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

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

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

Student code: 3082597 Session: 34 (2008-2013)

Can Tho, 2013

APPROVAL

SUPERVISOR STUDENT

TRAN THI XUAN MAI NGUYEN THANH HUY

Can Tho, May …, 2013

PRESIDENT OF EXAMINATION COMMITTEE

NGUYEN HUU HIEP

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ABSTRACT

Stevia (Stevia rebaudiana Bertoni), a natural caloric 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)

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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

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Chapter 4: Conclusions and Suggestions 17

4.1 Conclusions 17

4.2 Suggestions 17

References 18

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

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

2.2.1 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

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

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

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

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

Table 4 The results of callusing experiment after 21 days

S5 92.60a 6.92072 Profuse yellow callus

a

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)

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

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)

Table 5’ The results of shoot multiplication after 21 days

Green and hairy shoots, obovate leaves

Bright green and unhairy shoots, oblanceolate leaves

a

difference (p<0.05)

Figure 2 Shoots developed from nodal segments in 3

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

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

Table 6 The results of root induction after 25 days.

a

difference (p<0.05)

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

(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