Creating drought tolerance lines and isolating cystatin genes related to drought tolerance in peanut (Arachis hypogaea L.)

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 sc

MINISTRY OF EDUCATION AND TRAINING

THÁI NGUYÊN UNIVERSITY

PHD THESIS ABTRACT

Thai Nguyen - 2011

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:

"Creating drought tolerance lines and isolating cystatin genes related to drought

tolerance in peanut (Arachis hypogaea L.)”

2 Research objectives

culture of plant cells

of the peanut cultivars

3 Research contents

irradiation combined with dry blowing

RAPD technique

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-

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

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)

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

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

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,

seedling stage by artificial methods to cause limited by Le Tran Binh et al (1998)

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

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

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

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

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

Rate of regeneration

curly leaves edges

curly leaves edges

From the results of the evaluation survival and regeneration of scar tissue scar tissue after treatment with gamma irradiation we have identified the critical dose of scar tissue radiation dose 4krad la communication If you exceed the limit 4krad most dead scar tissue, can not produce enough of the line model for the further research, especially research on the selection line However, to obtain multiple mutations need to determine the appropriate dose

of irradiation, to have created the beneficial genetic variation, both the rate of the tissue regeneration and high survival Therefore, we selected the dose 2krad blower with 9 hours

to screen the tissue and the entire plant

Result of treatment of scar tissue in in vitro culture systems of peanut varieties L18 continuously blown by 9 hours and radiation dose in combined blown 2krad continuous 9 hours were obtained from 167 current and measuring 198 models communication lines are created

3.1.3 Agro-biological characteristics of the communication line in R0 and RM0

The study population lost R0 (plants regenerated from callus resistant blower), RM0 (Plant regeneration from callus under irradiation with blow dry) found that the communication line from the test tube to grow in the field (spring, 2008) that genetic variation is much larger than the original species in all study criteria (Table 3.7)

Table 3.7 Agronomic characteristics of the population selected communication R0, RM0

X

As such the process of selecting the line lost drought include: (1) Create callusand scar tissue; (2) regeneration of trees and Tree Planting complete (3) track and analyze the genetic lines

of selectivity

Scar tissue in the peanut processing plant in vitro culture, selective survival of scar tissue, regenerating plants and monitoring populations and RM0 R0 can draw some comments below: (1) The line comes from scar tissue dry wind bear up tree height than the original species, while the line is derived from scar tissue in combination with radiation suffered blow dry reduced plant height compared to the same original

(2) Populations of plants produced by tissue culture techniques of plant cells have some quick / plant than it resembles the original Military R0 can quickly reach 3 / tree, populations reached 6,75 RM0 fast / tree, like the original with rapid 8,33 / plant

(3) Population R0 is 22,80 fruit / tree, more than the same root (reached 110.30% from the same root); population RM0, with some troops may fruit / tree less like the original, only 14.00 fruit / tree (67.73% from the same root)

(4) Coefficient of variation for genetic research targets of the two forces can be created by plant tissue culture techniques of plant cells larger than the original species

From the evaluation results in generation of R0, RM0, we chose the 7 line is derived from the same communication L18, in which three lines originating from the scar tissue is influenced by the blower is R44, R46, R48 and 4 lines derived from scar tissue affected by radiation combined with blower is RM46, RM47, RM48, RM49 a starting material to review and filtering in the next generation

3.2 RESULT ANALYSIS OF SELECTED LINES THROUGH THE GENERATION

3.2.1 Assess the stability of agricultural biological characteristics of the selected contact in the first generation, the third generation

Compared to other crops, peanuts are difficult maintenance, and low germination when stored last time Number of fruit trees in the first generation touch is not large enough

to carry out research related to tolerance Therefore, the 7 whole grains in the military can communicate selectively R0, RM0 we look into the service next year looks Results assessing the stability of agronomic traits on the main stem height, number of branches / plant, number of fruits / plant, number of fruits make / tree generation R1, RM1 look at winter harvest in 2008 (Table 3.8 ); generation R3, RM3 look at winter harvest in 2009 (Table 3.9) showed the stability of agricultural biological characteristics of the communication line is derived from the scar tissue has contributed to confirm the value of carefully Arts tissue culture plant cells could create new strains and shortening breeding, increasing the efficiency of the process of breeding