Pyramided ph2 and ph3 genes for breeding tomato tolerant to late blight and evaluating the horticultural characteristics of some tomato lines in early winter season 2020 in red river delta

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF AGRONOMY GRADUATION THESIS Pyramided Ph2 and Ph3 genes for breeding tomato tolerant to late blight and evaluating the horticultural characteristics of some tomato lines in early winter season 2020 in Red River Delta Student : Nguyễn Xuân Khánh Student code : 611609 Class : K61KHCTT Department : Genetic and plant breeding Supervisors : Assoc Prof Vũ Thị Thu Hiền Dr Trần Ngọc Hùng Ha Noi, 2021 COMMITMENT I hereby commit that the research data and results in this paper are honest and have never been used in any reports before I declare that the references in the thesis are cited and not been published in any research Hanoi, 28th February, 2021 Student Nguyen Xuan Khanh i ACKNOWLEDGEMENTS In performing my undergraduate thesis, it is a successful one I had to take the help and guideline of some respected persons First and foremost, I would like to express my sincere gratitude to my supervisors, Assoc Prof Vũ Thị Thu Hiền -Head of Department of Genetic and Plant breeding, Vietnam National University of Agricultural (VNUA) and Dr Trần Ngọc Hùng-Head of Department of Biotechnolog, Fruit and Vegetable Research Institute, for the continuous support of my graduation thesis, for their patience, motivation, and immense knowledge Their devoted guidance helped me in all the time of research and writing of this thesis Besides my supervisors, I would like to thanks to The Fruit and Vegetable Research Institute for giving me an opportunity to access to the laboratory and research facilities In the other hand, I am also grateful to people working in here for sharing their experiences, time during my work Without their precious support, it would not be possible to conduct this thesis I would also like to express my indebtedness to all the teachers who taught me during the past studying and equipped me with the knowledge as the basis for conducting my thesis as well as in real life My sincere thanks also go to teachers in faculty of Agronomy, Vietnam National University of Agriculture for their insightful comments and encouragement Besides that, I am thankful to my dear classmates, class K61KHCTT, for all the fun, friendship we had in the last four years Last but not least, I would like to thank my family for supporting me spiritually throughout my studying and life in general Hanoi, 28th February, 2021 Student Nguyen Xuan Khanh ii TABLE OF CONTENT COMMITMENT ACKNOWLEDGEMENTS TABLE OF CONTENT LIST OF TABLES LIST OF FIGGURE ABSTRACT I II III VII VIII IX CHAPTER 1: INTRODUCTION 1.1 INTRODUCTION 1.2 PURPOSE 1.3 REQUIREMENTS CHAPTER 2: LITERATURE REVIEW 2.1 ORIGIN, DEVELOPMENT HISTORY, AND CLASSIFY 2.1.1 ORIGINS, DEVELOPMENTAL HISTORY 2.1.2 CLASSIFY 2.2 TOMATO VALUE 2.2.1 NUTRITIONAL AND MEDICAL VALUE 2.2.2 ECONOMIC VALUE 2.3 CHARACTERISTICS AND DEVELOPMENT CONDITIONS OF TOMATO 2.3.1 TOMATO CHARACTERISTICS 2.3.2 DEVELOPMENT CONDITIONS OF TOMATO 2.3.2.1 TEMPERATURE 2.3.2.2 HUMIDITY 2.3.2.3 LIGHT 2.3.2.4 SOIL AND NUTRIENT 2.4 THE SITUATION OF TOMATO PRODUCTION IN THE WORLD AND IN VIETNAM 2.4.1 THE SITUATION OF TOMATO PRODUCTION IN THE WORLD 11 11 iii 2.4.2 THE SITUATION OF TOMATO PRODUCTION IN VIETNAM 14 2.5 THE SITUATION OF TOMATO RESEARCH IN THE WORLD AND VIETNAM 17 2.5.1 THE SITUATION OF TOMATO RESEARCH IN THE WORLD 17 2.5.2 THE SITUATION OF TOMATO RESEARCH IN VIETNAM 19 CHAPTER 3: MATERIALS AND METHODS 26 3.1 PLACE AND TIME 26 3.2 MATERIALS 26 3.3 METHODS 26 3.3.1 EVALUATING THE HORTICULTURAL CHARACTERISTICS OF SOME TOMATO LINES IN EARLY WINTER SEASON 2020 IN RED RIVER DELTA 3.3.1.2 CULTIVATION TECHNIQUE 26 27 3.3.2 PYRAMIDING PH2 AND PH3 GENES FOR BREEDING TOMATO TOLERANT TO LATE BLIGHT 3.4 DATA COLLECTION 28 29 3.4.1 TIME TO COMPLETE THE GROWTH AND DEVELOPMENT STAGES 29 3.4.2 MORPHOLOGICAL CHARACTERISTICS OF PLANT STRUCTURE 29 3.4.3 MORPHOLOGICAL CHARACTERISTICS AND QUALITY OF THE FRUIT 30 3.4.4 PRODUCTIVITY AND YIELD COMPONENTS 30 3.4.5 EVALUATION OF RESISTANCE TO LATE BLIGHT DISEASE 30 CHAPTER 4: RESULT AND DISCUSSION 31 4.1 HORTICULTURAL CHARACTERS OF SELECTED PLANTS 32 4.1.1.TIME TO COMPLETE THE GROWTH AND DEVELOPMENT STAGES 4.1.1.1 TIME FORM SEEDLING TO FIELD PLANTING 32 34 iv 4.1.1.2 TIME FROM FIELD PLANTING TO FLOWERING 34 4.1.1.3 TIME FROM FIELD PLANTING TO FRUIT RIPENING 35 4.1.2 MORPHOLOGICAL CHARACTERISTICS OF PLANT STRUCTURE 36 4.1.2.1 GROWTH SHAPE AND PLANT HEIGHT 38 4.1.2.2 FLOWERING CHARACTERISTICS AND FLOWER CLUSTER FORM 4.1.3 MORPHOLOGICAL CHARACTERISTICS OF THE FRUIT 39 40 4.3 SOME FRUIT MORPHOLOGICAL CHARACTERISTICS OF THE EARLY WINTER CROP OF TOMATO VARIETIES IN 2020 40 4.1.3.1 FRUIT SHAPE 42 4.1.3.2 NUMBER OF SEED COMPARTMENTS / FRUIT 42 4.1.3.3 THICKNESS OF FLESH FRUIT (MM) 43 4.1.3.4 BRIX DEGREE 43 4.1.4 FACTORS CONTRIBUTING TO THE PRODUCTIVITY OF TOMATO VARIETIES IN EARLY WINTER SEASON 2020 44 4.1.4.1 NUMBER OF CLUSTER 46 4.1.4.2 TOTAL FRUIT 46 4.1.4.3 MASS/FRUIT 47 4.1.4.4 THEORETICAL PRODUCTIVITY AND REAL PRODUCTIVITY 48 4.2 EVALUATION OF THE RESISTANCE TO LATE BLIGHT DISEASE OF TOMATO VARIETIES CARRYING PH GENE 49 4.2.1 THE LATE BLIGHT RESISTANT CHARACTERISTICS OF TOMATO LINES CONTAINING PH2 AND PH3 GENES 49 4.2.2 HORTICULTURE AGRICULTURE CHARACTERISTICS OF F5 TOMATO LINES CARRYING PH2 AND PH3 GENES 52 CHAPTER 5: CONCLUSION AND PROPOSALS 55 5.1 CONCLUSIONS 55 5.2 PROPOSALS 55 v REFFERENCES 56 APPENDIX 59 vi LIST OF TABLES TABLE 2.1 SUMMARY OF MAIN FUNCTIONS OF PLANT NUTRIENTS: 10 TABLE 2.2 TOMATO ACREAGE, PRODUCTION AND PRODUCTIVITY OF DIFFERENT CONTINENTS IN 2014 12 TABLE 2.3 TOMATO ACREAGE, PRODUCTION AND PRODUCTIVITY OF DIFFERENT CONTINENTS IN 2014-2018 13 TABLE 2.4 TOMATO ACREAGE IN VIETNAM IN THE PERIOD 2012 2016 14 TABLE 2.5 TOMATO PRODUCTION IN VIETNAM IN THE PERIOD 2012 - 2016 16 TABLE 4.1 THE GROWTH AND DEVELOPMENT STAGES OF TOMATO VARIETIES IN THE EARLY WINTER 2020 SEASON 32 TABLE 4.2 SOME OF MORPHOLOGICAL CHARACTERISTICS OF PLANT STRUCTURE 37 TABLE 4.4 FACTORS CONTRIBUTING TO THE PRODUCTIVITY OF TOMATO VARIETIES IN EARLY WINTER SEASON 2020 44 TABLE 4.5 THE RESISTANCE TO LATE BLIGHT DISEASE OF TOMATO LINES AFTER PYRAMID PH2 AND PH3 GENE 51 TABLE 4.6 HORTICULTURE AGRICULTURE CHARACTERISTICS OF F5 TOMATO LINES CARRYING PH2 AND PH3 GENES 53 vii LIST OF FIGGURE FIGURE ELECTROPHORESIS IMAGE OF TOMATO LINE F5 HOMOZYGOUS FOR PH2 GENE BY UF-PH2-1 DIRECTIVE 49 FIGURE IMAGE ELECTROPHORESIS IDENTIFIED TOMATO LINES HOMOZYGOUS F5 GENE BY DIRECTIVE PH3 PH3-GSM1 50 FIGURE EXPRESSION OF RESISTANCE TO LATE BLIGHT DISEASE OF PH2 + PH3 GENES 52 viii ABSTRACT The study was carried out from August 2020 to the end of February 2021 at the Fruit and Vegetable Research Institute, Trau Quy – Gia Lam – Ha Noi We conducted two experiments In the first one, we studied to select plants carrying genes Ph2 and Ph3 governing Leaf Blight disease resistant Our result show that the the disease resistance of F5 tomato lines carrying both Ph2 and Ph3 genes was higher than that of parents which carrying only gene, and these lines are good materials for tomato breeding In the second experiment, we evaluated the horticultural characteristics of some tomato lines in early winter season Most of the varieties have good growth and development, especially experimental varieties such as TC-1, TC-2 and TC-3 have outstanding horticulture characteristics compared to the control varieties ix 4.1.4.4 Theoretical productivity and real productivity These is a decisive factor for tomato varieties Along with good care conditions, adequate nutritional source, individual productivity is directly decisive to the actual tomato yield in the future of tomato varieties In our experiment, we calculated two kind of productivity: Theoretical and Net The theoretical productivity is calculated by the formula: Tp = (T*M*D)/1000/10000 (kg) In which: Tp is theoretical productivity (kg) T is total fruit M is average mass of fruit (g) D is density of plot, calculated = 10000/0.5/1.2 (1.2 and 0.5 is the length, the width of plot) The theoretical productivity represents the potential of each variety Knowing this indicator gives a basis for developing technical measures to maximize the potential of the crop The result in Table 4.3 show that theoretical productivity has strong differences between varieties/combinations The two control varieties (VN390 and Tre Viet) have theoretical productivity of 75.13 and 70.8, respectively TC01 and TC-02 is two experiment combinations have theoretical productivity higher than both control varieties (95.99 and 77.25) Net productivity The net productivity is the goal of production Therefore, there are many varieties with high theoretical yield but low actual yield, it proves that such variety has not met practical production needs Net productivity is high or low depending on the characteristics of the variety and its adaptability to seasonal conditions 48 The result in Table 4.3 show that there is big difference between the theoretical productivity and net productivity in each variety/combination The net productivity of two control varieties (VN-390 and Tre Viet) are 35.81 and 32.45 kg TC-1 and TC-2 have more net productivity than both control varieties Despite having smaller theoretical yield, TC-2 has significantly higher net yield than TC-1 (59.19 to 51.13) Similarity happened with TC-3, which had lower theoretical yield than the Tre Viet control, but had higher net yield (46.14 and 45.07) 4.2 Evaluation of the resistance to late blight disease of tomato varieties/lines carrying Ph gene 4.2.1 The late blight resistant characteristics of tomato lines containing Ph2 and Ph3 genes Through hybridization (LA3151˟ CLN2037B) and selection, Ph2 and Ph3 genes were gathered into F5 tomato lines On electrophoresis image showed that all F5 lines are homozygous for both genes (Figure 1, 2) The disease index and the number of spores produced on the disease spot showed resistance to late blight to the mushroom samples collected in Hanoi and Lam Dong of new tomato lines Figure Electrophoresis image of tomato line F5 homozygous for Ph2 gene by UF-Ph2-1 directive 49 From left to right: M-ladder marker, 1-5- infected line; 6- LA3151; 7-12: F5 line is homozygous for the Ph2 gene Figure Image electrophoresis identified tomato lines homozygous F5 gene by directive PH3 PH3-gsm1 From left to right: M-ladder marker, 1- CLN2037B; 2-7: F5 line is homozygous for Ph3 gene, 8-12- infected line The sustainability of tomato varieties depends on the fungus sample in each growing area Tomato varieties with only Ph3 gene but exhibited good resistance in Tanzania (Ojiewo et al., 2010) and some East African countries when the mildew was US-1 (Pule et al., 2013) In Vietnam, although there are no research reports on the subgroup of the late blight mushroom samples, all the F5 tomato lines produced have a significantly lower disease index than the cultivars with only resistance gene when infected with the collected fungus samples in Lam Dong The number of spores formed on the pathogen of some strains of F5 was much lower than that of parental lines (Figure 3, Table 4.5) This showed that the convergence of both Ph2 and Ph3 genes significantly improved disease resistance This result is consistent with Chen et al (2008) that the Ph2 and Ph3 genes are both specific resistant and will complement each other to improve the resistance to late blight compared to the individual genes 50 Table 4.5 The resistance to late blight disease of tomato lines after pyramid Ph2 and Ph3 gene Hà Nội Lines/Varieties Genes Disease Lâm Đồng Spore index Disease Spore index LA3151 Ph2 3,7b 8,2b 4,5a 9,4b CLN2037B Ph3 2,3c 0,5c 2,6b 1,0c 20-TC-64 Ph2+Ph3 1,9c 0,0d 2,1c 0,5d 20-TC-65 Ph2+Ph3 1,8c 0,0d 2,2c 0,7cd 20-TC-66 Ph2+Ph3 2,1c 0,5c 2,0c 0,5d 20-TC-67 Ph2+Ph3 1,7c 0,5c 2,1c 0,5d 20-TC-68 Ph2+Ph3 1,8c 0,0d 2,2c 1,0c 20-TC-69 Ph2+Ph3 2,1c 0,0d 2,2c 0,5d PT18 None 5,3a 27,4a 5,6a 30,2a 51 Variety carries the Ph2 gene Variety carries the Ph3 gene Variety carries the Ph2 + Ph3 gene Variety infected late blight disease Figure Expression of resistance to late blight disease of Ph2 + Ph3 genes 4.2.2 Horticulture agriculture characteristics of F5 tomato lines carrying Ph2 and Ph3 genes 52 Table 4.6 Horticulture agriculture characteristics of F5 tomato lines carrying Ph2 and Ph3 genes Lines/ Variety Growth shape Mass/Fruit Brix Productivity (kg/plant) LA3151 Indeterminate 50,2±3,6 3,9 1,2±0,20 CLN2037B Determinate 85,2±2,6 4,7 1,8±0,15 20-TC-64 Indeterminate 79,4±2,8 4,2 1,9±0,22 20-TC-65 Indeterminate 68,2±3,9 4,5 1,5±0,17 20-TC-66 Semi determinate 56,1±1,9 4,3 1,4±0,21 20-TC-67 Determinate 63,8±3,2 4,6 1,3±0,11 20-TC-68 Semi determinate 83,7±2,6 4,4 1,7±0,12 20-TC-69 Semi determinate 57,9±2,2 4,1 1,2±0,17 Tomato growth form is a genetic trait, in which indeterminate growth is a dominant trait, determinate growth is a recessive trait Using opposing parental lines samples on growth morphology in crossbreeding resulted in dissociated posterior generation in this trait In addition to the parental line growth phenotype, F5 tomato line with semi-determinate growth appeared (20TC-66, 20TC-68, 20TC69) Fruit weight of the line 20-TC-64, 20-TC-68 belongs to the medium group (80 - 90 g), the other lines have smaller fruit The fruit weight of all lines is within the 53 limit of the parental line fruit, does not appear "superior" in this trait Similarly, the total dissolved solids (Brix) content also exhibited intermediate genetic traits of the parental line Due to the change in growth shape and fruit weight, the yield of F5 clones is also diverse The 20-TC-64 line had the same productivity as the excellent parental strain (Table 4.5) 54 Chapter 5: Conclusion and proposals 5.1 Conclusions - From the above research results, we can show some conclusions: Experiment 1: Most of the varieties have good growth and development, especially experimental varieties such as TC-1, TC-2 have outstanding horticulture characteristics compared to the control varieties VN-390 and Tre Viet The TC-3 variety also showed very good results Experiment 2: tomato lines carrying both Ph2 and Ph3 genes was higher resistance to late blight disease than that of parents which carrying only gene, and these lines are good materials for tomato breeding 5.2 Proposals - Quickly introduce good varieties to trial production - The selected plants could be used for disease-resistant tomatoes 55 REFFERENCES Nguyễn Hồng Minh (2000) Chọn giống cà chua Trong giáo trình chọn giống Nguyễn Văn Hiển chủ biên Nhà xuất giáo dục, 2000 Võ Văn Chi (1997) Từ điển thuốc Việt Nam NXB Y học Trịnh Khắc Quang, Trần Ngọc Hùng (2012) Xác định nguồn gien thích hợp phục vụ tạo giống cà chua chống chịu bệnh sương mai (Phytophthora infestans) Việt nam.Tạp chí Nơng nghiệp phát triển nơng thơn ISSN 1859-4581.16/2012 59-65 Trần Ngọc Hùng, Đặng Thị Mai (2010) Ảnh hưởng độ thành thục lá, tuổi thời gian buồng sinh trưởng đến lây nhiễm nhân tạo bệnh sương mai (Phytophthora infestans) phục vụ chọn tạo giống cà 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