Investigation of effective media for in vitro propagation of stevia rebaudiana bertoni

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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<0.05). Figure 1. Callus formed in treatment S5. (MS (including vitamins)+0.2mg/l NAA+0.15mg/l BAP) (observed under stereoscope at magnificent 10x1.6x1). 6 The combination of 2mg/l BAP and 1mg/l Kinetin used to be applied by Sairkar et al. (2009) in order to produce calli from stevia leaves. The results, nevertheless, were much different from this research, 88% compared to 33.33%. BAP and IAA were supplemented to MS (including vitamins) by Danh Xuyen (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 at earlier time, the end of second week, while it could be delayed to fourth week in S5. According to study of Gupta et al. (2010), MS medium with 2mg/l NAA was used for callus multiplication. However, all subcultured calli rapidly darkened and died after 1 week. Furthermore, addition of activated charcoal was applied as many studies done. Activated charcoal plays role in adsorption of inhibitory compounds from the medium, adsorption of growth regulators from the culture medium or darkening of the medium (Saad and Elshahed, 2012). In this research, activated charcoal was added to the medium at concentration 3g/l and all the other components unchanged. The results showed that activated could not maintain the callus growth since all explants continued to turn brown and died out. The maintenance of callus plays an important role in in vitro propagation through callusing. Good maintenance makes sure that globular structures have enough time to develop totally, creates a background for better shoot induction in the next 7 stage. In the research of Das et al. (2006), NAA and BAP added to 1/2MS medium resulted in good callus nourishment but the concentrations were used at 1mg/l and 1-2mg/l, respectively, much higher than 0.2mg/l and 0.15mg/l in this experiment. 3.2. Experiment 2: Study on the effects of different media on the multiplication of shoot from nodal segments. 100% of explants gave rise to new shoots after 3 days cultured in all 3 treatments. From table 5, the largest number of shoots per explant (3.24 shoots/explant) was found in treatment C3, significantly different from C1 and C2 (2.24 and 2.14, respectively) at 95% confidence level. Regarding the shoot length, however, C2 was the best with 1.15cm/shoot, much higher than C1 (0.36cm) and C3 (0.8cm). Furthermore, observation under stereoscope at magnification 1.6x10x0.65 clearly showed the morphological difference between 3 treatments. C2 possessed the most natural-like appearance (hairy green obovate leaves) while C3 developed unhairy bright green oblanceolate leaves. Although C1 also had obovate leaves, the leaf size was smaller than C2’s with yellowish green colour (figure 3). 8 Table 5’. The results of shoot multiplication after 21 days. C1 Number of 2.14 C2 b 2.24 C3 b 3.24a shoots/explant CV (%) Shoot length 7.89489 4.14186 8.28748 0.36c 1.15a 0.80b 11.1111 19.6624 2.61297 (cm) CV (%) Note Yellowish Green and Bright green green and hairy shoots, and unhairy unhairy shoots, obovate leaves shoots, narrowed oblanceolate obovate leaves leaves a b c , , : means followed by the same letters in the same row were not significant difference (p<0.05). 9 C3 C1 C2 Figure 2. Shoots developed from nodal segments in 3 treatments. (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). C1 C2 C3 Figure 3. Shoots from 3 treatments under stereoscope at magnification 1,6x10x0,65. (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) 10 Hossain et al. (2008) reported that MS medium added with 1mg/l BAP resulted in 1.8shoots/explant and 7.25-cm average length. At the concentration 3mg/l of BAP, the number of shoot increased to 3.4±0.58 while the length dropped to 6.51±0.76 (Jitendra et al, 2012). The type of cytokinine was the most important factor affecting shoot multiplication. The highest shoot multiplication rate was obtained from single stem node segment cultured on medium supplemented with BAP. Increasing BA concentration promoted shoot multiplication (Abd Alhady, 2011). However, as comparing to the results of C3, it is clear that the increase in BAP concentration could promote the rate of shoot proliferation but there was a certain limitation. Obviously, the number of shoot slightly decreased to 3.24 shoots/explant cultured at concentration 3.5mg/l BAP in this research. Moreover, the rising of shoot number might affect the length since the nutrients taken up were distributed to more shoots. In all 3 treatments, from a single nodal segment, there were only 2 shoots developing at the node after 1 week. The third and other shoots would appear later, at the end of second week. It was seen that shoots nearly stopped growing after 5 weeks cultured, they remained stunted regardless of subculturing or not. Due to such problem, 4-cm-and-above shoots were transferred to rooting medium in order to help the plants take up nutrients more efficiently. When those plantlets got stronger, they would be ready for a new round of shoot multiplication. 11 3.3. Experiment 3: Study on the effects of different media on root induction. 4-cm-and-above shoots were subcultured in rooting media. Root emerged at the end of second week in all 3 treatments. Root length in R2 developed the most rapidly among 3 treatments. After 25 days, this was also the treatment possessing the longest roots (7.02cm in average), significantly different from R1 and R3 (2.2cm and 1.39cm, respectively). Regarding the number of root per plant, nevertheless, R1 resulted in 9.57, much larger than 1.67 in R2 and 3.5 in R3. In addition, the highest rate of saplings responded to rooting, 83.33%, was recorded in R1, compared to 58.33% and 33.33% in R2 and R3, respectively. Furthermore, there were noticeable differences in the morphology of roots from 3 treatments. R1 and R3 tended to develop thick and hairy fibrous roots while R2 gave rise to far less hairy and thin taproots (Figure 5). Lastly, in all 3 treatments, especially R1, most of the plants grew faster in height and leaf size, many auxiliary shoots were elongated. 12 Table 6. The results of root induction after 25 days. Rooting R1 R2 R3 83.33a 58.33b 33.33c 9.9964 14.2886 25.005 9.57a 1.67c 3.50b 1.62049 12.3971 9.16515 2.20b 7.02a 1.39c 3.72061 1.60035 9.47622 responded (%) CV (%) Number of roots/plant CV (%) Root length (cm) CV (%) Note Hairy, thick Less hairy, thin Hairy, thick fibrous roots taproots fibrous roots a b c , , : means followed by the same letters in the same row were not significant difference (p<0.05). 13 R1 R3 R2 Figure 4. Roots in 3 treatments after 25 days. (R1: MS basal salt mixtures+0.5mg/l IAA, R2: 1/2MS basal salt mixtures+100mg/l activated charcoal, R3: MS including vitamins+0.5mg/l IAA). R1 R2 R3 Figure 5. Roots in treatments observed under stereoscope at magnification 1.6x10x4. 14 (R1: MS (basal salt mixtures)+0.5mg/l IAA, R2: 1/2 MS (basal salt mixtures)+100mg/l activated charcoal, R3: MS (including vitamins)+ 0.5mg/l IAA) MS medium supplemented with 0.5mg/l IAA was used by Ojha et al. (2010) for rooting but the obtained results were quite higher than R1 (98.1% compared to 83.33%). Such difference can be explained by the difference in length of the shoots cultured. The results of this study were also in agreement with Hossain et al.’ (2008), MS medium showed better capability to promote root growth in both number and length compared to 1/2MS. Besides, the experiments of Hossain et al. recorded that the majority of rooting plants was rapidly died after 30-day culturing in rooting media which were supplemented with NAA or BAP. In this research, such limitation was successfully solved as the type of auxin used was changed to IAA, after 50 days, plantlets still grew well, height and leaf size 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 day). Plastic bags were 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
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