i DEVELOPMENT OF DUCKWEED TRANSFORMATION TECHNIQUE FOR BIOLOGICAL APPLICATION AORNPILIN JAIPRASERT A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE MASTER DEGREE OF SCIENCE IN BIOLOGICAL SCIENCE FACULTY OF SCIENCE BURAPHA UNIVERSITY JULY 2018 COPYRIGHT OF BURAPHA UNIVERSITY ii iii ACKNOWLEDGEMENT In the success of this thesis, I would like to express my sincere gratitude and deep appreciation to my advisor, Dr. Salil Chanroj for support, attention, motivation, technical assistance, helpful suggestion and comment, and encouragement throughout my study. I would like to thank Assistant Professor Dr. Waranyoo Phoolcharoen from Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Dr. Wasinee Pongprayoon from Department of Biology, and Dr. Somchart Maenpuen, Department of Biochemistry, Faculty of Science, Burapha University for all of their guidance and valuable advice throughout the examination. Great appreciation is also given to Biological Science Graduate Program, Department of Biotechnology for laboratory facilities. I would like to thank Microscopic center, Faculty of Science for technical assistance on fluorescence microscope. I also would like to thank National Research Council of Thailand and Burapha University for the financial support. Finally, a great respect is brought to my parents and my family for their loving, take caring, attention, encouragement, guidance and supporting throughout my study. I specially thank to all lecturers of Department of Biotechnology and Department of Biology and all of my friends for their kindness, support, suggestion, encouragement, and friendship. Aornpilin Jaiprasert iv 56910051: MAJOR: BIOLOGICAL SCIENCE; M.Sc. (BIOLOGICAL SCIENCE) KEYWORDS: DUCKWEEDS/ IDENTIFICATION/ CULTIVATION/ FLOWERING/ TRANSFORMATION AORNPILIN JAIPRASERT: DEVELOPMENT OF DUCKWEED TRANSFORMATION TECHNIQUE FOR BIOLOGICAL APPLICATION. ADVISORY COMMITTEE: SALIL CHANROJ, Ph.D. 150 P. 2018. Plants are important as global energy and food sources, especially during the situation where the world population is rapidly increasing. Though the genetically modified crops such as maize and soybean are commercial available, these crops encounter several limitations, including long harvesting period, taking up land space, and requiring an extensive investment. Consequently, constructing genetically modified plants that can be cultivated in limited space and short-time life cycles is desirable. Duckweeds are one of the promising choices due to their rapid biomass duplication and high carbohydrate and protein contents. Nevertheless, the basic information and gene transformation technique in duckweeds are poorly described. Therefore, the main objectives of this research are studying the fundamental of duckweed biology, analyzing their biochemical composition and developing the simple method for genetic transformation of duckweeds. Three species of duckweeds found in Burapha University, Chon-Buri, Thailand, were characterized, including Spirodela polyrhiza, Lemna aequinoctialis and Wolffia globosa. They were subsequently surfaced sterilized using NaClO and successfully cultured axenically in the laboratory. When grown in Hoagland’s E medium, their doubling times were 2.4 days (L. aequinoctialis), 3.2 days (S. polyrhiza), and 3.6 days (W. globosa), respectively. Interestingly, of all duckweeds, S. polyrhiza was able to accumulate carbohydrate and protein content upto 40.7% and 31.4%, respectively. In contrast, the highest level of carbohydrate content in L. aequinoctialis was only 6.9% compensated for its fastest growth. Furthermore, addition of salicylic acid, a plant growth regulator, to the culture media triggered flowering in S. polyrhiza. The floral structure of S. polyrhiza was incomplete, lacking of petal and sepal, but was a perfect flower v having one pistil and two stamens. Agrobacterium-mediated transformation was performed to genetically modify S. polyrhiza by agroinfiltration of its turions, specialized fronds. Intriguingly, vacuum infiltration for 5 min yielded the highest transformation frequency as shown by the detection frequency of Bar and Egfp up to 75% and the observation of GFP fluorescence signals in the first generation of transgenic duckweeds (T1). Nevertheless, the transgenes were apparently lost during vegetative proliferation as shown by the reduction of the detection frequency of Bar and Egfp to 50% and 0%, respectively, in T2 (the second generation of transgenic duckweeds). Altogether, the striking superiority of S. polyrhiza, including rapid growth, high carbohydrate and protein content, inducible flowering, a simple transformation protocol, and an availability of its genomic DNA sequence, will make this duckweed as a versatile tool in biotechnology to cope with the world’s challenging problems, especially food and energy security, in the near future. vi CONTENTS Page ABSTRACT iv CONTENTS vi LIST OF TABLES xii LIST OF FIGURES xiii CHAPTER 1. INTRODUCTION 1 1.1 Objectives 2 1.2 Benefits expected to receive 2 1.3 Scope of the study 2 2. LITERATURE REVIEWS 2.1 Duckweeds 4 2.1.1 Classification 4 2.1.2 Growth 5 2.1.3 Reproduction 5 2.2 Molecular identification of duckweeds 6 2.3 Duckweeds surface sterilization 7 2.4 Tissue culture media 8 2.4.1 The composition of tissue culture media 8 2.4.2 Media formulas 9 2.5 The chemical compositions of duckweeds 12 2.6 Applications of duckweeds 13 2.7 Genetic transformation and transgenic plants 14 2.7.1 Physical gene transfer method 14 2.7.2 Chemical gene transfer method 15 2.7.3 Biological or vector gene transfer method 16 2.7.3.1 Gene transfer by Agrobacterium tumefaciens 16 vii CONTENTS (CONTINUED) Chapter Page 2.7.3.2 Agrobacterium mediated genes transfer in Monocotyldon and dicotyledon 18 2.8. Developing techniques for duckweed transformations 19 2.8.1 Flowering induction of duckweeds 19 2.8.1.1 Plant growth regulator 19 2.8.1.2 Photoperiodism 19 2.8.1.3 Stress conditions 20 2.8.1.4 Floral dip transformation 21 2.8.2 Callus induction 21 2.8.2.1 Factors affecting callus induction 22 2.8.2.2 Gene transfer into callus 23 2.8.3 Turions induction 24 2.8.3.1 Agroinfiltration 24 3. MATERIALS AND METHOD 26 3.1 Plant material preparation 28 3.2 Identification of duckweeds species 28 3.2.1 DNA extraction 28 3.2.2 DNA Amplification and Sequencing 29 3.3 Cultivation of duckweeds in the laboratory 29 3.3.1 Optimization of duckweeds surface sterilization 29 3.3.2 Optimization of media for duckweeds cultivation 30 3.4 Biochemical analyses 31 3.4.1 Carbohydrate analysis 31 3.4.2 Protein analysis 32 3.4.3 Oxalate analysis 33 3.5 Preparation of the transformation vector, Agrobactim 33 3.5.1 Growth analysis of transformed A. tumefaciens GV-3101 34 viii CONTENTS (CONTINUED) Chapter Page 3.5.2 Ceftriaxone tolerance of transformed A. tumefaciens GV-3101 34 3.6 Genetic transformation of duckweeds 35 3.6.1 Preparation of target tissues 35 3.6.1.1 Induction of flowering in S. polyrhiza (BUU1) 35 3.6.1.2 Induction of callus in S. polyrhiza and L. aequinoctialis 36 3.6.1.3 Induction of turion formation in S. polyrhiza 36 3.6.2 DNA transformation of duckweed 36 3.6.2.1 Herbicide (glufosinate) resistance of target tissue of duckweed 37 3.6.2.2 Turion transformation by Agroinfiltration 37 3.6.3 Verification of transgenic duckweeds 38 3.6.3.1 Genotyping by PCR technique 38 3.6.3.2 EGFP expression 39 3.7 Statistical analysis 39 4. RESULT 40 4.1 Identification of duckweeds species in Burapha University 40 4.1.1 Characterization of duckweed isolates 40 4.1.2 Identification of duckweed species by PCR technique 42 4.2 Cultivation of duckweeds in the laboratory 43 4.2.1 Optimization of surface sterilization regime 43 4.2.2 Optimal medium for culturing duckweeds 48 4.3 Carbohydrate, Protein and Oxalate content in duckweeds 50 4.3.1 Carbohydrate content in duckweeds 50 4.3.2 Protein content in duckweeds 51 4.3.3 Oxalate content in duckweeds 52 4.3.4 Biochemical composition of duckweeds 53 ix CONTENTS (CONTINUED) Chapter Page 4.4 Growth of Agrobacterium 54 4.4.1 Growth of A. tumefaciens GV-3101 54 4.4.2 Effect of ceftriaxone on growth of A. tumefaciens GV-3101 harboring pB7WG and pB7WG2D-X 56 4.5 Duckweed transformation 58 4.5.1 Preparation of target tissues 58 4.5.1.1 Induction of flowering in S. polyrhiza 58 4.5.1.2 Induction of callus 63 4.5.1.3 Induction of turions 65 4.5.2 Effect of herbicide on target tissues 67 4.5.2.1 Effect of glufosinate on growth of S. polyrhiza 67 4.5.2.2 Effect of glufosinate on growth of L. aequinoctialis 67 4.5.2.3 Effect of glufosinate on regeneration of turions from S. polyrhiza 67 4.5.2.4 Effect of glufosinate on regeneration of callus from L. aequinoctialis 68 4.5.3 Turions transformation by Agroinfiltration 72 4.5.4 Verification of transgenic duckweeds 73 4.5.4.1 Genotyping by PCR technique 73 4.5.4.2 Observation of Egfp expression 75 5. DISCUSSION AND CONCLUSIONS 77 5.1 Species of duckweeds in Burapha University 77 5.2 Surface sterilization of duckweeds 78 5.2.1 Surface sterilization of S. polyrhiza and L. aequinoctialis 78 5.2.2 Surface sterilization of W. globosa 79 5.3 Culture of duckweeds in the laboratory 79 5.3.1 Hoagland’s E is a universal medium for duckweeds 79 x CONTENTS (CONTINUED) Chapter Page 5.3.2 L. aequinoctialis is the fastest growing duckweed in the laboratory 80 5.4 Carbohydrate, Protein and Oxalate content in duckweeds 80 5.4.1 S. polyrhiza and W. globosa have high carbohydrate and protein content 80 5.4.2 L. aequinoctialis has high protein content but low in carbohydrate 81 5.4.3 All three species of duckweed have significant amount of oxalate 81 5.4.4 Duckweeds as alternative food and energy sources 82 5.5 Optimization of Agrobacterium growth and growth inhibition 82 5.6 Salicylic acid induces flowering in S.