Individual plant yield (gram/plant) I II III IV 87,5 67,4 27,6 20,6 95,5 86,2 62,4 23,4 15,6 92,4 90,4 78,5 26,6 16,4 93,2 86,4 86,0 23,5 16,0 90,1 4,5 6,7 4,7 6,5 8,9 6,6 4,4 4,5 CT2: treatment under drought stress, CT1: treatment under well-watering I, II, III, IV: stress timing Due to the experimental conditions in pots and net houses, it was only possible to measure the shelling percentage and 100 kernel weight of individuals Table 3.26 showed that the trait of shelling percentage was closely related to grain yield If severe drought took place at the 10 leaf stage (V10)whorl stage - post tassel emergence (corresponding to drought stress timing IV, III, II), the growth and development of maize plants were harshly affected, anthesis-silking interval (ASI) was extended, leading to a reduction in kernal formation, shelling percentage and finally grain yield In regard of stress time points, drought at the 10 leaf stage (IV), it was the most reduction in grain yield due to the lowest shelling percentage, with only 5-9 kernels per row on average The trait of 100 kernel weight of individuals decreased in order from drought stress timing of I - II - IV – III The transgenic maize lines K3-ZmDREB2A and K7-ZmDREB2A shown a better drought tolerance They possessed a lower reduction in yield components under drought stress compared to wild-type plants However, statistically significantly differentce was observed only in K7- ZmDREB2A Moreover, in artificial drought stress, these transgenic maize lines grew better, kernel development and dry matter accumulation in kernels were higher than that in K7 and K3 lines The results in table 3.26 figured out that the productivity of materials was different between conditions (drought stress and well-watering) This result is consistent with previous conclusions by Zaidi (2002) and Le Quy Kha (2005): drought causes severe damage to grain yield due to shortening physiological maturity, increasing leaf scenescence speed, reducing the dry matter accumulation of kernels, also photosynthesis area, utilization of total solar radiation and finally harvest index [13] and [189] The results of this thesis showed that Vietnam can fully use gene transfer engineering in the development of materials with precious characteristics for breeding high-yield and high-quality crop varieties 23 CONCLUSION AND SUGGESTION Conclusion 1.1 Three maize lines K1, K3 and K7 possessed a high regeneration rate, equivalent to 52.8%, 45.9%, and 45.7%, respectively In addition, medium and early maturity of 103-105 days in the Autumn season, or 113-115 days in the Spring season They were also resistant to stem borers (point 1), northern leaf blight and southern leaf blight (point 1-2), sheath blight (0-1.5%), drought (point 3-4) These lines had a fairly high and stable grain yield (24.8 -27.5 quintals/ha) Therefore, they are considered to be materials for receiving the transgene 1.2 The vector pCAMBIA1300 that contained ZmDREB2A-S type sequences and hygromycin resistant gene as selective marker, was designed This construction was integrated into the genome of K1, K3, and K7 lines by Agrobacterium-mediated method There were 77 T0 transgenic maize individuals including 15 plants of K1, 19 of K3 and 43 of K7 with the transgenic efficiency of three materials of 0.17%, 0.71% and 0.81%, respectively 1.3 The maize lines K3 and K7 in the T0 generation carrying ZmDREB2A sequences were successfully maintained through the generations of T1, T2 and T3 Analysis was performed by PCR, sequencing (to compare with original sequences), Southern hybridization, and RT-PCR Protein ZmDEB2A, that was approximately 37 kDa, was detected in K3 and K7 transgenic plants 1.4 Compared to wild-type, transgenic plants possessed higher shoots/roots length and weight, total dry mass, and chlorophyll (Chl), proline, non-structural carbohydrate contents, which are indicators for drought tolerance Regarding the Chl a/Chl b ratio, in K3 and transgenic K3 plants, it was about in normal condition, but 5.4 in drought stress In K7 and transgenic K7 plants, the Chl a/Chl b ratio increased from around 3.4 (normal condition) to 5.3 and 5.9 (drought stress), respectively The proline acumulation in K3 and transgenic K3 plants in drought increased 2.5 times and times, respectively, compared to normal condition K7 and K7 transgenic plants under drought stress possessed 3.55 times and 4.5 times higher in proline content than normal condition Compared with the wild-type under artificial drought, ZmDREB2A transgenic plants also had a higher non-structural carbohydrate content Although drought at any stage of maize growth can affect plant growth and grain yield, ZmDREB2A drought-tolerant transgenic lines showed enhanced tolerance to drought at different stages Compared to wild-type plants, transgenic plants possessed higher shoots/roots length and weight The total maize biomass of K7-ZmDREB2A and K3-ZmDREB2A were 16.6% and 16.3% higher than those of K7 and K3 Suggestions - Maintaining the K3 and K7 transgenic maize lines overexpressing ZmDREB2A to develop stable drought-tolerant maize lines - Developing hybrids combinations from these transgenic lines and testing their drought tolerance - More researches on transfering of other genes into maize for maize breeding program in the future 24 PUBLISHED RESEARCH WORKS RELATED TO THESIS Thao Doan Thi Bich, Thang Nguyen Xuan, Anh Le Tuan, Tung Le Cong, Hoai Nguyen Thi Thu, Hai Nong Van, Cuong Bui Manh (2019) “Evaluation of biochemical parameters and drought resistance in ZmDREB2A transgenic maize at the seedling stage” Vietnam Journal of Science, Technology and Engineering, 61(5): 55 – 59 Thao Doan Thi Bich, Thang Nguyen Xuan, Luc Le Cong, Hoai Nguyen Thi Thu, Dung Ta Thi Thuy, Tung Le Cong, Cuong Bui Manh, Hai Nong Van (2017) “Sequencing, evaluating the similarity between the transformed sequences and the designed expression sequence of ZmDREB2A in maize” Jounal of Vietnam Agricultural Science and Technology, (78): 32-37 Thao Doan Thi Bich, Thang Nguyen Xuan, Hoai Nguyen Thi Thu, Dung Ta Thi Thuy, Tung Le Cong, Cuong Bui Manh, Hai Nong Van (2016) “Transformation of Drought-Tolerance Gene ZmDREB2A into Vietnamese Maize Mediated by Agrobacterium Tumefaciens” Jounal of Vietnam Agricultural Science and Technology, 3(64): 7-12 Hue Huynh Thi Thu, Minh Bui Manh, Thao Doan Thi Bich, Hai Nong Van, Cuong Bui Manh (2015) “Construction of binary vector contained artificial ZmDreb2A-S gene and transformation into Agrobacterium tumefaciens” Vietnam Journal of Science, Technology and Engineering, 2(9): 44-48 25 ... evaluation of the T1 generation 3.3 EVALUATION OF THE STABILITY OF THE TRANSGENE ZmDREB2A IN T1, T2, T3 GENETATIONS 3.3.1 Evaluation of the presence of ZmDREB2A gene in T1-generation transgenic maize... objectives (1) Developing ZmDREB2A transgenic maize lines; (2) Identifying the presence, stability, and expression of ZmDREB2A gene through generations; (3) Testing ZmDREB2A transgenic maize lines for... transgene was unstable in the transgenic maize genome, because the transgene location appears to be crossexchanging, resulting in a change in the rate of gene transfer across subsequent generations,