VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS SUBJECT: “IDENTIFICATION AND SEQUENCE ANALYSIS OF PARTIAL 16S rRNA OF ENDOPHYTIC BACTERIA ISOLATED FROM POTATO ROOT” Hanoi - 2022 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS SUBJECT: “IDENTIFICATION AND SEQUENCE ANALYSIS OF PARTIAL 16S rRNA OF ENDOPHYTIC BACTERIA ISOLATED FROM POTATO ROOT” Student: NGUYEN THI NGOC ANH Student’s Code: 620514 Class: K62CNSHE Department: PLANT BIOTECHNOLOGY Supervisor: Ph.D DINH TRUONG SON Hanoi – 2022 COMMITMENT I guarantee that the graduate thesis: “Identification and sequenceanalysis of partial 16S rRNA of endophytic bacteria isolated from potato root” is my own research wich is done with the instruction of Mr. Dinh Truong Son, PhD. The parts where I use references in the thesis have been mentioned in the references section. The data and results presented in the thesis are completely honest.
If wrong, I will take responsibility and all the discipline that the faculty and the academy offer. Hanoi, September 11, 2022 Student Nguyen Thi Ngoc Anh i ACKNOWLEDGEMENT In order to complete this graduation thesis, in addition to my own efforts, i would like to express my sincere thanks to: Thanks to the Vietnam National University of Agriculture for supporting me about facilities with modern library system, diverse types of documents that are convenient for my research. I would like to express my deepest gratitude to my instructor, Mr. Dinh Truong Son, whose sincerity and encouragement I will never forget.
This thesis would not have been possible without Mr. Dinh Truong Son, whose guidance from the initial step in research enabled me to develop an understanding of the subject. I am thankful for the extraordinary experiences he arranged for me. It is an honor to learn from Mr.
Dinh Truong Son. I would also like to thank some students at the Department of Plant Biotechnology, Faculty of Biotechnology, Vietnam National University of Agriculture for creating favorable conditions for me to complete this thesis. Finally, i look forward to receiving comments and suggestions from the teachers to make my graduation thesis more complete. Hanoi, September 11, 2022 Student Nguyen Thi Ngoc Anh ii INDEX COMMITMENT.
iii LIST OF ABBREVIATIONS. vi LIST OF TABLES. vii LIST OF FIGURES. viii PART I: INTRODUCTION.
Rationale of the Study. 2 PART II: LITERATURE REVIEW. General introduction of potato. Overview of potato.
Morphological characteristics of potato. Growth stages of potato. Factors affecting the growth and development of potato. Value of potato.
Definition of endophytic bacteria. Origin of endophytic bacteria. The role of plant endophytes. Methods for identifying microorganisms.
ITS fragment sequencing. 16s rRNA gene sequencing technique. 18s rRNA gene sequencing technique. 26s rRNA gene sequencing technique.
Some studies using 16s rRNA. Cultivation-independent population analysis of bacterial endophytes in three potato varieties based on eubacterial and Actinomycetes-specific PCR of 16S rRNA genes. Analysis of endophytic bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S rDNA based PCR fragments. MATERIALS AND METHODS.
Study place and time. Contents and methods. Quantification of DNA by using spectrophotometer. 18 Part 4: RESULTS AND DISCUSSION.
19 iv Part V: CONCLUSIONS AND RECOMMENDATIONS. 37 v LST OF ABBREVIATION CTAB Cetyltrimethylammonium bromide DNA Deoxyribonucleic acid RNA Ribonucleic acid EDTA Ethylendiamin Tetraacetic Acid et al. et alii (Latin), and others ml Milliliter PCR Polymerase chain reaction PVPP Polyvinylpyrrolidone TAE Tris-acetate EDTA °C Degree Celsius % Percent spp. Species vi LIST OF TABLES Table 1: List of chemicals used in the study Table 2: Components of each PCR reaction Table 3: PCR produre Table 4: Identification of microorganisms Table 5: Secondary structure of 12 species of microorganisms determined by Unafold (https://www.org/) and R2DT (https://rnacentral.
vii LIST OF FIGURES Figure 2. Picture of potato Figure 2. Detailed image of potato Figure 4. PCR results Figure 4.
Phylogenetic tree Figure 4. Nucleotide alignment and diversity viii PART I: INTRODUCTION 1. Rationale of the Study Potato is a short-term agricultural crop, grown for its starchy tubers. Potato is a food crop with high nutritional value, a valuable export item, and high economic efficiency.
In the world, potatoes are considered as one of the most important crops ranked 5th after rice, maize, wheat, and soybean. Potatoes provide nutrients for humans and animals and are valuable raw materials for many industries. In Vietnam, potatoes are grown quite widely in the winter in the North and play a major role as food. Potato cultivation in Vietnam is being focused on yield and quality.
Therefore, research to improve the growth and development of potato plants is focused. The endophytic bacteria of the potato plant play an important role in the development of the potato plant. So, the study of the endogenous microflora, especially the endosymbiotic group in potato plants, can contribute to better farming solutions. Numerous microbial communities can be found in the plant endosphere.
It is widely acknowledged that these microbial populations significantly influence plant health. The study of plant-microbe interactions is still being developed today using both conventional microbiology methods as well as recently embraced genomic technologies. Endophytes are microbial symbionts that spend the majority of their life cycle inside the host plant without causing any harm to it. It is possible to increase crop output while minimizing agriculture's negative effects on the environment by using these natural symbionts.
Through nitrogen fixation, phytohormone synthesis, nutrient uptake, and giving tolerance to abiotic and biotic challenges, endophytes support plant growth. Endophyte colonization is essential for giving the host plant these advantages. Endophytic colonization is the process through which endophyte populations enter, develop, and multiply within the host 1 plant. The study of the plant microbiome has received a lot of interest lately, but it is still unclear how plants attract endophytes (Kandel, Joubert, and Doty 2017) Endophytes were traditionally extracted from surface-sterilized plant tissue and grown in nutrient-rich media.
Previously, Nguyen Dac Tien successfully isolated some isolates from potato root and 14 isolates were used to evaluate the genetic diversity. The results conducted by Nguyễn Đắc Tiến showed the high genetic diversity of the isolates; however, the identification of those isolates is need. Therefore; I decided to do this research: “Identification and sequenceanalysis of partial 16S rRNA of endophytic bacteria isolated from potato root”. Research Objectives Identification and sequence analysis of partial 16S rRNA of endophytic bacteria isolated from potato root.
Research requirements Identification of 12 endophytic bacteria isolated from potato root. Sequence diversity analysis by prediction of the secondary structure of endophytic microorganisms by R2DT and Unafold tools. 2 PART II: LITERATURE REVIEW 2. General introduction of potato 2.
Overview of potato The scientific name of the potato plant is Solanum tuberosum. L, belongs to the Solanaceae family. Picture of potato 2. Morphological characteristics of potato Potato plants are herbaceous perennials growing to about 60 cm in height, dying after flowering.
Detailed image of potato 3 Potato flowers are white, pink, red, blue, or purple, with yellow pistils. Potatoes are pollinated mainly by insects, bumblebees carry pollen from one plant to another. After the potatoes flower, some varieties produce green cherries-like fruit, which can contain 300 seeds. Potatoes contain a large amount of toxic alkaloids, solanine, so they are not edible.
All new potato varieties are grown from seed as distinct from tubers. Cut the potatoes and soak them in water, the seeds split and sink to the bottom after a day of soaking. Any type of potato can be grown with tubers or pieces of tubers. Some commercial potato varieties are not produced all from seed (because the variety is not favorable for flowering) but are grown from tubers, pieces of which are called varietal.
(Kumari et al. Growth stages of potato The resting period: usually freshly harvested potatoes are not able to sprout; We call it the phenomenon of sleep. The resting period is long or short depending mainly on the breed, besides external factors such as mechanical rubbing, the impact of chemicals are also factors affecting the sleeping period. After the dormancy period is over, or when being treated, the potato tubers are able to sprout.
Germination period: This is the first period in the growth cycle of the potato plant. The basic feature of this period is that the sprouts in the tubers gradually develop into young plants. The most suitable time to plant is when the tubers have many sprouts and the sprouts have high vigor. Stem and leaf growth period: after planting, sprouts develop into stems.
The main stem grows directly from the seed tuber, the secondary stem grows from the main stem. Underground stem formation: tuberous rays are formed about 30-40 days after planting. Tuberous rays are white, growing horizontally with the ground. The 4 tip of the tuber has the ability to grow strongly in size and quantity, accumulating nutrients to form tubers later.
The period of tuber development, flowering, fruiting and ripening: followed by tuber ray formation is the period when tuber rays are enlarged. Nutrients are transported to the tubers to make them grow faster. With the development of the tuber, inflorescences form. After pollination, the fruit grows and moves into the period of fruit and seed ripening.
Leaves turn yellow and die. Factors affecting the growth and development of potato Temperature: Potatoes prefer a warm and temperate climate, both heat and cold are not tolerated. Requires different temperatures at different growth stages. In the period of growth of stem and leaves, suitable temperature: 20-25°C.
The appropriate temperature for tuber formation and development is 17-20°C, but for starch accumulation, the appropriate temperature is 16-18°C. Light: Potato is a light-loving plant, the light intensity is suitable for high yield from 40,000 to 60,000 lux. Potato plants require appropriate light according to each growth period. The period of tuber formation and tuber development requires a short time of day light of 10-12 hours/day.
Water: Potatoes have shallow roots, so they need to be watered regularly. According to research, during the growing process, potatoes need about 500- 700mm of water with a humidity of about 60-86%. Soil, pH and nutrients: the best soils for potatoes are those with light mechanical composition such as alluvial soil, loamy soil, sandy soil. The suitable pH for potato plants is 5-7, the most suitable is 6-6.
Nutritional requirements need to be full of macronutrients (N, P, K, .) and trace elements (Mo, Ca, .) for good growth and development of plants. Value of potato Potato is a versatile tuber, relatively cheap, easy to grow, easy to care for and has a high nutrient content, so many households in Vietnam have chosen potatoes as a main dish in daily meals. Nutritional value: Potatoes cooked in their skinless state are a great source of many essential vitamins and minerals, such as vitamin C or potassium. Potatoes are mostly water, in addition, the main components of potatoes include carbs, protein and a moderate amount of fiber, especially potatoes have almost no fat.
Research history The study of non-pathogenic bacteria colonizing plant tissues began in 1926. Since 1940, there have been numerous reports of native endophytic bacteria residing in the tissues of many other plants each other, including seeds, ovules, tubers, roots, stems, and fruits (Quadt-Hallmann, Kloepper, and Benhamou 1997) Subsequently, endophytic bacteria studies mainly focused on their beneficial and ecological effects (Ryan et al. 2008)to better understand the mode of interaction of endophytic bacteria with parasites their owner. Recently, scientists have focused on screening endogenous bacterial strains and studying their effectiveness on plant growth and development and limiting environmental pollution to produce medicinal products biology for agriculture (Kumar 2011) 2.