Selection of iaa9 gene edited t1 tomato lines by crispr cas9

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY    UNDERGRADUATE THESIS TITLE: SELECTION OF IAA9 GENE-EDITED T1 TOMATO LINES BY CRISPR/CAS9 HANOI – 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY    UNDERGRADUATE THESIS TITLE: SELECTION OF IAA9 GENE-EDITED T1 TOMATO LINES BY CRISPR/CAS9 Student name : NGUYEN THUY LINH Class : K63CNSHE Student code : 637416 Department : BIOTECHNOLOGY Supervisor : TONG VAN HAI, M.S PHAM THI DUNG, Ph.D HANOI – 2022 DECLARATION I hereby declare that the graduate thesis work is mine All research results have been results during the implementation of the topic The results and the data are completely true and never appeared in any scientific report I also guarantee that the references and useful information for the topic are cited and all help is appreciated Hanoi, December 22nd, 2022 Student Nguyen Thuy Linh i ACKNOWLEDGEMENTS During the process of implementing my graduation project, I received a lot of attention and help from individuals and groups First of all, I would like to express my respect and deep gratitude to Tong Van Hai, M.S and Pham Thi Dung, Ph.D for allowing me to carry out this work, and for their huge efforts, enthusiasm, and support throughout the duration of the undergraduate thesis Secondly, I would like to thank the teachers in the Faculty of Biotechnology who have helped and taught me during my training at the university Especially the teachers of the Molecular biology and Applied Biotechnology department who helped and gave me useful advice during carrying out Finally, I would like to sincerely thank my family members and friends who always trust, support, and encourage me to complete this report Sincerely thank! Hanoi, December 22nd, 2022 Student Nguyen Thuy Linh ii CONTENTS DECLARATION i ACKNOWLEDGEMENTS ii CONTENTS iii LIST OF TABLES v LIST OF FIGURES vi LIST OF ABBREVIATIONS vii PART I INTRODUCTION 1.1 Preface 1.2 Objective and Requirements 1.2.1 Objective 1.2.2 Requirements PART II LITERATURE REVIEW 2.1 The situation of tomato production in the world and Vietnam 2.1.1 The situation of tomato production in the world 2.1.2 The situation of tomato production in Vietnam 2.2 Genome editing 2.3 CRISPR/Cas9 gene-editing system in plants 11 2.4 Improvement of tomato physiological by CRISPR/Cas9 15 2.4.1 Colors and pigments content in tomato fruits 15 2.4.2 Tomato fruit ripening and shelf life 16 2.4.3 Production of parthenocarpic tomato fruits 18 2.4.4 Generation of dwarfed tomato cultivars 19 2.4.5 Characterization of senescence-related genes in tomato 19 2.4.6 Architecture of tomato inflorescence 20 2.4.7 Flowering time in tomato 20 2.4.8 Development of abscission zone in tomato fruit 20 2.4.9 CRISPR/Cas9 and metabolic pathways studies in tomato plants 21 2.4.10 CRISPR/Cas9 decreases the impact of environmental stress on tomato 21 2.4.11 Role of CRISPR/Cas9 in tomato resistance to biotic stress 22 2.4.12 CRISPR/Cas9 can produce herbicide-resistant tomato plant 24 2.4.13 CRISPR/Cas9 can identify genes functions in tomato plant 24 PART III MATERIAL AND METHODS 25 3.1 Location and time for research 25 3.1.1 Research location 25 iii 3.1.2 Time for research 25 3.2 Material 25 3.2.1 Subjects 25 3.2.2 Key equipment 31 3.2.3 Chemicals 31 3.3 Methods 32 3.3.1 DNA extraction 32 3.3.2 Selection of mutant T1 plants by molecular markers 32 3.3.3 Evaluation of agro-biological characteristics of tomato lines 33 PART IV RESULTS AND DISCUSSION 35 4.1 Selection of editing plants in the T1 generation 35 4.1.1 DNA extraction 35 4.1.2 Selection of T1 plants containing homozygous edited gene by molecular markers 38 4.2 Agro-biological characteristics of the edited T1 lines 42 4.2.1 Growth pattern 42 4.2.2 Growth stages 43 4.2.3 Some characteristics of leaf morphology and plant structure 44 4.2.4 Some morphological characteristics, flower structure and bloom characteristics 47 4.2.5 Yield and yield components 49 PART V CONCLUSION AND SUGGESTION 51 5.1 Conclusion 51 5.2 Suggestion 51 REFERENCES 52 iv LIST OF TABLES Table 2.1 The situation of tomato production in the world Table 2.2 Tomato production of the 10 largest producing countries in the world from 2016 to 2020 Table 2.3 Area, yield, and output of tomatoes in Vietnam from 2015-2019 Table 2.4 Area, yield, and output of the top 10 provinces and cities nationwide in 2017 Table 2.5 Summary of gene-edited crop species using CRISPR/Cas9 system 13 Table 3.1 Regeneration efficiency and morphological and genotypic results of T0 generation IAA9 gene-edited plants of two tomato lines TP1-1 and TP195-2 29 Table 4.1 OD measurement results of tomato DNA 36 Table 4.2 Growth stages and some morphological characteristics of plant structure 46 Table 4.3 Inflorescence structure, flowering characteristics 48 Table 4.4 Yield and yield components 50 v LIST OF FIGURES Figure 2.1 Tomato production by regions of the world Figure 2.2 The workflow of CRISPR/Cas9-based gene editing in plants 12 Figure 2.3 Data on research articles published on CRISPR/Cas9 from 2016 to 2020 14 Figure 2.4 The number of genes modified using the CRISPR/Cas system with the aim of crop improvement 14 Figure 2.5 Carotenoid biosynthesis pathway in tomato plant in rectangular shapes 16 Finger 3.1 The 12,068 bp IAA9 lv2 vector was transformed into Agrobacterial tumerfacies GV3101 for IAA9 gene editing 27 Finger 3.2 Samples of shoot and shoot formation of T0 tomato lines TP195-2 28 Finger 3.3 T-DNA testing on T0 generation IAA9 gene-edited plants (TP1-1) 29 Finger 3.4 T-DNA testing on T0 generation IAA9 gene-edited plants (TP195-2) 29 Finger 3.5 PCR product multiplying exon gene IAA9 region of large plants of TP1-1 parents 30 Finger 3.6 PCR product multiplying exon gene IAA9 region of large plants of TP195-2 parents 30 Finger 3.7 Sequencing exon gene IAA9 30 Finger 4.1 Total DNA electrophoresis of T1 lines (408) 37 Finger 4.2 Total DNA electrophoresis of T1 lines (701) 37 Finger 4.3 Total DNA electrophoresis of T1 lines (704) 38 Finger 4.4 PCR products multiplying the IAA9 gene region of plants (408 and 704) 38 Finger 4.5 Cutting site of enzyme HindIII 39 Finger 4.6 Cut PCR products of T1 individuals (408) of parental origin from TP1-1 line by HindIII enzyme (600 and 370 bp) 39 Finger 4.7 PCR products of individuals T1(701) have parental origin from line TP195-2 40 Finger 4.8 Cutting site of enzyme AluI 41 Finger 4.9 Cut PCR products of T1(704) individuals of parental origin from TP195-2 line with AluI enzyme (560 and 410 bp) 41 Finger 4.10 T1-701 plants with developed cotyledons 42 Finger 4.11 Leaf form of T1-408 line and parental line 45 Finger 4.12 Simple inflorescence in T1-408 tomato line 47 vi LIST OF ABBREVIATIONS alc Alcobaca ALS Acetolactate synthase ARFs Auxin Response Factors BeYDV Bean yellow dwarf virus Blc Beta-lycopene cyclase Cas9 CRISPR-associated protein-9 nuclease CBEs Cytidine base editors CP Coat protein CRISPR Clustered Regularly Interspaced Short Palindromic Repeats crRNA CRISPR RNA CRTISO Carotenoid isomerase CVYV Cucumber vein yellowing virus D Determinant ERF Ethylene-responsive factor GABA Gamma-aminobutyric acid gRNA Guide RNA ID In-determinant j Jointless j2 Jointless2 JAs Jasmonates LCY E Lycopene cyclase LCYB1 Lycopene beta cyclase MAPK3 Mitogen-activated protein kinase3 MDH Malate dehydrogenase MeJA Methyl jasmonate MLO Mildew resistance locus O NarCh Naringenin chalcone nor Non-ripening NPR1 No express or pathogenesis-related vii PAM Protospacer adjacent motif PG2a Polygalacturonase 2a PL Pectate lyase PRSV-W Papaya ring spot mosaic virus-W PSY1 Phytoene synthase I rep Replicase R-genes Resistance genes rin Ripening inhibitor RNP Ribonucleoprotein RTDS Rapid Trait Development System SBPase Sedoheptulose-1,7-bisphosphatase SD Semi-determinant S-genes Susceptibility genes sgRNA Single guide RNA SlAGL6 SlAGAMOUS-LIKE SlCBFs C-repeat binding factors SP5G Self-Pruning 5G TMF Terminating Flower tracrRNA Trans-activating crRNA TYLCV Tomato yellow leaf curl virus VIGS Virus-induced gene silencing ZYMV Zucchini yellow mosaic virus viii continuously and has the potential for high yield Although considered to have a higher yield potential than the determinant pattern, in temperate countries the yield potential of the in-determinant variety is not higher than that of the determinant variety due to the presence of frost and snow cover In tropical and subtropical countries, the potential yield of in-determinant varieties is also not really higher than that of determinant varieties due to the strong destruction of diseases Therefore, tomato varieties with a determinant growth pattern have more advantages and are preferred over in-determinant varieties As a result of observing the growth, height and flowering characteristics of the 12 samples studied, we found that all plants of the T1-408 line and of the T1-704 lines had determinant growth similar to parental line (Table 4.2) 4.2.2 Growth stages Like other plants, to complete the life cycle, tomato plants have to go through different growth periods Each period has a direct or indirect impact on yield and fruit quality Evaluation of the growth periods of tomato plants helps to determine the basic characteristics of the variety such as early or late ripening, and short or long growth time, thereby planning suitable planting arrangements The main stages in the development cycle of the tomato plant include: planting to flowering, first fruit harvesting, and the end of harvest The results of the evaluation of the growth stages of T1-408 and T1-704 tomato lines are presented in Table 4.2 - Time from planting - flowering: This is the period when the plant completes the vegetative growth phase and enters the reproductive growth phase Usually, if the plant flowers early, the fruit ripens early, and early harvest is a very important feature in production The results of monitoring 12 samples of tomato plants showed that: samples of T1-408 plants had the time from planting to flowering in the range of 31 - 35 days and samples of T1-704 plants had the time from planting to flowering in about 30 - 38 days 43 - Time from planting - harvesting the first fruits: This time is an indicator of early or late ripening of the variety Good, preferred tomato varieties must be early ripening varieties for early harvest - Time from planting to end of harvest: This indicator is studied on determinant varieties However, because the studied tomato lines are only fruiting, two indicators: the time from planting to harvesting the first fruits and the time from planting to the end of harvest cannot be evaluated 4.2.3 Some characteristics of leaf morphology and plant structure - Green levels of leaves: Leaves are the main photosynthetic organ, providing energy for physiological and biochemical processes of plants The greener the plants, the more favorable the photosynthesis This is also one of the characteristic morphological features of the plant Results of monitoring variety T1-408 have samples of green leaves and sample of dark green leaves; variety T1-704 have sample of dark green leaves and samples of green leaves - Leaf characteristics: Depending on the degree of leaf lobes, leaves are distinguished into two forms feathery compound leaves and potato leaves Plants with potato leaf form often have a small number of leaves on the plant, long, large leaves, and shorter petioles than normal leaves Observation results showed that all plants of the edited lines had potato leaves (Figure 4.11a), while the parent line has feathery compound leaves with small leaf surface, many lobed, short leaves (Figure 4.11b) 44 a b Finger 4.11 Leaf form of T1-408 line and parental line - Height from the base to the first inflorescence: The height from the base to the first inflorescence depends on the number of nodes from the base to the first inflorescence, internode length, external conditions, and cultivation and care techniques If the first inflorescence is too high will affect the position, number, and development of the next inflorescence, thereby reducing the number of fruits/plant, and affecting the yield of varieties The study showed that the plants of the T1-408 line had a height from the base to the first inflorescence ranging from 30.5 to 49.0 cm, the plants of the T1-704 line had a height from the base to the first inflorescence ranging from 35.6 - 43.5 cm (Table 4.2) - The number of nodes from the base to the first inflorescence is correlated with the time from planting to flowering and with the time from planting to fruit ripening Specifically, the smaller the number of nodes under the first inflorescence, the shorter the time from sowing to flowering and to ripening, thus allowing earlier harvest The evaluation results show that the T1 408 line has the number of nodes to inflorescence ranging from 5.0 to 9.0 nodes, the T1-704 line ranges from 8.0 to 10.0 nodes (Table 4.2) 45 Table 4.2 Growth stages and some morphological characteristics of plant structure No Name Growth pattern Day of flowering (day) Leaf color Leaf features Height from base to inflorescence (cm) Number of nodes under the first inflorescence (node) Height of the main stem (cm) P (408) D 33 Green Feathery compound 36.8 10.2 55.6 T1 (408-3) D 34 Green Potato 35.8 8.0 47.5 T1 (408-8) D 31 Dark green Potato 49.0 7.0 58.6 T1 (408-10) D 33 Dark green Potato 30.5 5.0 53.5 T1 (408-12) D 34 Dark green Potato 36.7 7.0 55.0 T1 (408-14) D 33 Green Potato 43.2 9.0 56.0 T1 (408-16) D 35 Green Potato 44.4 9.0 64.3 P (704) D 34 Green Feathery compound 37.5 8.9 60.7 T1 (704-2) D 35 Green Potato 35.6 8.0 63.0 10 T1 (704-8) D 32 Dark green Potato 36.5 8.0 62.5 11 T1 (704-9) D 35 Green Potato 39.1 9.0 58.0 12 T1 (704-10) D 34 Green Potato 43.5 9.0 63.5 13 T1 (704-15) D 38 Green Potato 37.6 10.0 59.0 14 T1 (704-18) D 30 Green Potato 41.1 10.0 61.2 46 - The height of the main stem depends on the characteristics of the variety and the care conditions as well as the external conditions With good care conditions, no pests and diseases, favorable weather, the plant height is higher Samples with determinant and semi-determinant growth, after the final inflorescence, the plant stops growing in height, so there is a height limit depending on the variety characteristics According to author Ta Thu Cuc (2009), in determinant varieties, when the plant produces the last inflorescence, the plant stops growing in height and the main stem height is less than 65 cm The results of the assessment of the main stem height of T1-408 trees ranged from 47.5 to 64.3 cm, and T1-704 plants ranged from 58.0 to 63.5 cm (Table 4.2) Thus, compared with Ta Thu Cuc's study, the plant samples with determinant growth pattern had the same height as studied 4.2.4 Some morphological characteristics, flower structure and bloom characteristics The characteristics of inflorescence structure include inflorescence type, bloom characteristics, number of flowers/inflorescence There are types of inflorescences including: Simple form with a main axis, staggered form on the axis; The intermediate form has major axes and the complex form has many major axes The results show that both T1-408 and T1-704 tomato lines have simple inflorescence form (Figure 4.12) Finger 4.12 Simple inflorescence in T1-408 tomato line 47 Flowering characteristics are related to the ripening characteristics of the fruit The variety blooms synchronously, the fruit ripens in a concentrated manner, which is convenient for harvesting, especially for harvesting by machine, when the flowers bloom scatteredly, the fruit ripens scatteredly, which is convenient for spreading the crop The evaluation results showed that 12 plant samples all had the characteristics of concentrated flowering, so the ripe fruit was concentrated, which was convenient for harvesting The number of flowers/inflorescence is one of the factors that determine the yield of varieties The number of flowers/ inflorescence is high, the fruit setting rate is high, leading to a large number of fruits, usually higher yield The results show that the T1-408 line has the number of flowers/inflorescence ranging from 6.0 - 9.4 flowers, the T1-704 lines range from 5.4 - 8.2 flowers/ inflorescence (Table 4.3) Table 4.3 Inflorescence structure, flowering characteristics No Line Inflorescence form Flowering characteristic Number of flowers/inflorescence P (408) Simple Concentrated 7.3 T1 (408-3) Simple Concentrated 6.5 T1 (408-8) Simple Concentrated 9.4 T1 (408-10) Simple Concentrated 8.3 T1 (408-12) Simple Concentrated 6.0 T1 (408-14) Simple Concentrated 6.5 T1 (408-16) Simple Concentrated 7.6 P (704) Simple Concentrated 7.8 T1 (704-2) Simple Concentrated 5.4 10 T1 (704-8) Simple Concentrated 7.0 11 T1 (704-9) Simple Concentrated 7.1 12 T1 (704-10) Simple Concentrated 6.2 13 T1 (704-15) Simple Concentrated 6.9 14 T1 (704-18) Simple Concentrated 8.2 48 4.2.5 Yield and yield components Yield and yield components are indicators used to more fully assess the adaptability of varieties to external conditions In addition, along with fruit quality, yield is one of the two top important factors that producers pay attention to in deciding whether to put varieties into production or not The yield of tomato plants is controlled by genetic characteristics of the variety and is affected by environmental conditions, nutritional regimes as well as cultivation techniques There are many factors to make up the yield such as: number of flowers/inflorescence, number of fruits/bunch, fruit setting rate, number of fruit bunches/plant, average weight of fruit, number of fruits/plant (Table 4.4) The number of fruits/bunch depends on the number of flowers/inflorescences and the rate of fruit set Varieties with a large number of flowers/inflorescences and high fruiting rates have a large number of fruits/bunch, which is a direct determinant of the yield of the variety The evaluation results showed that the number of fruits/bunch of the T1-408 line ranged from 5.6 – 8.3 fruits, the T1-704 line ranged from 5.0 – 7.5 fruits/bunch The number of fruits/plant and the average fruit weight are two factors that directly determine the yield of the variety The higher the number of fruits and the higher the fruit weight, the higher the yield potential of the variety Research results show that the T1-408 line gives the number of fruits/plant ranging from 21.0 – 30.0 fruits, T1-704 ranges from 23.0 - 27.0 fruits/plant 49 Table 4.4 Yield and yield components No Line Number of fruits/bunch Number of fruits/plant P (408) 6.8 27.5 T1 (408-3) 6.4 27.0 T1 (408-8) 8.3 25.0 T1 (408-10) 7.7 22.0 T1 (408-12) 5.6 23.0 T1 (408-14) 5.8 21.0 T1 (408-16) 7.2 30.0 P (704) 7.1 28.3 T1 (704-2) 5.0 27.0 10 T1 (704-8) 6.5 26.0 11 T1 (704-9) 6.5 24.0 12 T1 (704-10) 5.8 25.0 13 T1 (704-15) 6.3 24.0 14 T1 (704-18) 7.5 23.0 Individual yield is an indicator that helps to determine the yield potential of a variety and is determined by two factors: number of fruits/plant and average weight of fruit If the variety has a large number of fruits/plant and average fruit weight, the individual yield is high and vice versa Therefore, in order to have a large individual yield, it is necessary to harmoniously combine the two factors of number of fruits/plant and average weight of fruit Due to the limited research time, the studied tomato lines have not yet developed to the ripe fruit stage, so the average fruit weight and individual yield have not been evaluated 50 PART V CONCLUSION AND SUGGESTION 5.1 Conclusion DNA of T1 tomato lines was extracted successfully to serve PCR reaction to select mutant lines of the IAA9 gene By PCR, multiplying the IAA9 gene region and restriction enzyme HindIII for the T1-408 line, AluI for the T1-704 line, homozygous edited individuals were selected Through evaluation of homozygous edited tomato lines in terms of agrobiological characteristics, it was found that: T1-701 line only developed cotyledons T1-408 and T1-704 lines had growth time, some characteristics of stem and fruit similar to the parental line In particular, in leaf characteristics, the edited lines had potato leaves while the parental lines have feathery compound leaves 5.2 Suggestion Due to the limited time in the research process, the report has some limitations Because of this, we would like to give some recommendations: Continue to monitor agro-biological characteristics of homozygous gene-edited tomato lines T1-408 and T1-704 such as yield and yield components, and fruit characteristics Research on evaluation of T2-generation tomato lines, from which there is a basis to create pure tomato lines and put them into production to help 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