MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES UNDERGRADUATE THESIS EFFECT OF COBALT, COPPER AND MOLYBDENUM TRIOXIDE NANOPARTICLES ON EFFICIENT MICROPROPAGATION OF STRAWBERRY (Fragaria x ananassa Duch.) Major : BIOTECHNOLOGY Student : HO CAM KHANH HOA Student ID : 18126049 Academic year : 2018-2023 Thu Duc City, 08/2023 MINISTRY OF EDUCATION AND TRAINING NONG LAM UNIVERSITY-HO CHI MINH CITY FACULTY OF BIOLOGICAL SCIENCES UNDERGRADUATE THESIS EFFECT OF COBALT, COPPER AND MOLYBDENUM TRIOXIDE NANOPARTICLES ON EFFICIENT MICROPROPAGATION OF STRAWBERRY (Fragaria x ananassa Duch.) Advisor Student Do Manh Cuong, Ph.D Ho Cam Khanh Hoa Hoang Dac Khai, M.Sc Thu Duc City, 08/2023 ACKNOWLEDGEMENT I would like to express my deepest gratitude to my academic advisor, Prof. Duong Tan Nhut, Ph. Do Manh Cuong, and M. Hoang Dac Khai for their invaluable guidance, patience, and encouragement throughout the course of this study.
Their expertise and insightful feedback have been instrumental in shaping the research. They dedicatedly guided me with a lot of knowledge, directions, corrections, support, and created favorable conditions for me during the process of making my thesis. I am thankful to Nong Lam University for providing a conducive academic environment, state-of-the-art facilities, and access to resources that were indispensable for the successful completion of this study. I wish to thank the Board of Directors of Nong Lam University - Ho Chi Minh City, the administrators of the Faculty of Biological Sciences, and all the lecturers for providing me with the necessary resources to pursue my studies there.
Thanks to guidance and teaching, I can apply it to solve problems related to the major during the thesis. I would like to thank Ph. Phan Dang Thai Phuong - academic mentor of class DH18SHC for taking care of me during 4.5 years of university. Special thanks go to the members of the Department of Molecular Biology and Plant Breeding of Tay Nguyen Institute for Scientific Research, Vietnam Academy of Science and Technology for their collaboration and assistance during the data collection and analysis process.
Their valuable contributions significantly enriched this research. Lastly, I am deeply grateful to my family and friends for their unwavering support, understanding, and encouragement throughout my academic journey. This thesis 1s like a great exercise to help me improve myself and prepare for the next phase of my career 1n scientific research. Sincere thanks to all! AFFIRMATION AND COMMITMENT My name is Ho Cam Khanh Hoa, student ID: 18126049, class: DHISSHC of Biotechnology, Nong Lam University, Ho Chi Minh City.
This is my undergraduate thesis which was conducted by myself under the guidance of Ph. Do Manh Cuong and M. Hoang Dac Khai. This research was supported by Lam Dong Department of Science and Technology and Vietnam Academy of Science and Technology under grant number NCXS01.
All the data and information are completely honest and objective. I take full responsibility in front of the committee for these commitments. Thu Due City, August 28th, 2023 Student’s signature Ho Cam Khanh Hoa ii ABSTRACT Nanotechnology is indeed a rapidly growing field of science and technology that has significant implications for various aspects of modern life. Nanomaterials (NPs) can be readily absorbed, metabolized, and accumulated in cells.
Substituting inorganic salts with nanomaterials in MS (Murashige and Skoog) media is a trend of interest to researchers in the field ofplant cell, tissue and organ culture. In this study, traditional inorganic salts (CoCl2.5H2O and Na2MoOy4.2H20) in MS medium were replaced with cobalt nanoparticles (CoNPs), copper nanoparticles (CuNPs) and molybdenum trioxide nanoparticles (MoO3NPs) to improve strawberry (Fragaria x ananassa Duch. This study showed that CoNPs, CuNPs and MoO3NPs had positive effect on the root formation and in vitro growth of strawberry. Accordingly, the results indicated that replacing 0.6H20 in MS medium with 0.0062 mg/L CoNPs gave the best growth indicators after 30 days of culture.
The growth indicators of 0.0062 mg/L CoNPs such as plant height, number of leaves, number of roots, root length, fresh weight, dry weight and chlorophyll index (4.80 cm; 13 leaves/plantlet; 15.70 nmol/cm?; respectively) were higher than the basal MS medium (without CoNPs). Among the CuNPs concentrations investigated, 0.0064 mg/L CuNPs treatment showed the number of roots (16.67 roots/plantlet) and root length (2.5 cm) was highest compared to the control (without CuNPs) and other treatments after 30 days of culture. In terms of MoO3NPs, MS medium added of 0.149 mg/L MoO3NPs was the most optimal with plant height (6.36 cm), fresh weight (452.33 mg), dry weight (47.49 mg), and SPAD (45.23 nmol/cm?), number of roots (20.33 roots/plantlet) and root length (3.01 cm) in comparison to the control (without MoO3NPs) after 30 days of culture. The findings from this study are promising, as well as it opens up exciting possibilities for further research and advancements in strawberry micropropagation.
Keywords: Fragaria x ananassa Duch., metal nanoparticles, mineral elements, MS medium, rooting ili TÓM TAT Công nghệ nano đã và đang thực sự là một lĩnh vực khoa học và công nghệ phát triển nhanh chóng và có ý nghĩa quan trọng đối với nhiều khía cạnh khác nhau trong cuộc sống. Vật liệu nano (NPs) có thé dé dàng hap thụ, chuyên hóa và tích lũy trong tế bào. Thay thế muối vô cơ bằng vật liệu nano trong môi trường MS (Murashige và Skoog) thực sự là một xu hướng được các nhà nghiên cứu trong lĩnh vực nuôi cấy mô thực vật và vi nhân giống quan tâm. Trong nghiên cứu này, các muối vô cơ truyền thống (CoCls.5H20 và Na;MoO¿.2H2O) trong môi trường MS được thay thé bằng nano coban (CoNPs), nano đồng (CuNPs) và nano molybdenum trioxide (MoO3NPs).
Nghiên cứu này đã chứng minh tác động tích cực của CoNPs, CuNPs và MoOsNPs đối với sự hình thành rễ và sự phát triển in vitro của cây dâu tay (Fragaria x ananassa Duch. Kết quả cho thấy việc thay thé 0.6H2O trong môi trường MS bằng 0.0062 mg/L CoNPs cho các chỉ tiêu sinh trưởng tốt nhất sau 30 ngày nuôi cấy. Thật vậy, các chỉ số sinh trưởng như chiều Cao cây, số lá, số rễ, chiều dài rễ, trọng lượng tươi, trọng lượng khô và chỉ số diệp lục (4,80 cm; 13 lá/cây; 15,67 ré/cay; 1,97 cm; 248,33 mg; 25,56 mg; 48,70 nmol/cm?; tương ứng) cao hon môi trường MS cơ bản (không có CoNPs). Trong số các nồng độ CuNPs được khảo sát, nghiệm thức 0.0064 mg/L CuNPs cho SỐ lượng rễ (16,67 rễ/cây) và chiều dài rễ (2,5 cm) cao nhất so với đối chứng (không có CuNPs) và các nghiệm thức khác sau 30 ngày nuôi cấy.
Liên quan đến MoOaNPs, môi trường MS bồ sung 0.149 mg/L MoO3NPs là tối ưu nhất với chiều cao cây (6,36 cm), trọng lượng tươi (452,33 mg), trọng lượng khô (47,49 mg) và SPAD (45,23 nmol/cm?), số lượng rễ (20,33 rễ/cây) và chiều dài rễ (3,01 cm) so với đối chứng (không có MoO3NPs) sau 30 ngày nuôi cây. Những phát hiện từ nghiên cứu này thực sự đầy hứa hẹn và mở ra những khả năng thú vị cho các nghiên cứu sâu hơn và những tiến bộ trong vi nhân giống dâu tây. Từ khóa: Fragaria * ananassa Duch., môi trường MS, nano kim loại, nguyên tố khoáng, tạo rễ iv TABLE OF CONTENTS Page ACKNOW LEDGEMEN T sassgesegõngg in kHD011004300003880800588808S2tU19018,G08BLIS3/3GãR0H843030 30LG83HEH8S5H883gỂÄ 1 AFFIRMATION AND COMMITMENT .eccecceeceeeesceeceeeeeseeeeeseeseenseeseeeseeseeseess i ABS TRAG DD sassssscecsai161001806886220800560068-033588006301550830/3030053838596143893200488i5558i039090003303008136509930071480388 11 TOM TAT.ÔỎ iv TABLE OF CGONTENTS.- ác in s0 HH HH1 ng n0 hở ng 08m0 0x e0 V LIS] OF ABBREVIATIONS soscisissoiasiigix2ES080859.3ã: sưuvil LIST OF TABLES esesecupeeeuccieresnresunane ere sewerceunmeen eal neaeeene naar tenet urenmees inact Vill LIST OF FIGURES scsscassossesnsecenemanasneesuseinsnanenus aun ssweetesen esas tsecour ssetuugnce mennwaenest 1X CHAPTER 0090)09. 1 IN Problera Statement .c2-2nccnnsanesorennesvesansenrennsantenseaonpensanserseenrannasennnneganensnanennsssenens 1 1L Oe) (lee ee ee ee ee ee ee ee 3 13.
Content iss cove sesass cease Saeco Ss RE Ee 2 CHAPTER.LITERA TURE REV IE W cisssnseccveoessvnvesssxonessrereensnuiverssnereeseesennsesievearees 3 2. Overview of the strawberry (Fragaria x ananassa Duch. SELSHMHG CLASSI CATION os, oz cs: asus xp vs ounassreaseneasesanenn xasnasisn seni 3043034014036 36 355ã3.2, Physical chatacters OS ecsnasweemercnare eee EER ees 3 21:1L, 3a he vallmeso tt stray DEDDNiossce-cesrsesoeEbikuseBdikBuiedfBbsEkgindskisi-GE03-ggi.Hđ8 0000085000008 D00 4 Zo Sue 2L1ÌG/IUEEIHGIBI/WBUeseseinnssnebennidediotitatiilBoStg0EGIAI-SSBI-SEGSSESUDAEEBHRHHBBISGBEESIS0800000. The role of Cobalt, Copper and Molybdenum in plant physiology, growth and ES Le\fel (0) 0) 8016) | eee ee ee ee ee ee 6 252 Lhe tOle OF Cobalt plants ox ccnccsessnconss sp ancousssennments amave siuemandahaatedastasnenseruisansanantenies 6 2.
The role of Copper in pÏATIES. The role of Molybdenum in pÏaI{S. Cobalt, Copper and Molybdenum nanoparticles in plant tissue culture. Overview of metal nanopaTICÌ€S.
Cobalt nanoparticles in plant tissue cuÏfUTe. Copper nanoparticles in plant tissue CuÏfUTG. Molybdenum trioxide nanoparticles in plant tissue culture. Recent related studies of CoNPs, CuNPs, and MoO3NPs in domestic and in the "x01.1: DOMESUO SỈUOTEisensnseseaoieiigrodoltsisoitgSISESDMXSSSSEVIAIEEHESEISEHIĐESESSHSSSIHSNSNQIESSESSIGESSS032 11 251220.
NNOHÌCIBEUIHĐBissec.ecsvoisseargBkexsoodrdkeoklsiitcsolg93014iu8iddaxSin0SuL.iiyS03gu26881seoog818ÐsaalzgbSugEx 13 CHAPTER 3. MATERIALS AND METHODS.-- -----+55+c<+<+c+sc+ezsesersreee 14 ENJuno. 14 Be Mgt ila Sex, sss 5 de Re a Ss a a Se Oa cates 668 14 o2: Lại Plant Materials coiasssesdbneoiEneose Bhisagitl0SSagVSEBSsgS2LSESSSSSXSBSg/A001103R01303838001g2100153053g08 14 3.5 BEUIPI Sits ANG LOIS: s:cserasmuvenwse-enaeeratennne meme sea ue trannies tienen mepntceavanteetties 14 3. Culture media oo.
in vitro culture CONdINON s66iii55416211115 0651143141155555453855ES83XE3LLSEESX4AS5SSX3S4G144885448338438538 15 0, MSO OS sac 111512452 5001000E01A1B6L28031528938048g359473938/30303591330858300193090303021-104g38g22z100598 15 3. Plant cell, tissue and organs culture. Effects of CoNPs on the rooting and in vitro growth of strawberry. Effects of CuNPs on the rooting and in vitro growth of strawberry.
Effects of MoO3NPs on the rooting and in vitro growth of strawberry. Statistical aia lV S18 sci cncwretemnaneessmesreevense mise BruiggglitxriucpeiididrbritigiickdcrsrsSdSdrdbbsonidiaetrke 16 CHAPTER 4. RESULTS AND DISCUSSION. Effects of CoNPs on the rooting and in vitro growth of strawberry.
Effects of CuNPs on the rooting and in vitro growth of strawberry. Effects of MoO3NPs on the rooting and in vitro growth of strawberry. Abnormal root morphology at high concentrations of CoNPs, CuNPs, and Vu Lỗ scons sevens se assune tars en saisaeinn nis ct :48H0018303BQEx330Đ0538. CONCLUSIONS AND SUGGESTIONS.--72-c+ccccssce2 29 Siglo CONCIUSIONS sosnsssssssessi8146515305593059635660V433395508S83383550455SEA04ESE4ASE5SET5SS4345S515326481G1823898 29 900 AS UG OSU ODS seesuzesscskesodedigntdalgiisrgsieaddigttoptrgBi2sgitoauigisiigixii,oxgiirtšgigooktgcsieiiogkisosgzzti203:gdS100-mgu8:guingg00u.0 29 REP ERENCES ssoosssoninsseoitittbtiGGEE0SIEHÀ S08 88.88088 30 APPENDIX VI LIST OF ABBREVIATIONS CoNPs: Cobalt nanoparticles CuNPs: Copper nanoparticles MoO3NPs: Molybdenum trioxide nanoparticles CoCl2.6H20: Cobalt chloride hexahydrate CuSOx.5H›O: Copper sulphate pentahydrate NaaMoOx.2H›O: Sodium molybdate dihydrate MS-CoNPs: CoNPs was used to replace CoCl2.6H20 in MS medium MS-CuNPs: CuNPs was used to replace CuSO4.5H20 in MS medium MS-MoO3NPs: MoO3NPs was used to replace Naz2MoOu4.2H20 in MS medium MS: Murashige and Skoog NAA: 1-Naphthaleneacetic acid SPAD: Soil Plant Analysis Development (Total chlorophyll content) FAOSTAT: Food and Agriculture Organization Corporate Statistical Database vil LIST OF TABLES Page Table 2.
Scientific classification of strawberry (Fragaria x ananassa Duch. Effect of different concentrations of CoNPs replacing CoCl2.6H20 on in vitro growth and rooting of strawberry plantlets after 30 days of culfure. Effect of different concentrations of CuNPs replacing CuSO4.5H20 on in vitro growth of strawberry plantlets after 30 days of culture. Effect of different concentrations of MoO3NPs replacing NazMoOx.2H2O on in vitro growth of strawberry plantlets after 30 days of cuÏtUre.-- -------s<-+2 24 Vili LIST OF FIGURES Page Figure 4.
Effect of different concentrations of CoNPs replacing CoCl2.6H20 on in vitro growth of strawberry plantlets after 30 days of culfure. Effect of different concentrations of CuNPs replacing CuSO4.5H2O on in vitro growth of strawberry plantlets after 30 days of cuÌture. Effect of different concentrations of MoO3NPs replacing NaaMoOx.2H2O on in vitro growth of strawberry plantlets after 30 days of culture. Normal roots and abnormal roots morphology.
Problem statement Strawberry (Fragaria x ananassa Duch.) belonging to the Rosaceae family, 1s one of the most popular and widely cultivated fruit trees globally. It is known for its delicious and sweet taste, vibrant red color, and aromatic fragrance. The economic importance and increasing demand for high-quality strawberry plants in the agricultural industry, but the conventional methods of strawberry propagation such as runner division and seed germination are limited by slow multiplication rates and susceptibility to diseases.