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VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - GRADUATION THESIS “COMPLETE THE MULTIPLICATION PROCESS OF PAULOWNIA FORTUNEI IN VITRO” HANOI, 3/2021 VIETNAM NATIONAL UNIVERSITY OF AGRICULTURE FACULTY OF BIOTECHNOLOGY -*** - GRADUATION THESIS “COMPLETE THE MULTIPLICATION PROCESS OF PAULOWNIA FORTUNEI IN VITRO” Student’s name : Vu Duc Canh Class : K61CNSHE Faculty : Biotechnology Supervisor : Dr Nguyen Thi Lam Hai HANOI, 03/2021 STATEMENT OF ORIGINAL AUTHORSHIP The work contained in this thesis has not been previously submitted to meet requirements for an award at this or any other education institution To the best of my knowledge and belief, the thesis contains no material previously published or written by another person except where due reference is made Hanoi, February 23rd, 2021 Student Vu Duc Canh i ACKNOWLEDGEMENTS To complete this graduation thesis, I would like to thank Nguyen Thi Lam Hai.Dr, and the teachers in the Department of Plant – Faculty of Biotechnology, who have always been helpful and guided me throughout the process of implementing the topic I would like to express my sincere thanks to all of the students working in the Department of Plant – Faculty of Biotechnology for creating the best conditions to help me during the time of the thesis I would like to express my sincere thanks to the teachers of the Department of Biotechnology, Vietnam National University of Agriculture for imparting new knowledge to me, and having a passion for scientific research during my time studying at the university Finally, I sincerely extend my gratitude to my family and friends who have always supported and encouraged me during my studies and scientific research Hanoi, February 23rd, 2021 Student Vu Duc Canh ii CONTENTS STATEMENT OF ORIGINAL AUTHORSHIP i ACKNOWLEDGEMENTS ii Content of table v Content of figure vi LIST OF ABBREVIATIONS vii ABSTRACT viii PART I INTRODUCTION 1.1 Preface 1.2 Objectives 1.3 Requirements PART II LITERATURE REVIEW 2.1 General introduction 2.1.1 The origin of the research object 2.1.2 Distribution 2.1.3 Scientific classification 2.1.4 Botanical characteristics of Paulownia fortune 2.1.5 Advantages and application 2.1.6 Requires external scene 10 2.2 Reacher Paulownia fortunei trees in the world and in Vietnam 11 2.1.1 Research overview in the world 11 2.2.2 Overview of domestic research 12 PART 3: MATERIALS AND RESEARCH METHODS 14 iii 3.1 Object, material, location, time of culture 14 3.1.1 Research materials 14 3.1.2 Location, time of culture 14 3.2 Content and reseach methods 14 3.3 Tracking indicators 18 PART 4: CONCLUSION AND RECOMMENDATION 21 4.1 Conclusion 21 4.1.1 Effects of BA and Kinetin on shoot multiplication 21 4.1.2 Effects of BA and IAA on shoot multiplication 23 4.1.3 Effects of BA and IBA on shoot multiplication 25 4.1.4 Effects of coconut milk on shoot multiplication 27 4.1.5 Effects of iron content in MS on shoot photosynthesis, thereby affecting the shoot multiplication 29 4.1.6 Effects of Vitamin content in MS on shoot metabolism and shoot multiplication 31 4.1.7 Effects of Vitamin B2 on growth and development of cultured shoots 33 4.1.8 Effect of the MS concentration to the rooting (non-sterile - free sugar and liquid medium) 35 4.1.9 Effect of α-NAA on rooting (non-sterile and liquid medium) 36 4.2 Recommendation 36 REFERENCES 37 iv CONTENT OF TABLE Table 1: The statistic result of effects of BA and Kinetin on shoot multiplication 21 Table 2: The statistic result of effects of BA and IAA on shoot multiplication 23 Table 3: The statistic result of effects of BA and IBA on shoot multiplication 25 Table 4: The statistic result of Effects of coconut milk on shoot multiplication 27 Table 5: The statistic result of effects of iron content in MS on shoot photosynthesis, thereby affecting the shoot multiplication 29 Table 6: The statistic result of effects of Vitamin content in MS on shoot metabolism and shoot multiplication 31 Table 7: The statistic result of effects of Vitamin B2 on growth and development of cultured shoots 33 Table 8: The statistic result of effect of the MS concentration to the rooting (non-sterile free sugar and liquid medium) 35 v Content of figure Figure Paulownia fortune shoot Figure Paulownia fortune leaves Figure The inflorescence of Paulownia fortune Figure Fruit of Paulownia fortune Figure Root system of Paulownia fortune Figure 6: Effects of BA and Kinetin on shoot multiplication (after weeks) 23 Figure 7: Effects of BA and IAA on shoot multiplication (after weeks) 24 Figure 8: Effects of BA and IBA on shoot multiplication (after weeks) 26 Figure 9: Effects of coconut milk on shoot multiplication (after weeks) 28 Figure 10: Effects of iron content in MS on shoot photosynthesis, thereby affecting the shoot multiplication (after weeks) 30 Figure 11: Effects of Vitamin content in MS on shoot metabolism and shoot multiplication (after weeks) 32 Figure 12: Effects of Vitamin B2 on growth and development of cultured shoots (after weeks) 34 Figure 13: Effect of the MS concentration to the rooting (non-sterile - free sugar and liquid medium) (after weeks) 36 vi 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 NAA 1-naphthaleneacetic acid vii ABSTRACT Paulownia fortunei is a tree that has a lot of economic value and has high growth rate, good wood quality, and less pest and disease The project was conducted to find the best formula for multiplication in vitro paulownia fortunei and the best rooting recipe with low economic cost With the use of disease-free samples and in vitro culture, transfer to different culture media obtained the following results: the highest shoot multiplier was obtained on MS medium supplemented with 0.5 mg / L BAP (2.31 cm / plant and 2.14 shoots /plant) tall, fat, strong shoots With the MS formula supplemented with 0.5 mg / L BAP and coconut milk, the multiplier was low, however green plants, and healthier and fresher roots were suitable for complete tree formation With the formula of iron supplementation by changing the amount of iron in the MS ingredient by 1.5 times, the multiplier ratio of 2.53 shoots / plant and the average height of 1.98 cm / plant is obtained, the plants are greener and healthier With the formula of changing the amount of Vitamins by increasing their content in MS and Vitamin B2 supplementing formula, the shoot multiplication efficiency is not increased, the multiplier factor is not high culture plants from culture different medium to non-sterile liquid root medium, medium containing 1/3 of the non-sterile liquid MS is best The best substrate for planting plants in the garden is peat moss viii 4.1.3 Effects of BA and IBA on shoot multiplication Table 3: The statistic result of effects of BA and IBA on shoot multiplication IBA Shoot height Number of shoots (mg/l) (cm / shoot) (buds / plant) shoot quality F1: control 0.00 2.22 1.52 ++- F2 0.1 1.67 1.80 + F3 0.2 1.87 1.68 + F4 0.3 1.35 2.2 + F5 0.4 0.96 1.48 + LSD0,05 0.21 0.64 CV% 0.97 1.59 Similar to IAA, the incorporation of IBA into MS + 3% succrose + g / l agar + 0.5 mg / l BA basal culture medium to cultivate Paulownia fortunei was somewhat inferior to the addition of kinetin and addition of IAA In the formula with 0.3 mg / l IBA, the highest coefficient was 2.2 shoots / plant and 1.35 cm / shoot, but the quality of the plants was poor and weak Whereas in the control formula, although the coefficient was slightly lower with the coefficient of 1.52 shoots / plant and 2.12 cm / shoot, respectively, but the plants were bigger and stronger In the remaining formulas, the index is also much lower and the quality of the tree is not stable 25 Control 0.1 IBA 0.2 IBA 0.3 IBA 0.4 IBA Figure 8: Effects of BA and IBA on shoot multiplication (after weeks) 26 4.1.4 Effects of coconut milk on shoot multiplication The addition of industrial coconut milk to the study of the development of the lime is aimed at replacing the natural amount of coconut water In natural coconut water, the most important factor in tissue culture is the myo-inositol content This is an indispensable substance for the process of shoot generation during rapid multiplication However, this content in each period of the coconut has different content and high cost, so the experiment aims to test and provide a solution to provide more organic matter in tissue culture to reduce expense Table 4: The statistic result of Effects of coconut milk on shoot multiplication Coconut Shoot height Number of shoots milk (cm / shoot) (buds / plant) shoot quality (ml/l) F1: control 0.00 2.30 1.87 ++- F2 25 2.31 2.01 +++ F3 50 2.18 2.22 +++ F4 75 2.40 2.18 +++ F5 100 2.36 2.10 +++ LSD0,05 0.21 0.64 CV% 2.22 2.17 From the results after culturing with coconut milk after weeks, we can see that the coefficients between the formulas are not much different, the shoot rate is stable, the tree is large, strong and for better development when compared with the control formula The coefficients are all from 2.01 shoots / plant and the average height is 2.18 cm / shoot 27 Control 25 ml/ L coconut milk 50 ml/ L coconut milk 75 ml/ L coconut milk 100 ml/ L coconut milk Figure 9: Effects of coconut milk on shoot multiplication (after weeks) 28 4.1.5 Effects of iron content in MS shoot multiplication Table 5: The statistic result of effects of iron content in MS shoot multiplication MS Number of concentration shoots (shoots / Shoot height (cm / shoot) shoot quality (ml/l) plant) F1: control 2.30 1.87 ++- F2 7.5 2.53 1.98 +++ F3 10 1.73 1.28 ++- F4 12.5 1.00 1.20 + LSD0,05 0.49 0.98 CV% 1.32 1.52 Iron is a fairly important component in photosynthesis, so iron supplementation also contributes to the formation and development of shoots during tissue implantation Since the leaves of Paulownia fortunei cradle under long term transplant conditions often lead to yellowing and wilting of the leaves, I added iron salary for verification Results after weeks of culture, with the addition of iron to the plants no longer yellowing compared to the control, the leaves were greener and darker With the concentration of 7.5 ml / l MS 3, the plants not only achieved the quality, but the rapid multiplication rate and height were also much higher than the other formulas with the number of shoots of 2.53 shoots / plant and the average height of 1.98 cm / tree however, with higher jaws, shoot generation and height were also reduced and decreased compared to the control 29 Control 7.5ml/L MS 10 mg/L MS 12.5 mg/L MS Figure 10: Effects of iron content in MS on shoot multiplication (after weeks) 30 4.1.6 Effects of Vitamin content in MS shoot multiplication Table 6: The statistic result of effects of Vitamin content in MS and shoot multiplication MS Number of concentration shoots (shoots / Shoot height (cm / shoot) shoot quality (ml/l) plant) F1: control 2.30 1.87 ++- F2 7.5 1.98 2.24 +-+ F3 10 2.55 1.77 + F4 12.5 2.52 1.67 + LSD0,05 0.89 0.98 CV% 1.36 3.67 Vitamins are very important components in the process of tissue culture Through the results we see that the addition of Vitamin concentration is not necessary although the rate of shoots is higher than that of the control: 2.55 shoots / plant; 2.52 shoots / plant But the quality of the tree is very poor Leaves are chlorotic and have many signs of death after weeks Shoots in the different formulas develop the same, but after weeks, plants with 10 ml / l and 12.5 ml / l did not develop at all compared to the third week after culture 31 Control 7.5 ml/L MS 10 ml/L MS 12.5 ml/L MS Figure 11: Effects of Vitamin content in MS on and shoot multiplication (after weeks) 32 4.1.7 Effects of Vitamin B2 on growth and development of cultured shoots Table 7: The statistic result of effects of Vitamin B2 on growth and development of cultured shoots Vitamin B2 Number of concentration shoots (shoots / Shoot height (cm / shoot) shoot quality (mg/l) plant) F1: control 2.30 1.87 ++- F2 2.13 1.67 + F3 10 2.17 1.35 + F4 25 0.60 1.01 + F5 50 0.63 0.96 - LSD0,05 0.26 0.46 CV% 1.34 1.54 With the added vitamin B2 content after week of culture, the medium loses its orange color (the color of Vitamin B2) gradually, in the formula 5mg / l B2 almost completely lost Over a 4-week observation period, the larger the amount of Vitamin B2, the harder it is for plants to grow the leaves are yellow, some leaves are nearly opaque white, the leaves are small and the death rate is higher However, with the concentration of 5mg / l of vitamin B2, the plant was still growing well and almost equal to the control, the leaves were also greener than the control, although the early cultivation process was very weak 33 Control mg/L B2 10 mg/ B2 25 mg/L B2 50 mg/L B2 Figure 12: Effects of Vitamin B2 on growth and development of cultured shoots (after weeks) 34 4.1.8 Effect of the MS concentration to the rooting (non-sterile - free sugar and liquid medium) Table 8: The statistic result of effect of the MS concentration to the rooting (non-sterile - free sugar and liquid medium) Shoot MS Number of concentration root (root / Root length (cm /root) height (cm shoot / shoot) quality (ml/l) plant) F1: control 1.25 0.44 1.35 + F2 1/2 1.72 0.26 0.94 + F3 1/3 3.19 1.17 1.76 ++- F4 1/4 3.48 0.55 1.21 + F5 1/6 2.6 0.97 0.76 + LSD0,05 0.8 0.35 0.61 CV% 1.36 2.35 2.03 With the static liquid culture without non-sterilization the medium contribute to reducing the cost and time in culturing complete plants From the above data, we obtained at a concentration of 1/3 MS, the phta plants grow very stably and have good roots Besides, the height development and the number of leaves also develop stably with the number of roots is 3.19 roots / plant, the root length is 1.17 cm / tree and the plant height is 1.76cm 35 Figure 13: Effect of the MS concentration to the rooting (non-sterile - free sugar and liquid medium) (after weeks) 4.1.9 Effect of α-NAA on rooting (non-sterile and liquid medium) Although the concentration of MS at a concentration of 1/3 times is suitable for both solid and liquid roots, the addition of root growth regulators leads to poor plant development and very high death Experiment was performed times and both of them failed to show any results Mortality rate reaches 100% The explants were all dead and did not arise with the appearance of callus, but the plant was still dead 4.2 Recommendation - Optimization of the fast multiplication process to produce large quantities of Paulownia fortunei - Conducting experiments on the effects of seasons on the survival and development of Paulownia fortunei 36 REFERENCES Documents in the country Lê Tấn Đức (2003): Tạo giống hông chuyển gen kháng sâu thông qua vi khuẩn Agrobacterium tumefaciens (Hội thảo Cơng nghệ sinh học tồn quốc, 2003) Lê Tấn Đức (2003) Nghiên cứu hệ thống tái sinh in vitro Hông (Paulownia fortunei) ảnh hưởng tác nhân chọn lọc để tạo chuyển gen (Hội nghị CNSH Toàn quốc, 2003) Thái Xuân Du (2001) Quy trình trồng chăm sóc hơng sau giai đoạn ống nghiệm (Tạp chí Cơng nghệ sinh học Nông nghiệp sinh thái bền vững Nhà xuất Nông nghiệp, 2001) Lưu Việt Dũng (2002) Nhân giống vô tính Hơng (Paulownia fortunei) phương pháp ni cấy mơ tế bào thực vật (Tạp chí Sinh học, tập 24, số 2) Bùi Duy Ngọc, Nguyễn Đình Hợi, Nguyễn Thị Minh Xuân (2007) bước đầu nghiên cứu nâng cao hối lượng thể tích gỗ Hơng (Paulownia fortunei) (Tạp chí khoa học Lâm nghiệp số 1/2007, trang 291-296) Đoàn Thị Ái Thuyền, Vũ Ngọc Phương, Thái Xuân Du Nguyễn Văn Uyên (2001) nghiên cứu nhân giống Hông (Paulownia fortunei) phương pháp nuôi cấy mơ, kết đăng tạp chí Sinh học số 23 (3) trang 46-50 Nguyễn Thị Trà My (2012): Nghiên cứu nhân nhanh giống Hông ( Paulownia fortunei) phương pháp nuôi cấy mô tế bào thực vật Foreign documents Bergman B A & Moon H K In vitro adventitious shoot production in Paulownia fortunei, Plant Cell Rep, 16, 315-319 Venkateswarlu B, Mukhopadhyay J, Sreenisavan & Moses Kumar V (2001) Micropropagtion of Paulownia fortunei through in vitro axillary shoot proliferation Indian Journal of Experimental Biology Vol 39, June 2001 Pp 594 – 599 Burger D W., Lin L & Wu L (1985) “Rapid micropropagation of Poulownia tomentosa, Hort Sci 20-4, pp760” 37 Song S L., Sato T., Saito A & Kihachiro O (1989) “Meristematic culture of seven Paulownia species” J Jpn For Soc 71-11 pp456-462 Rao C D., Goh C-J & Kumar P P (1996) “High frequency adventitious shoot regenation from excised leaves of Paulownia spp cultured in vitro Plant Cell Rep 16 Pp204-223 Ho C K., Jacobs G & Donald D G M (1994) “Organo genetic capacities of different explants of four Paulownia species” Taiwan for Res Inst New Series 9-4(1994) pp 397-405 Shtereva, L., et al (2014) “Micropropagation of six Paulownia genotypes through tissue culture” Central European Agriculture 15(4): 147-156 Po˙zoga, M., et al (2019) “In Vitro Propagation Protocols and Variable Cost Comparison in Commercial Production for Paulownia tomentosa Paulownia fortunei Hybrid as a Renewable Energy Source” MPDI 9(11): 2272-2281 Documentation on Website http://nhipcaudautu.vn/article.aspx?id=3845 http://***********/xem-tai-lieu/chuong-ii-cong-nghe-nuoi-cay-mo-te-baothuc.573762.html http://baigiang.violet.vn/present/same/entry_id/374963 http://www.scribd.com/doc/73485861/Thuc-Hanh-Nuoi-Cay-Mo http://www.dostbinhdinh.org.vn/KyYeu/GiaiDoan2001-2005/cnsh/Kyyeu_T66T69.htm http://***********/xem-tai-lieu/bai-giang-nuoi-cay-mo-te-bao-thuc-vat-bang-tebao-tran-va-ki-thuat-chuyen-gen.492239.html https://khoahoc.tv/paulownia-loai-cay-cuu-rung-8121 https://sites.google.com/site/cayhongvietnam/home https://tailieu.vn/doc/nghien-cuu-khoa-hoc-huong-dan-ky-thuat-trong-cay-hongpaulownia-fortunei 1100480.html 38 10 https://vi.wikipedia.org/wiki/H%C3%B4ng_hoa_tr%E1%BA%AFng#/media/T%E 1%BA%ADp_tin:PaulowniaFortunei.jpg 11 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