MINISTRY OF EDUCATION & TRAINING CAN THO UNIVERSITY BIOTECHNOLOGY RESEARCH & DEVELOPMENT INSTITUTE SUMMARY BACHELOR OF SCIENCE THESIS THE ADVANCED PROGRAM IN BIOTECHNOLOGY INVESTIGATION OF EFFECTIVE MEDIA FOR IN VITRO PROPAGATION OF STEVIA REBAUDIANA BERTONI SUPERVISOR STUDENT MSc. TRAN THI XUAN MAI NGUYEN THANH HUY Student code: 3082597 Session: 34 (2008-2013) Can Tho, 2013 APPROVAL SUPERVISOR TRAN THI XUAN MAI STUDENT NGUYEN THANH HUY Can Tho, May …, 2013 PRESIDENT OF EXAMINATION COMMITTEE NGUYEN HUU HIEP ABSTRACT Stevia (Stevia rebaudiana Bertoni), a natural noncaloric sweetener, can be used as sugar replacement for patients suffering from diabetes, obesity, hypertension or on-diet people. In this research, in vitro propagation of stevia, was carried out in 4 experiments (callusing, shooting, rooting, and acclimatization). The results supported that MS medium (including vitamins) supplemented with 0.2mg/l NAA and 0.15mg/l BAP was the best formula for not only callusing with 92.6% of explants forming profuse calli but also shooting, 100% of nodal segment developed healthy shoots (1.15cm in average length) with hairy and obovate leaves after 21 days. In rooting stage, MS medium (basal salt mixtures) supplemented with 0.5mg/l IAA had resulted in 83.33% responses with fibrous roots (9.7 roots/explant and 2.2cm/root). Maximum percentage (100%) of plantlets was successfully acclimatized in the mixture of 1 soil: 1 sand: 1 decomposed rice straw (v/v) after 3 weeks. (Keywords: BAP, IAA, in vitro, NAA, stevia) i CONTENTS Page Approval........................................................................................ Abstract ........................................................................................i Contents ..................................................................................... ii Chapter 1: Introduction............................................................. 1 Chapter 2: Materials and Methods ........................................... 2 2.1. Materials ................................................................. 2 2.2. Methods ................................................................... 2 2.2.1. Experiment 1: Study on the effects of different media on callusing of stevia leaves. .... 2 2.2.2. Experiment 2: Study on the effects of different media on the multiplication of shoot from nodal segments. ......................................... 3 2.2.3. Experiment 3: Study on the effects of different media on root induction ....................... 3 2.2.4. Experiment 4: Acclimatization................. 4 2.2.5. Statistical method ..................................... 4 Chapter 3: Results and Discussion ........................................... 5 3.1. Experiment 1: Study on the effects of different media on callusing of stevia leaves. .............................. 5 3.2. Experiment 2: Study on the effects of different media on the multiplication of shoot from nodal segments. ........................................................................ 8 3.3. Experiment 3: Study on the effects of different media on root induction .............................................. 12 3.4. Experiment 4: Acclimatization ........................... 15 ii Chapter 4: Conclusions and Suggestions ............................... 17 4.1. Conclusions ........................................................... 17 4.2. Suggestions ............................................................ 17 References ................................................................................. 18 iii CHAPTER 1 INTRODUCTION Stevia (Stevia rebaudiana Bertoni), also known as sugar leaf, honey leaf, or sweet weed, is perennial shrub belonging to genus Stevia, family Asteraceae. Stevia is said to be a natural sweetener due to stevioside compounds found mostly in leaf content. Steviosides can be used as sugar replacement for patients suffering from diabetes, obesity, hypertension, or on-diet people since they are 300 times sweeter than sugarcane, delicious, and non-caloric. The demand on stevia is increasing in recent years accompanying with the significant rising rate of mentioned diseases. Consequently, large-scale production of this valuable herb is expanded in many provinces of Vietnam requiring much investment for not only growing techniques but also large number of homogeneous and disease-free plantlets. Propagation by seeds, however, is very poor and usually results in great variability in features like sweetening levels and composition. Moreover, vegetative propagation can be done from stem nodes but limitation in number is a facing problem (Guruchandran and Sasikumar, 2013). Plant tissue culture is thus an alternative way for rapid and mass production of stevia. Objective This research, “Investigation of effective media for in vitro propagation of Stevia rebaudiana Bertoni”, was aimed to find out a complete process for plant tissue culture of stevia, starting from callusing, shoot multiplication to rooting and finally acclimatization. 1 CHAPTER 2 MATERIALS AND METHODS 2.1. Materials In vitro grown stevia from Ha Noi Chemicals and equipments in Plant Genetics Engineering laboratory, Biotechnology Research and Development Institute, Can Tho University. 2.2. Methods All media were prepared with 20g/l sucrose and solidified with 8g/l agar. The pH was adjusted to 5.8 before autoclaving at 121oC and 1atm pressure for 20 minutes. All the cultures were placed under stable conditions, at temperature 27±1oC, light intensity 1000lux, 16-hour illumination per day. 2.2.1. Experiment 1: Study on the effects of different media on callusing of stevia leaves. This experiment was set up to test the appropriate medium and supplemented phytohormones for leaf-derived callus formation. Placing 0.3-0.5cm2 leaf explant on different media. It was noted that the dorsal side should be in contact with the medium surface. There were totally 6 treatments with 3 repetitions. S1: MS (Basal salt mixtures)+1mg/l Kinetin+2mg/l 2,4-D S2: MS (Basal salt mixtures)+0.75mg/l NAA+1mg/l 2,4-D S3: MS (Basal salt mixtures)+3mg/l 2,4-D S4: MS (Basal salt mixtures)+0.5mg/l BAP S5: MS (Including vitamins)+0.15mg/l BAP+0.2mg/l NAA S6: MS (Including vitamins)+0.2mg/l BAP+0.5mg/l IAA 2 There were 9 leaf explants cultured on each Petri dish and 3 Petri dishes for a repetition. For each treatment, the ratio of explants forming callus was recorded. 2.2.2. Experiment 2: Study on the effects of different media on the multiplication of shoot from nodal segments. This experiment was carried out to obtain the most effective medium for shoot multiplication from stem nodes. 1-cm nodal segments were cultured on different media. There were totally 3 treatments with 3 repetitions. C1: MS (Basal salt mixtures)+0.2mg/l NAA+0.15mg/l BAP C2: MS (Including vitamins)+0.2mg/l NAA+0.15mg/l BAP C3: MS (Basal salt mixtures)+3.5mg/l BAP There were 4 explants cultured on each Petri dish and 3 Petri dishes for a repetition. For each treatment, the ratio of shooting explants, the number of shoots per explant, and average shoot length were recorded. 2.2.3. Experiment 3: Study on the effects of different media on root induction. This experiment aimed to find out the best medium for root induction. 4-cm-and-above shoots were subcultured in various media for rooting There were totally 3 treatments with 3 repetitions. R1: MS (Basal salt mixtures)+ 0.5mg/l IAA. 3 R2: 1/2MS (Basal salt mixtures)+100mg/l activated charcoal. R3: MS (including vitamins)+0.5mg/l IAA. There were 4 explants cultured on each Petri dish and 3 Petri dishes for a repetition. For each treatment, the ratio of plantlets emerging roots, the number of roots per plantlet, and average root length were recorded. 2.2.4. Experiment 4: Acclimatization This experiment was to harden the rooted plants from in vitro environment to nursery. After 50 days cultured on the rooting medium, rooted plants were taken out of the bottles and removed agar under tap water. The plantlets were then transplanted to plastic glasses containing mixture of soil, sand and decomposed rice straw (1:1:1 v/v/v). During the first week, plantlets were covered by plastic bags and kept under well-managed conditions (temperature 27±1oC, light intensity 1000lux, 16-hour illumination per day). In the second week, plastic bags were bored to allow air flow passing inside. From the third week, non-covered plantlets were transferred to the greenhouse. The survival rate was recorded after 3 weeks. 2.2.5. Statistical method Data were stored in Microsoft Office Excel 2003 and analyzed by Statgraphics Centurion XV. 4 CHAPTER 3 RESULTS AND DISCUSSION 3.1. Experiment 1: Study on the effects of different media on callusing of stevia leaves. Among 6 treatments, only 3 of them formed callus after 3 weeks cultured. In table 4, it could be clearly seen that S5 was the most effective formula with 92.6% of explants developing profuse yellow calli, statistically different from the others at 95% confidence level. Under stereoscopic magnifier (10x1.6x1), differentiation to form globular structures was occurring quite well. S1 resulted in the growth of white calli but the amount was poor and only 1/3 of explants responded. In addition, observation under stereoscope showed no differentiation. Following S1, S4 was the last treatment, of three, which got just 3.7% of explants forming callus. Although the colour of calli in this treatment was nearly the same as that of S5, yellow, the amount in the latter was far less than in the former and gathered mainly at the leaf edges. 5 Table 4. The results of callusing experiment after 21 days. Treatment Callus CV (%) Note responded (%) S1 33.33b 11.115 Poor white callus S2 0.00d - Unswollen leaves S3 0.00d - Unswollen leaves S4 3.70c 0 Poor yellow callus S5 92.60a 6.92072 S6 0.00d - Profuse yellow callus Swollen leaves a , b, c, d: means followed by the same letters in the same column were not significant difference (p[...]... decreased to 82.14% as being transplanted to mixture including 1 soil: 1 sand: 1 farm yard manure) 16 CHAPTER 4 CONCLUSIONS AND SUGGESTIONS 4.1 Conclusions The research Investigation of effective media for in vitro propagation of Stevia rebaudiana Bertoni was carried out and successfully obtained the complete process for plant tissue culture of Stevia MS medium (including vitamins) supplemented with... Newly Introduced Sweetener Plant (Stevia rebaudiana Bertoni. ) in Bangladesh American-Eurasian Journal of Scientific Research 2(2): 121-125 Das, A., S Gantait and N Mandal 2011 Micropropation of an Elite Medicinal Plant: Stevia rebaudiana Bert International Journal of Agricultural Research 6(1): 4048 Das, K., R Dang and P E Rajasekharan 2006 Establishment and maintenance of callus of Stevia rebaudiana Bertoni. .. Micropropagation of stevia Int J Sustain Crop Prod 3(4):1-9 Jitendra, M., S Monika, S.D Ratan, G Priyanka, S Priyanka and D.J Kiran 2012 Micropropagation of an Anti diabetic Plant- Stevia rebaudiana Bertoni, (Natural Sweetener) in Hadoti Region of South-East Rajasthan, India Journal of Biological Sciences 1(3): 37-42 Ojha, A., V.N Sharma and V Sharma 2010 An efficient protocol for in vitro clonal propagation. .. phytohormones in order to find out more effective media for the callusing of stevia leaves as well as good maintenance of callus The research in shoot induction from callus, an important stage in propagation via callus, need to be carried out to comple the whole process 17 REFERENCES Vietnamese Danh Xuyên 2010 Khảo sát hiệu quả tái sinh của cà chua (Lycopersicum esculentum Miller) in vitro Luận văn... nghệ Sinh học Viện Nghiên cứu và Phát triển Công nghệ Sinh học Trường Đại học Cần Thơ English Abd Alhady, M.R.A 2011 Micropropagation of Stevia rebaudiana Bertoni A new Sweetening Crop in Egypt Global Journal of Biotechnology & Biochemistry 6 (4): 178-182 Ahmed, M.B., M Salahin, R Karim, M.A Razvy, M.M Hannan, R Sultana, M Hossain and R Islam 2007 An Efficient Method for in vitro Clonal Propagation of. .. propagation of natural sweetener plant (Stevia rebaudiana Bertoni) African Journal of Plant Science 4(8): 319-321 Sairkar, P., M.K Chandravanshi, N.P Shukla and N.N Mehrotra 2009 Mass production of an economically important medicinal plant Stevia rebaudiana using in vitro 19 propagation techniques Journal of Medicinal Plants Research 3(4): 266-270 Saad, A.I.M and A.M Elshahed, 2012 Recent Advances in Plant in. .. (2010) to obtain callus from in- vitro- grown tomato leaves; 85.53% was recorded but no explants responded as carrying on stevia Such variations in the results may be due to the endogenous phytohormone contents in plants, their uptake, type of Auxins and Cytokinins used and their mode of action (Gupta et al., 2010) A concerned problem was the browning of callus In treatments such as S2 or S3, browning occurred... continued to increased 3.4 Experiment 4: Acclimatization After 50 days cultured in rooting media, plantlets with strong root systems were taken out and washed off all adhering agar under tap water Saplings were then transplanted into plastic glass containing mixture of 1 soil: 1 sand: 1 decomposed rice straw (v/v/v), put in laboratorial conditions (27±1oC, 1000lux light intensity, 16-hour illumination per... Sharma and S Saxena 2010 Callusing in Stevia rebaudiana (Natural Sweetener) for Steviol Glycoside Production International Journal of Agricultural and Biological Science 1(1): 30-34 Guruchandran, V and C Sasikumar 2013 Organogenic plant regeneration via callus induction in Stevia rebaudiana Bert International Journal of Current Microbiology and Applied Sciences 2(2): 56-61 Hossain, M A., A H M Shamim Kabir,... used to cover saplings during the first week In the second week, plastic bags were bored to allow air flow passing inside, new plants could gradually get familiar with the ex vitro conditions At the end of the third week, plantlets were moved to greenhouse and 15 totally removed plastic bags Maximum survival rate (100%) was obtained after 3-week hardening After attaining the rapid in vitro multiplication