Study on in vitro propagation of lavandula dentata

VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY GRADUATION THESIS TITLE: STUDY ON IN VITRO PROPAGATION OF LAVANDULA DENTATA Instructor: Assoc Prof Dr NGUYEN THANH HAI (Deputy Head, Department of Plant Biotechnology) Student: VU TRONG HIEU Student code: 610615 Class: K61-CNSHE Hanoi – 2021 DECLARATION The thesis entitled "study in in vitro propagation of Lavandula dentata" is conducted under the supervision of Assoc Prof Dr NGUYEN THANH HAI and Master PHAM THI THU HANG I declare that the information reported in the current thesis is the result of my own work, except where the reference is made I declare that all information and references in this thesis have been clearly stated the origin, name of author, name of work, time and place of study I accept full responsibility for this declaration Hanoi, January, 2021 Signature Vũ Trọng Hiếu i ACKNOWLEDGEMENTS This study has been completed thanks to the help of many individuals and organizations from whom I have received support I would like to express my appreciation to all of them First of all, I would like to send my sincere thanks to the Vietnam National University of Agriculture for facilitating my graduation thesis, sincerely thank the Dean of the Faculty of Biotechnology and all the teachers in the Department of Plant Biotechnology- Faculty of Biotechnology for their wholehearted guidance, knowledge addition and assistance during the internship at the laboratory In particular, I would like to express my gratitude to Assoc Prof Dr NGUYEN THANH HAI and Master PHAM THI THU HANG in the Department of Plant Biotechnology- Faculty of Biotechnology- Vietnam National University of Agriculture for taking the time and enthusiasm to directly guide, help throughout the process of graduation I also thank all those who are doing research at the laboratory for helping and facilitating me to complete my thesis well Hanoi, January, 2021 Signature Vũ Trọng Hiếu ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ii TABLE OF CONTENTS iii LIST OF ABBREVIATIONS v LIST OF TABLES vi LIST OF FIGURES vii SUMMARY viii CHAPTER I INTRODUCTION 1.1 Abstract 1.2 Objective CHAPTER OVERVIEW OF REFERENCE 2.1 Introduction of Lavandula genus 2.1.1 The distribution of Lavandula genus 2.1.2 The classification of Lavandula genus 2.2 Introduction of Lavandula dentata 2.2.1 Classification and distribution 2.2.2 Characteristics 2.3 Traditional propagation methods of Lavandula dentata 10 2.3.1 Cutting Propagation 11 2.3.2 Layering Propagation 11 2.3.3 Division Propagation 11 2.3.4 Seed Propagation 12 2.4 Research and application of tissue culture in propagation of Lavandula dentata in Vietnam and around the world 12 2.5 Overview of plant cells and tissue culture 14 2.5.1 Introduction 14 2.5.2 Plant tissue culture medium 15 iii CHAPTER III SUBJECTS, MATERIALS, CONTENT AND METHODOLOGY OF RESEARCH 19 3.1 Subjects and materials 19 3.1.1 Subjects 19 3.1.2 Materials 19 3.1.3 Place and time 19 3.2 Contents and methodology of research 19 3.2.1 Contents of research 19 3.2.2 Methodology of research 22 3.2.3 Tracking criteria 22 3.2.4 Data processing methods 22 PART IV RESULT AND DISCUSSION 23 4.1 Effect of BA on the multiplication of Lavandula dentata shoots 23 4.2 Effect of BA and Kinetin on the multiplication of Lavandula dentata shoots 25 4.3 Effect of BA and IBA on the multiplication of Lavandula dentata shoots 27 4.4 Effect of BA and α - NAA on the multiplication of Lavandula dentata shoots 30 4.5 Effect of BA and IAA on the multiplication of Lavandula dentata shoots 32 4.6 Effect of IBA and α - NAA on rooting of Lavandula dentata shoots 34 CONCLUSIONS AND RECOMMENDATIONS 38 REFERENCE 39 APPENDIX: TABLES OF DATA PROCESSING 41 iv LIST OF ABBREVIATIONS Abbreviations Annotate MS Murashige and Skoog medium CT Formula CV Coefficient of variation LSD5% Least significant differences BA Benzylidenes IAA Indole-3-acetic acid IBA Indole-3-butyric acid α - NAA 1-naphthaleneacetic acid v LIST OF TABLES Table Several major growth regulators are used in plant tissue culture… 17 Table 2: Results and data processing of experiment 23 Table 3: Results and data processing of experiment 25 Table 4: Results and data processing of experiment 28 Table 5: Results and data processing of experiment 30 Table 6: Results of data processing of experiment 33 Table 7: Results of data processing of experiment 35 vi LIST OF FIGURES Figure 1: Lavandula genus (source: Internet) Figure 2: The distribution of Lavandula dentata Figure 3: Lavandula dentata; Figure 4: Effect of BA on the multiplication of Lavandula dentata shoots (after weeks) 24 Figure 5: Chart shows the effect of BA on the multiplication of Lavandula dentata shoots 24 Figure 6: Effect of BA and Kinetin on the multiplication of Lavandula dentata shoots (after weeks) 26 Figure 7: Chart shows the effect of BA and Kinetin on the multiplication of Lavandula dentata shoots 26 Figure 8: Effect of BA and IBA on the multiplication of Lavandula dentata shoots (after weeks) 28 Figure 9: Chart shows the effect of BA and IBA on the multiplication of Lavandula dentata shoots 29 Figure 10: Effect of BA and α - NAA on the multiplication of Lavandula dentata shoots (after weeks) 31 Figure 11: Chart shows the effect of BA and α - NAA on the multiplication of Lavandula dentata shoots 31 Figure 12: Effect of BA and IAA on the multiplication of Lavandula dentata shoots (after weeks) 33 Figure 13:Chart shows the effect of BA and IAA on the multiplication of Lavandula dentata shoots 34 Figure 14: Effect of IBA and α - NAA on rooting of Lavandula dentata shoots (after weeks) 36 Figure 15: Chart shows the effect of IBA and NAA on rooting of Lavandula dentata shoots 36 vii SUMMARY Tissue culture has been used for diverse purposes, being the regeneration of explants, during in vitro establishment, a decisive stage to development of this technique One of the difficulties encountered in the initial stage, besides contaminations by microorganisms, is the regeneration of the excised explants, which normally requires the exogenous source of plant growth regulators The aim of this research was to evaluate the effect of plant growth regulators on the regeneration of Lavandula dentata shoot tips Shoot samples were excised and placed on MS culture medium containing 30.0 g/L sucrose, 7.0 g/L agar The experimental design used was complete randomize design (CRD) with replicates of 10 explants per plot The largest multiplication rate was 8.43 obtained on medium containing 1.0 mg/L BA The largest height of shoots was recorded on medium containing 1.0 mg/L BA The most suitable medium for rooting of Lavandula dentata was MS medium supplemented with 1.0 mg/L NAA Plant growth regulators combinations initial development of the shoot tips is the presence of 1.0 mg/L BA and 0.2 mg/L IAA viii CHAPTER I INTRODUCTION 1.1 Abstract Lavender is a sort of blossoming plant that has a place in the mint family There are in excess of 30 types of lavender that original from Mediterranean area, northern and eastern pieces of Africa and southwestern Asia Lavender fills in dry, radiant regions on all around depleted soils Synthetic compounds separated from lavender have useful impact on the human wellbeing Lavandula dentata are some of the most popular ornamental and medicinal plants with great economic values These species are vegetative propagated by stem cuttings However, the poor rooting ability and vulnerability of plantlets to contamination are major limiting factors for propagation In vitro culture methods are suitable to overcome these limitations In Vietnam, only grown in Da Lat and known by many people through perfumes, aromatic bags and medicinal products The demand for using Lavender flowers in medicine, beauty, cuisine in Vietnam is increasing However, the domestic supply is not guaranteed due to difficulties in the process of cultivation and care Especially the quality of seedlings This graduate thesis presented of in vitro propagation of Lavandula dentata towards development of additional and alternative sources of material 1.2 Objective Determine effect of growth regulators on in vitro multiplication of Lavandula dentata Determine the appropriate rooting medium for the complete Lavandula dentata 1 NL 566534E-01 283267E-01 7.45 0.015 CT 3.99491 998726 262.70 0.000 * RESIDUAL 304141E-01 380176E-02 -* TOTAL (CORRECTED) 14 4.08197 291570 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN1 18/ 1/21 16: - :PAGE Data analysis of experiment MEANS FOR EFFECT NL -NL NOS 5 SC 4.98000 5.08000 4.86000 CC 2.31600 2.35000 2.46000 SE(N= 5) 0.761579E-01 0.275745E-01 5%LSD 8DF 0.248343 0.899176E-01 -MEANS FOR EFFECT CT -CT NOS 3 3 3 SE(N= 5%LSD 3) 8DF SC 2.93333 5.33333 8.43333 4.83333 3.33333 CC 3.16667 2.30333 2.66000 2.11333 1.63333 0.983194E-01 0.355985E-01 0.320609 0.116083 42 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN1 18/ 1/21 16: - :PAGE Data analysis of experiment F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE |NL |CT | GRAND MEAN | (N= 15) STANDARD DEVIATION C OF V SD/MEAN | | | NO BASED ON BASED ON % | OBS TOTAL SS RESID SS 0.17029 3.4 0.61658E-01 2.6 | | | | SC 0.1851 4.9733 2.0225 15 2.3753 0.53997 0.0000 CC 0.0151 15 0.0000 Experiment 2: Effect of BA and Kinetin on the multiplication of Lavandula dentata shoots BALANCED ANOVA FOR VARIATE SC FILE TN2 18/ 1/21 17: - :PAGE Data analysis of experiment VARIATE V003 SC LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF SQUARES SQUARES LN 43 MEAN ========================================================== =================== CT 33.4092 11.1364 93.02 0.000 NL 135000 675000E-01 0.56 0.600 * RESIDUAL 718335 119723 -* TOTAL (CORRECTED) 11 34.2625 3.11477 -BALANCED ANOVA FOR VARIATE CC FILE TN2 18/ 1/21 17: - :PAGE Data analysis of experiment VARIATE V004 CC LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 9.67776 3.22592 631.98 0.000 NL 407582E-01 203791E-01 3.99 0.079 * RESIDUAL 306268E-01 510447E-02 -* TOTAL (CORRECTED) 11 9.74914 886286 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN2 18/ 1/21 17: - :PAGE Data analysis of experiment 44 MEANS FOR EFFECT CT -CT NOS 3 3 SC 8.43333 6.50000 4.30000 8.26667 CC 2.66000 3.10333 1.42333 0.885667 SE(N= 3) 0.199769 0.412491E-01 5%LSD 6DF 0.691032 0.142687 -MEANS FOR EFFECT NL -NL NOS 4 SC 6.95000 6.72500 6.95000 CC 1.96925 1.98500 2.10000 SE(N= 4) 0.173005 0.357228E-01 5%LSD 6DF 0.598451 0.123571 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN2 18/ 1/21 17: - :PAGE Data analysis of experiment F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE |CT |NL | GRAND MEAN | (N= 12) DEVIATION C OF V SD/MEAN | | NO | STANDARD BASED ON | 45 BASED ON % | OBS | TOTAL SS RESID SS | 0.34601 5.0 0.71446E-01 3.5 | SC 0.0001 6.8750 1.7649 12 2.0181 0.94143 0.5998 CC 0.0000 12 0.0788 Experiment 3: Effect of BA and IBA on the multiplication of Lavandula dentata a shoots BALANCED ANOVA FOR VARIATE SC FILE TN3 18/ 1/21 17:31 - :PAGE Data analysis of experiment VARIATE V003 SC LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 32.9292 10.9764 288.43 0.000 NL 111667 558334E-01 1.47 0.303 * RESIDUAL 228336 380561E-01 -* TOTAL (CORRECTED) 11 33.2692 3.02447 -BALANCED ANOVA FOR VARIATE CC FILE TN3 18/ 1/21 17:31 - :PAGE Data analysis of experiment VARIATE V004 CC 46 LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 3.95982 1.31994 415.73 0.000 NL 672167E-01 336083E-01 10.59 0.011 * RESIDUAL 190500E-01 317500E-02 -* TOTAL (CORRECTED) 11 4.04609 367827 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN3 18/ 1/21 17:31 - :PAGE Data analysis of experiment MEANS FOR EFFECT CT -CT NOS 3 3 SC 8.43333 5.56667 5.00000 3.96667 CC 2.66000 2.61000 1.49000 1.48333 SE(N= 3) 0.112629 0.325320E-01 5%LSD 6DF 0.389603 0.112533 -MEANS FOR EFFECT NL -NL NOS SC 47 CC 4 5.87500 5.70000 5.65000 2.02500 1.99250 2.16500 SE(N= 4) 0.975398E-01 0.281736E-01 5%LSD 6DF 0.337406 0.974569E-01 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN3 18/ 1/21 17:31 - :PAGE Data analysis of experiment F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE |CT |NL | GRAND MEAN | (N= 12) STANDARD DEVIATION C OF V SD/MEAN | | | | | | NO BASED ON BASED ON OBS TOTAL SS RESID SS 0.19508 3.4 0.56347E-01 2.7 SC 0.0000 5.7417 1.7391 12 2.0608 0.60649 | | 0.3033 CC 0.0000 12 % 0.0114 Experiment 4: Effect of BA and NAA on the multiplication of Lavandula dentata shoots BALANCED ANOVA FOR VARIATE SC FILE TN4 18/ 1/21 17:54 - :PAGE Data analysis of experiment VARIATE V003 SC 48 LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 75.1633 25.0544 875.69 0.000 NL 416668E-01 208334E-01 0.73 0.524 * RESIDUAL 171667 286112E-01 -* TOTAL (CORRECTED) 11 75.3767 6.85242 -BALANCED ANOVA FOR VARIATE CC FILE TN4 18/ 1/21 17:54 - :PAGE Data analysis of experiment VARIATE V004 CC LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 3.05127 1.01709 102.77 0.000 NL 350000E-03 175000E-03 0.02 0.984 * RESIDUAL 593830E-01 989717E-02 -* TOTAL (CORRECTED) 11 3.11100 282818 49 TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN4 18/ 1/21 17:54 - :PAGE Data analysis of experiment MEANS FOR EFFECT CT -CT NOS 3 3 SC 8.43333 3.03333 2.40000 2.60000 CC 2.66000 2.10000 1.37667 1.54333 SE(N= 3) 0.976579E-01 0.574374E-01 5%LSD 6DF 0.337814 0.198685 -MEANS FOR EFFECT NL -NL NOS 4 SC 4.20000 4.07500 4.07500 CC 1.92250 1.92500 1.91250 SE(N= 4) 0.845742E-01 0.497423E-01 5%LSD 6DF 0.292556 0.172066 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN4 18/ 1/21 17:54 - :PAGE Data analysis of experiment F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - 50 VARIATE |CT |NL | GRAND MEAN | (N= 12) STANDARD DEVIATION C OF V SD/MEAN | | | | | | NO BASED ON BASED ON OBS TOTAL SS RESID SS 0.16915 4.1 0.99485E-01 5.2 SC 0.0000 4.1167 2.6177 12 1.9200 0.53181 | | 0.5239 CC 0.0001 12 % 0.9836 Experiment 5: Effect of BA and IAA on the multiplication of Lavandula dentata shoots BALANCED ANOVA FOR VARIATE SC FILE TN5 18/ 1/21 18:26 - :PAGE Data analysis of experiment VARIATE V003 SC LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 47.7092 15.9031 160.82 0.000 NL 180000 900000E-01 0.91 0.454 * RESIDUAL 593335 988892E-01 -* TOTAL (CORRECTED) 11 48.4825 4.40750 51 BALANCED ANOVA FOR VARIATE CC FILE TN5 18/ 1/21 18:26 - :PAGE Data analysis of experiment VARIATE V004 CC LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 6.11609 2.03870 234.41 0.000 NL 288167E-01 144083E-01 1.66 0.267 * RESIDUAL 521838E-01 869730E-02 -* TOTAL (CORRECTED) 11 6.19709 563372 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN5 18/ 1/21 18:26 - :PAGE Data analysis of experiment MEANS FOR EFFECT CT -CT NOS 3 3 SE(N= 5%LSD 3) 6DF SC 8.43333 6.06667 4.20000 3.20000 0.181557 0.628036 52 CC 2.66000 2.41667 1.24333 1.01667 0.538433E-01 0.186253 -MEANS FOR EFFECT NL -NL NOS 4 SC 5.62500 5.32500 5.47500 CC 1.87250 1.76500 1.86500 SE(N= 4) 0.157233 0.466297E-01 5%LSD 6DF 0.543895 0.161299 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN5 18/ 1/21 18:26 - :PAGE Data analysis of experiment F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE |CT |NL | | | | | STANDARD DEVIATION C OF V SD/MEAN | NO BASED ON BASED ON % | OBS TOTAL SS RESID SS 0.31447 5.7 0.93259E-01 5.1 | | SC 0.0000 12 5.4750 2.0994 12 1.8342 0.75058 0.4538 CC 0.0000 GRAND MEAN | (N= 12) 0.2674 Experiment 6: Effect of IBA and NAA on rooting of Lavandula dentata shoots BALANCED ANOVA FOR VARIATE 15:33 SR 53 FILE TN6 20/ 1/21 - :PAGE Data analysis of experiment VARIATE V003 SR LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 610.097 152.524 160.33 0.000 NL 196000 979999E-01 0.10 0.903 * RESIDUAL 7.61062 951328 -* TOTAL (CORRECTED) 14 617.904 44.1360 -BALANCED ANOVA FOR VARIATE CD FILE TN6 20/ 1/21 15:33 - :PAGE Data analysis of experiment VARIATE V004 CD LN SOURCE OF VARIATION F RATIO PROB ER DF SUMS OF MEAN SQUARES SQUARES LN ========================================================== =================== CT 80.8442 20.2110 544.42 0.000 NL 238800E-01 119400E-01 0.32 0.737 * RESIDUAL 296991 371239E-01 54 -* TOTAL (CORRECTED) 14 81.1650 5.79750 -TABLE OF MEANS FOR FACTORIAL EFFECTS FILE TN6 20/ 1/21 15:33 - :PAGE Data analysis of experiment MEANS FOR EFFECT CT -CT NOS 3 3 SR 5.13333 6.76667 22.8667 14.7000 10.1333 CD 2.23000 2.43333 8.06667 2.39333 2.03667 SE(N= 3) 0.563125 0.111241 5%LSD 8DF 1.83629 0.362747 -MEANS FOR EFFECT NL -NL NOS 5 SR 12.0200 11.9800 11.7600 CD 3.45400 3.46600 3.37600 SE(N= 5) 0.436194 0.861671E-01 5%LSD 8DF 1.42239 0.280982 -ANALYSIS OF VARIANCE SUMMARY TABLE FILE TN6 20/ 1/21 15:33 - :PAGE 55 Data analysis of experiment F-PROBABLIITY VALUES FOR EACH EFFECT IN THE MODEL SECTION - VARIATE |CT |NL | STANDARD DEVIATION C OF V SD/MEAN | | | | | | SR 0.0000 NO BASED ON BASED ON OBS TOTAL SS RESID SS % | | 15 11.920 6.6435 0.97536 8.2 15 3.4320 2.4078 0.19268 5.6 0.9028 CD 0.0000 GRAND MEAN | (N= 15) 0.7367 56