0 MINISTRY OF EDUCATION AND TRAINING THÁI NGUYÊN UNIVERSITY VŨ THỊ THU THỦY CREATING DROUGHT TOLERANCE LINES AND ISOLATING CYSTATIN GENES RELATED TO DROUGHT TOLERANCE IN PEANUT (Arachis hypogaea L.) Major: Genetics Code: 62.42.70.01 PHD THESIS ABTRACT Thai Nguyen - 2011 1 INTRODUCTION 1. Reasons for choosing the topic Groundnut is a valuable crop in many aspects and many countries around the world wishing to expand and develop groundnut production. Groundnut plant belongs to the bean group with low drought tolerance. Compared to many other crops, groundnut specially needs water as its roots do not have absorbing hair, groundnut fruit is shaped underground. Statistical results show that groundnut production accounts for 40% of total cultivated land area of short-term industrial crops, of which 2/3 depends on the rain. For many localities, groundnut is the main crop, investment in the development of groundnut production however has not yet been commensurate with its inherent potential. There are many methods of improving plant varieties of which tissue culture of plant cells is an effective technique, allowing applications and improvement of many features of the plant. During the culture, the cells can be genetically modified because of the influence of the environment. If mutation generated agents are combined, the mutation generated frequencies will be significantly increased. This is significant in creating the source material for breeding. Effectiveness of application of plant cells technologies to improve the tolerance to externally adverse conditions is continuously confirmed. In Vietnam, the birth of two rice varieties DR1 and DR2 which resist the drought and the cold primarily demonstrates the possibility. Following is the research to improve drought tolerance and salt-resistance of sugarcane by Yadav et al. (2006) and of wheat by Abdelsamad et al. (2007), etc. Drought tolerance of the plant is characteristics specified by multiple genes therefore the search and analysis of genes related to drought tolerance are studied by many scientists. Several genes related to drought tolerance of plants has been isolated and published such as LEA genes in soybean and green beans, P5CS gene in soybean, cystatin genes in green bean plants, DREB genes in Arabidopsis, etc. Cystatin gene (Cys) of vegetation was first published on rice by Abe et al. (1987), so far Cys gene has been isolated in many species of higher plants in both monocots and dicotyledonous (Cys in green beans, Cys gene in potato gene, Cys genes in maize). Studies of the cystatin gene have been widely discussed focusing on its relationship to resistance to drought, cold and salt etc. With the above mentioned reasons and from the practical needs of groundnut breeding optimism towards improving drought tolerance, we have carried out the thesis topic: 2 "Creating drought tolerance lines and isolating cystatin genes related to drought tolerance in peanut (Arachis hypogaea L.)”. 2. Research objectives - Create groundnut strain with drought tolerance higher than original breed using tissue culture of plant cells. - Compare cystatin gene sequence of groundnut strain selected from scar tissue with that of the peanut cultivars. 3. Research contents - Screening scar tissue strain resistant to water loss affected by dry blowing and gamma irradiation combined with dry blowing. - Analyzing the fluctuation of the number of selective lines through the generations. - Making comparison and defining the discrepancy in genome of the line selected by RAPD technique. - Amplifying, separating and defining the sequence of the cystatin gene of selective groundnut line and the original line. 4. New contribution of the topic i) The thesis is a systematic research on application of technologies in plant cells to improve and enhance drought tolerance of groundnut, from creating scar tissue, processing scar tissues that create somatic mutation, selecting cell lines resistant to dehydration, reviving plants, generating plants and analysis, assessment through generations, selecting lines of groundnuts preeminent by drought tolerance and some biological and agricultural features. ii) Callus were treatment of scar tissue by gamma rays has been reduced in height and rate of regeneration plants, change colors and leaf shapes. The findings are five indicators specific RAPD two lines of contact selectively RM47, RM48: RM48/OPA07- 750bp; RM48/OPA08-500bp; RM48/OPB05-900bp; RM48/UPC348-200bp; RM47/OPH08-200bp. Discovery of peanut cystatin genes of group I phytocystatin, the most closely related to cystatin green beans, farthest to the cystatin of kiwi fruit (42.9%). The gene contains an intron and two exons coding for 98 amino acid protein. Cystatin of RM48 line derived from scar tissue is processed by gamma rays associated with the blower has 7 positions of amino acid differences compared to the same original L18. iii) Determine the difference compared to the same original L18 on drought tolerance of three lines lost RM48, RM47, R46 can be derived from scar tissue dehydration is treated by gamma rays (2krad) with blower continuous 9 hours in in vitro culture systems. 3 5. The meaning of scientific and practical 5.1. Scientific Significance i) The results of the thesis provide scientific data to guide application of plant cells to improve drought tolerance of groundnut. The technique selected cell lines under dehydration and treatment of scar tissue to increase the frequency of mutations arising. The approach and methods to evaluate the difference of the selected line compared to just touch base in terms of morphology, expression yield component traits, drought tolerance, grain characteristics and biochemical differences in the genome. ii) Provide information on the cystatin and cystatin genes in peanut. Handling of scar tissue by radiation combined with water is blown causing agent appeared cystatin gene mutations as evidenced by differences in genetic sequences of the RM48 (derived from scar tissue to be irradiated with blow dry) than the R46 line (derived from scar tissue be blown) and like the original L18. 5.2. Practical significance The results of a comprehensive review of drought tolerance of groundnut extent of scar tissue, germinating seeds and young plants as a basis to assess and apply measures to improve drought tolerance of groundnut. Results bred three communication lines derived from scar tissue under treatment by irradiation and the blown advantage of drought tolerance Vietnamese and some characteristics of agricultural biology, biochemistry, can foster the seeds of new varieties or material for hybridization. 6. The structure of the thesis The thesis consists of 128 pages (including references), divided into sections: Introduction includes 3 pages; Chapter 1: Overview document, 34 pages; Chapter 2: Materials and Methods, 14 pages; Chapter 3: Results and discussion Comment, 54 pages; The conclusions and proposals: 2 pages; works were published concerning the thesis: 1 page. References: 15 pages; thesis has 22 tables, 20 pictures. There are 145 references in Vietnamese and English. Chapter 1. DOCUMENT OVERVIEW The thesis has reference materials and review 34 domestic and 98 foreign documents with related content, including: (1) Groundnuts and drought-resistant properties of the peanut, (2) Advanced research drought tolerance of crops by plant cell technology, (3) 4 Analysis and evaluation of selected lines derived from callus culture (4) Genes related to drought tolerance in groundnut; (5) Cystatin and the role of cystatin in plants. With the lead data collected, the analysis has confirmed that peanut valuable and important position in the economy of many countries around the world. The trend of climate change that changed the elements of environmental conditions and a growing number of evaluation studies, breeding optimism towards higher resistance is made. Like other crops, the methods used in breeding, including hybridization communication, breeding, mutant selection from the population and use of modern biotechnology. The application of modern techniques of biotechnology to improve plant resistance was conducted in two directions, which are selected lines and transgenic somatic mutations. The success of gene transfer techniques have been published on several crops and several properties related to the drought tolerance of plants has improved. The technique selected cell lines in higher plants based application understanding of the omnipotence of the cell; heterogeneity of tissue or cultured cell populations; influence of culture medium in forming a complete body make cultured cell populations can be considered a population of plant cells, thereby screening the individual will be faster and more efficient than conventional breeding methods applied on intact plants. Select the line by increasing resistance to the unfavorable factors of the external environment has been successful on several subjects such as rice, dry rice, wheat, tobacco These are our suggestions for choosing in vitro culture techniques to improve drought resistance in peanut. Drought tolerance of plants is due to multiple gene traits decision. Search trends drought gene research is a major concern of many authors. The genes involved in drought tolerance of groundnut published in recent years such as LEA genes related to cellular dehydration. Su's research et al. (2010) on the same communication Luhua 14 detected at least eight genes LEA. AhNCED gene encoding synthetic 9-cis epoxycarotenoid dioxygenase was also confirmed related to the resistance of plants, the peanut AhNCED isolated by Wan et al. (2005) size 2486bp, encoding a protein consists of 610 amino acids. PLD gene encoding phospholipase D was also confirmed related term was found in two types of communication is through AhPLD1 and AhPLD2 Nakazwa team (2006) Cystatin phytocystatin in plants is called, consists of two groups, different in size, mass and areas associated with the cysteine proteinase. Since 1987, cystatin genes in rice were isolated, the cystatin gene has been isolated in many plants, but the peanut was little known. Study the function of cystatin, many authors discuss the relationship of cystatin with tolerance of plants. The complexity of the structure of the cystatin genes, the ability of gene expression in different growth stages of crops and in relation to the resistance of the disadvantages of 5 the external Some published on gene expression of cystatin-related drought tolerance has been demonstrated cove on bean plants, vegetables genes, sea mustard, winter wheat is the basis for our continued search and analysis on peanut cystatin genes. Chapter 2. MATERIAL AND METHODS 2.1 Plant materials The thesis using 10 peanuts cultivars as the materials research. In particular, 8 varieties (L05, L16, L18, L23, L24, V79, MD7, MD9) offered by the Center for research and development Legum, Institute of Food Crops Vietnam. SD30 variety provided by Center of Agricultural Extention Nam Dinh province and red peanut (red BG) due to Bean Development Center Legum Viet Yen district, Bac Giang Province has to offer. 2.2 Chemicals and equipment Using the chemical purity and dedicated derived from reputable companies such as the drug company's growth Sigma; Taq-polymerase, EDTA, SDS, agarose Invitrogen's. 2.3. Research methodology 2.3.1. Cultured in vitro method To create the technical communications in vitro culture, the culture of the steps: creating scar tissue completely in the dark (10 days) to conduct the airflow caused by dehydration of the box sterile culture and irradiated and then combined to cause dehydration by blowing dry recycling plant, creating a complete tree as described by Nguyen Thi Tam et al. (2006). 2.3.2. Field research methodology Mark individual plant lines regenerated from callus. To make the planting and care as directed by the Ministry of Agriculture and Rural Development. Track indexes and agronomic performance of plants at maturity. 2.3.3. Method of physiological, biochemical Quantification of soluble protein by the method of Lowry; quantitative lipid Soxhlet - amylase activity by the method of Heinkel; The methodαmethod; Define of sugar analysis. Assessing dehydration tolerance of scar tissue by staining of cells et al. Towill (1975). Assessing drought-resistant seed germination stage method caused due to sorbitol -amylase, sugar.α7% defined tolerance by determining the activity of Assessing drought tolerance at seedling stage by artificial methods to cause limited by Le Tran Binh et al. (1998). 6 2.3.3. Methods in molecular biology Total DNA extraction method Gawell et al. (1991). DNA polymorphism analysis by RAPD technique. Isolation of genes by PCR technique. Separation of the gene by the method of Sambrook et al. (2001). Gene sequence was determined on the ABI PRISM 3100 Avant automatic Analizer Genetic, Institute of Biotechnology. 2.3.4. Methods of data analysis Analysis of statistics with Excel software as Chu Hoang Mau (2008) Data analysis by RAPD NTSYSpc software version 2.0; Results of genetic analysis using BioEdit and DNAstar software. 2.4. Study location The experiment was conducted at the University of Pedagogy; University of Science; Institute of Life Sciences-University of Thai Nguyen. Irradiation gamma ray irradiation at the National Center, Tu Liem, Hanoi. DNA sequences were determined at the Institute of Biotechnology. Field experiments are located at the Forest cane, Quang Vinh Ward-Thai Nguyen city. Chapter 3. RESULTS AND DISCUSSION 3.1. RESULTS CREATED THE DROUGHT TOLERANCE BY TECHNICAL CALLUS TREATMENT IN THE IN VITRO CULTURED SYSTEM 3.1.1. Screening lines callus subjject blow dry 3.1.1.1. Ability to create scar tissue and the growth of scar tissue studied 10 breeds optimism For the purpose of assessing scar tissue of the same communication in in vitro culture systems as the basis for the research to create drought-tolerant line of communication with the plant cell technology, we surveyed the ability to create scar tissue from the embryo of the seed cells and speed the growth of scar tissue in 10 varieties studied communications. Research results show that rate of scar tissue from embryos of the 10 varieties ranged from 82.71% optimistic (L18) to 98.55% (V79). The same communication rate of scar tissue over the same 97% MD7, MD9, V79. Same can create scar tissue ratio lower than 90% include the same L18, L23, Red BG. In particular, the proportion of scar tissue created at least the same L18 (82.71%). The volume of the same communication scar tissue is formed ranged 7 from 115.00 mg (L18) to 198.00 mg (V79). The same L05, L23, MD7, MD9, SD30, V79 mass of scar tissue over 180mg. Four varieties L16, L18, L24 and Red BG mass of scar tissue formed by more than 160 mg. The results showed the ability to meet the growth of scar tissue and scar tissue of the 10 varieties of communication, be sure to use for subsequent studies in selected cell lines. 3.1.1.2. Dehydration tolerance of scar tissue the same communication research Dehydration tolerance of scar tissue is determined by the dehydration, the results of rapid assessment of scar tissue viability by TTC staining method and determine the survival of scar tissue after treatment by blowing dry. The results determine the survival rate of scar tissue after treatment with blown shown the stamina of cells and this is the basis for screening cell lines. The scar tissue dehydration under which we obtained the materials to make recycling plant for selecting drought-tolerant line of communication. Results of studies of tree regeneration under dehydration scar tissue that, like us all participated in the study were able to regenerate plants from callus survived. The models for survival rate from 83.33% tree reborn to 100.00%. Based on research and results, we choose the time scale of 84% water loss from the initial fresh weight and tissue survival rate of about 10% to 20% is the threshold to filter the line. Scar tissue processing and technical communications dry wind scar tissue, we identified peanut varieties L18 resistant dehydration and choose the best cream puff dry 9 hours threshold to filter. Results of assessment of dehydration tolerance of scar tissue, associated with evaluated tolerance of 10 seed lost in the period when the seed germinates and physiological drought period when young trees have shown that artificial limit , in 10 varieties studied communications, L18 resistant varieties dehydration least, have - amylase and sugar content in the lowest group inαthe activity of the day when the seed germinates term, the lowest drought index . This result is consistent with published by the Center for beans, Institute of Food crops and trees provide food Vietnam. 3.1.2. The effect of gamma rays associated with dry wind to the survival and regeneration of plants like communications L18 Like peanut varieties L18 is defined as strains resistant to drought, low water loss under dehydration among 10 varieties of research communications. In addition, L18 is the same high-yield varieties. So, with the goal of improving drought tolerance of the varieties have lost tolerance cream, we conducted surveys combined effects of gamma irradiation for the subject screening dehydration using the technique for blowing dry peanut varieties L18 with scar tissue. 8 Results exploring the effects of gamma-rays are performed on the fifth dose level radiation: 0.5 krad; 1.0 krad; 2.0 krad; 3.0 krad; 4.0 krad blown with scar tissue in 9 hours . The results determine the rate of tissue regeneration of tree survival showed regeneration of scar tissue plants affected by irradiation combined with blown ranged from 21,30% to 88,87%. Compared to the regeneration of plant tissue to survive only be blown, then the process of scar tissue by irradiation with blower reduces the rate of tree regeneration. Significant difference of plant regeneration is affected by irradiation combined with the blower that is, tree regeneration of a change in morphology, particularly, variation in color and leaf morphology. With low-dose irradiation (0.5 krad; 1krad; 2krad), the leaves are still green as the tree control. But with high-dose irradiation as 4krad 3krad and greens of the leaves appear in about 1-2 weeks after the leaves begin to yellow UA. Small leaf morphology and leaf curling occurs. Plant growth speed is inversely proportional to the intensity of the radiation dose. At higher doses of radiation phenomena plants gradually died, relatives barren. Regenerating tree height is much lower than the control plants. Table 3.6. The effect of irradiation combined with blower 9 am to survival and regeneration of tissue in the same communication tree L18 Radiation dose combination blower 9 hours Rate of survived tissue (% ) Rate of regeneration (% ) Patterns of regenerated trees 0,5krad 34,23 ± 0,37 47,63 ± 2,37 Normal 1,0krad 33,65 ± 0,52 88,87 ± 5,57 Normal 2,0krad 28,42 ± 0,43 38,87 ± 5,54 Normal 3,0krad 18,87 ± 0,59 26,17 ± 4,97 Small, yellow and fallen leaves, curly leaves edges 4,0krad 12,51 ± 0,42 21,30 ± 6,03 Small, yellow and fallen leaves, curly leaves edges 9 A B C D E G Figure 3.3. Some pictures of the same tree regeneration touch L18 6-week-old stage A: Plant regeneration not blown; B: Plant reproduction under blower 9 hours; C: irradiation dose 0.5 krad + blower 9 hours D: irradiation dose 2.0 krad + blower 9 hours; E: irradiation dose 3.0 krad + blower 9 hours; G: irradiation dose 4.0 krad + blower 9 hours [...]... dehydrated and callusinfluenced irradiation combined with handle causing dehydration we selected 3 lines has outstanding features, including: (1) The R46 has a mass of 100 fruit and seeds; (2) Lines of RM47 protein content higher; (3) Line of RM48 lipid content higher 3.2.2.2 Results evaluated tolerance of the selective in the fifth generation Drought tolerance of plants in general and in particular peanut. .. L18, L23 and 7 selective communication line near 500 bp in size However, to affirm correctly that cystatin genes, we performed separate line, identifying the sequence comparison with cystatin gene sequence was published We perform a separate line and determine the order of 2 cystatin genes dong R46, RM48 (two lines have the highest tolerance) , similar to the original L18 (drought- resistant cream) and similar... cream) and similar to L23 (with tolerance good) for comparison purposes cystatin gene sequence of lines and varieties 3.3.2 Results of cloning and sequencing of genes cystatin Result of splitting the colonies carrying cystatin genes were tested by clonyPCR reaction Defining the nucleotide sequence obtained from the plasmid of the sample is treated with DNAstar software shows that cystatin genes of the same... encoding cystatin be classified into two groups: Group I sub-divided into three large groups and small groups of women includes the same communications and other crops, including communications lines and similar distribution in the same group Group and the same communication lines (R46, RM48, L18, L23, AY722693) is closely related to cystatin with green beans (number AM712476), then the seed of rice cystatin- number... asparagine 35 (N35) is a pair of amino acids encoded by the first three exon junction 2 of the cystatin Position corresponding to position it as arginine 34 (R34) and asparagine 35 (N35) of cystatin RM48 line Glutamine and arginine at position 34 is two different amino acid structure characteristics of the original R, in which glutamine is the amide of glutamic acid; different from the amino acid arginine... OPH08 primer 200bp in size (RM47/OPH08-200bp) 5 Amplification and separation of the cystatin gene from DNA into the genome of a selected communication line and the same root Cystatin gene of peanut has 461 nucleotides, with two exons and one intron, the first group of phytocystatin, the most closely related to cystatin green beans, farthest to the cystatin of kiwi fruit (42.9%) The protein encoded by the... amino acid control group This difference is related to molecular structure and activity of cystatin in the inhibition of cysteine proteinase activity, is suggested for further study of cystatin in groundnut Besides differences in amino acid position 34, resulting in amino acid sequence comparison of the same communication L23, L18 and R46 2 lines, RM48 we found more variation in the position 6 amino... changes in the peanut genome structure leads to a change of phenotypic larger than just the scar tissue processed by the dry wind RECOMMENDATION 1 Continue to monitor, analyze and communicate the advantages refreshing Vietnamese 3 is R46, RM47, RM48 to introduce assaying 2 Design vector carrying cystatin gene and moved on peanuts and other crops as well as to study the effects of cystatin drought resistant... fruit (42.9%) The protein encoded by the gene has 98 amino acid, including 45.92% hydrophobic amino acid, amino acid 54.08% and 28.57% hydrophobic amino acid essential to the total amino acid protein of cystatin 6 Compare the order of the rows of peanut protein cystatin select and breed seeds have been detected in seven amino acid changes, including co dong RM48 biggest change This has affirmed the... and Correlation Assessing drought tolerance at seedling stage of the selection fifth generation by determining the rate of withered tree, the tree recovered in the first 5 days time limit and determine the relative drought indices for RM48 line with the highest drought index 13 (10,089.20%), then to the R46 and L23 cultivar At least the current R44, RM49, and like the original L18 In the seventh line . by plant cell technology, ( 3) 4 Analysis and evaluation of selected lines derived from callus culture ( 4) Genes related to drought tolerance in groundnut; ( 5) Cystatin and the role of cystatin. MINISTRY OF EDUCATION AND TRAINING THÁI NGUYÊN UNIVERSITY VŨ THỊ THU THỦY CREATING DROUGHT TOLERANCE LINES AND ISOLATING CYSTATIN GENES RELATED TO DROUGHT TOLERANCE IN. separate line and determine the order of 2 cystatin genes dong R46, RM48 (two lines have the highest tolerance) , similar to the original L1 8 (drought- resistant cream) and similar to L2 3 (with tolerance