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Sugar Tech DOI 10.

1007/s12355-013-0228-7

RESEARCH ARTICLE

In Vitro Propagation and Synthetic Seeds Production: An Efficient Methods for Stevia rebaudiana Bertoni
Ahmed Abbas Nower

Received: 13 March 2013 / Accepted: 7 June 2013 Ó Society for Sugar Research & Promotion 2013

Abstract Germplasm can be effectively stored in the form of synthetic seeds. Shoot tips obtained from in vitro shoot cultures of Stevia rebaudiana Bertoni were encapsulated in 4 % calcium alginate. The present work discussed the role of components of culture medium on morphogenic response of Stevia rebaudiana Bertoni encapsulated buds to various MS strengths and sugar alcohol (Mannitol or sorbitol) in different concentrations for long term storage. Germination ability of the synthetic seeds was investigated. Shoots were regenerated from nodal explants of Stevia through axillary shoot proliferation. The induction of multiple shoots from nodal segments was the highest in MS medium supplemented with 1.0 mg l-1 BA. Maximum shoot formation was obtained with fructose at 20 and 40 g l-1 fructose, while with fructose at 20 g l-1 gave the highest leaves number/explants). The longest shoot length obtained with sucrose and fructose than other sugar. Different media type (NN and WPM) were suitable for best shoot number of Stevia, leaf number and shoot length than other media. Growth of shoot was increased and observed in capsules stored for 5 weeks on MS than other MS strengths. The growth of capsules dependent on mineral concentration and storage time. The most suitable conversions of capsules was using 0.05 M mannitol after 6 weeks from storage of synthetic seeds of Stevia. For rooting, when Stevia shoots cultured on MS medium supplemented with IAA, at 0.2 mg l-1 resulted in the maximum number of roots/ explant while, IBA at 1.0 and 2.0 mg l-1 resulted in longest root/plant and gave the same length of root.
A. A. Nower (&) Department of Plant Biotechnology, Genetic Engineering and Biotechnology Research Institute (GEBRI), Menofyia University, Sadat City, Egypt e-mail: Ahmed_newer@yahoo.com

Keywords Germplasm conservation Á Multiplication Á Rooting Á Stevia rebaudiana Bertoni Á Synthetic seeds

Introduction Stevia rebaudiana Bertoni, is a perennial herb belongs to the Asteraceae family. It is a natural sweetener plant known as ‘‘Sweet Weed’’, ‘‘Sweet Leaf’’, ‘‘Sweet Herbs’’ and ‘‘Honey Leaf’’, which is estimated to be 300 times sweeter than cane sugar (Chalapathi and Thimmegowda 1997; Liu and Li 1995). The leaves of stevia are the source of diterpene glycosides, viz. stevioside and rebaudioside (Yoshida 1986). Stevioside is regenerated as a valuable natural sweetening agent because of its relatively good taste and chemical stability (Yamazaki and Flores 1991; Toyoda and Matsui 1997). An interesting feature of this plant is intense sweetness of leaves and aqueous extracts. Steviosides, sweet crystalline diterpene glycosides, which gives sweet taste to the plant are noncaloric and 200–300 times sweeter than sugar (Midmore and Rank 2002). Diet conscious and diabetic persons with hyperglycemia can use steviosides as an alternative sweetener (Din et al. 2006). Stevioside can also be used as an antihyper glycaemic (Gregersen et al. 2004), antihypertensive (Ferri et al. 2006), anti-tumor (Yasukawa et al. 2002) drug. Seeds of Stevia show a very low germination percentage (Felippe and Lucas 1971, Toffler and Orio 1981) and vegetative propagation is limited by lower number of individuals (Sakaguchi and Kan 1982). Micropropagation which can provide genetically uniform plants in large numbers appears as an alternative technique for rapid multiplication of Stevia plants (Sairkar et al. 2009). The highest per cent response (92.10 %) was noticed on MS medium containing 1.0 mg l-1 BAP and 0.5 mg l-1 IBA, and thereafter the nodal explants produced approximately two

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2008). glucose. Synthetic seed technology can be considered as effective alternative conservation technique for endangered rare plants like Stevia which using effectively to conservation and micropropagation explants. spices. medicinal plants and wood yielding forest trees etc. orchids. and 121 °C for 20 min. The potential advantages of this technology include genetically identical. Effect of Different MS Salt Strengths on Capsules Germination Finally. The explants were then inoculated aseptically into MS medium (Murashige and Skoog 1962). Synthetic seed consists of encapsulation matrix such as sodium alginate and vegetative part of plant like shoot tips. long term storage and low cost of production (Ghosh and Sen 1994). 30. ’. and MS media supplemented with 0. The droplets containing the explants were held for at least 30 min to achieve polymerization of the sodium alginate. The cultures were incubated at a constant temperature of 25 ± 1 °C and 16 h photoperiod (2. 0.1 % mercuric chloride for 5 min followed by rinsing them five times with double distilled water inside the Laminar Air flow chamber. 0. industrially important crops. Effect of Sugar Types and Concentrations on Shoot Regeneration Shoot tips about 2–4 mm in length were cultured on MS media supplemented with 1 mg l-1 BA and different sugar types (sucrose.0. (Chandrasekhara Reddy et al.1 m long) and then were treated with 1 % savlon for 5–6 min with constant shaking and washed thoroughly with distilled water. However. plantation crops. axillary buds and somatic embryos. The explants were cut into small pieces (about 0.5 mg l-1) adding 30 g l-1 sucrose and 0. The culture vessels (350 ml) were incubated at 25 ± 1 °C and 16 h photoperiod. the artificial seeds were cultivated in a germination medium in jars (250 ml) contained . DKW (Chu 1978). 123 . The pH of the medium was adjusted to 5. LS (Driver and Kuniyuki 1984). sorbitol or mannitol) and various concentrations (15. 2006).7 before adding 7 g l-1 agar. Encapsulation of germplasm along with micropropagation can be used for in vitro conservation of endangered species (West et al. ornamental plants. The culture vessels (350 ml) were incubated at 25 ± 1 °C and 16 h photoperiod. The synthetic seed technology was developed in different economically important plant species such as vegetable crops. 2012).5. Synthetic seed technology is an advancement in micropropagation (Naik and Chand 2006).Sugar Tech shoots within 2 weeks (Laribi et al. Storage of Synthetic Seeds Materials and Methods The explant nodal segments were collected from the farm of GEBRI. ‘. N6 or (Lloyd and McCown 1980) WPM supplemented with 1 mg/l BA and 30 g l-1 sucrose.7 % Difco Bacto-agar. The pH of the medium was adjusted to 5. virus free germplasm.25.1 NaOH before autoclaving at 1. Subcultures were done every 21 days interval. beads were collected and rinsed with sterile distilled water to wash away Ca (NO3)2Á4H2O residues.7 with 0. 1 and 1. Effect of Different Media on Shoot Regeneration Shoot tips about 2–4 mm in length were cultured on MS (Nitsch and Nitsch 1969). Encapsulation was accomplished by mixing the shoot tip explants into the alginate and dropping these into the Ca (NO3)2Á4H2O solution. NN (Linsmier and Skoog 1965). The main objective of this study was to develop efficient system for synthetic seed production and storage of Stevia because it is don’t gave seeds under Egyption conditions and germination it into plants for further propagation.25 mg l-1 BA and 7 g l-1 agar. ease in transportation. Nodal segments from the proliferated shoots were subcultured again for further multiple shoot induction.06 kg cm G2 Artificial Seeds Preparation Sodium alginate (4 %) was prepared using MS liquid medium and 100 mM of Ca (NO3)2Á4H2O solution for complexation and autoclaved. They were then left in the growth chamber at a temperature of 20 °C in complete darkness. forage legumes. Synthetic seed technology is most effective alternation for the conservation of those plant species which produce non viable seeds and difficult to propagate by other means (Daud et al. axillary buds and shoot tips can also be used for synthetic seeds (Sarka and Naik 1998). They were then surface sterilized with 0. 45 or 60 g l-1). The pH of the medium was adjusted to 5. The formation of shoots was determined after 4 weeks of culture. Effect of Some Plant Growth Regulators on Shoot Regeneration Shoot tips cultured in MS medium at concentrations of growth regulators benzyl adenine (BA).000 lux). kinetin (KN) and Thidiazuron (N-pheny1-N0 -1. 2012).3-thiadiazol-5yl-urea (TDZ) (0.7 before adding 7 g l-1 agar. fruit crops.2. The formation of shoots was determined after 4 weeks of culture. Data were taken after 6 weeks. cereals.

and 0.50 18.87 3.25 mg l-1 BA and 7 g l-1 agar. Maximum number of leaves obtained at the concentration of 1.5 LSD at 5 % level 4. The growth limitation has been achieved by mannitol or sorbitol.50 81.Sugar Tech Effect of Sugar Alcohol (Mannitol or Sorbitol) on Capsules Germination Attempts in the in vitro storage of synthetic seeds have focused on imposing some sort of stress on the cultures in order to achieve reduced growth.75 13.50 1.25 22. 0. Shoot length (cm) BA 0. Root Induction Regenerated multiple shoots were cut and individual shoots were placed in 350 ml jars MS medium containing different concentrations 0. All experiments were designed in factorial design and data were compared according to method described by Snedecor and Cochran (1989).25 6. 0.25 1.2.00 5.0 1.37 5.5 or 1. Since the germplasm remains viable.5 mg l-1 TDZ and 1.12 7.00 8.2. 0.00 52. Layout of the Experiments The all experimental design was completely randomized with 5 replicates in treatment each replicate had 5 explants (shoot tip or artificial seeds). 1.25 3.00 16.0.00 3.50 15. Surpassed shoot length on MS medium supplemented with 0.00 112.50 1.37 Results and Discussion Effect of Some Plant Growth Regulators on Shoot Regeneration The effect of different plant growth regulators on shoot proliferation has shown in Table 1 and Fig. (mg l-1) Shoot no.59 4.50 22.50 13.0 mg l-1 (22.00 8.00 47.00) compared to the other concentrations.00 2.50 1.05 level Fig.25 4.0 and 2 mg l-1 of IBA.25 0. 0. it can be grown again into shoots.00 6.75 4. rebaudiana Cytokinin conc.37 cm respectively) than Mean values followed by the same letter are not significantly different at ** p \ 0. 1 Effect of different concentrations of BA on shoot proliferation of S. Table 1 Effect of some plant growth regulators on shoot proliferation of S.25 0.00 59.87 7. Capsules were cultured on MS medium supplemented with mannitol or sorbitol at 0.25 20. They were kept in the culture chamber at a temperature of 20 °C in complete darkness for 6 weeks to slow growth.0 1.25 5.3 M.500 19. However.05. lower responses were obtained with TDZ at all concentrations. Finally. Leave no.0 0.00 62. the higher percentage of cultures producing shoot number were achieved when placed on media with higher concentrations of BA at 0.00 24.5 TDZ 0.50 89.1. the artificial seeds were cultivated in a germination medium in 250 ml jars on MS medium supplemented with 0.0 1.0 mg l-1 KN (81.50 57.25 and 7. In general. 1. 0.25 0.75 respectively).075 4.00 5.5 KN 0.5 mg l-1 KN (8.5.05 according to LSD test: F-test significant at 0.410 31. rebaudiana 123 .1.75 72. NAA and IAA for root induction.50 12.25 and 22.75 5.

80). the result showed that NN medium was suitable for formation number of shoot (21. DKW and WPM solid medium containing 1 mg l-1 BA and 30 g sucrose were tested.80) and shoot length (7. rebaudiana Media type N6 NN MS LS DKW WPM LSD at 5 % Shoot no.00 85.00 8.66 respectively). 2).00 and 86. 2010).10 1.40 21. some beads appeared to be dry and did not generate while some of the others did not regenerate Mean values followed by the same letter are not significantly different at p \ 0.80 34. Maximum shoot formation was obtained with fructose at 20 and 40 g l-1. Effect of Different Sugar Types on Regeneration The effect of different types and concentrations of sugar on regeneration of Stevia.10 4. MS.20 7. The highest number of shoots (21. which is in agreement with the finding of (Murashige and Skoog 1962.33) (Table 3).029 Effect of Different MS Salt Strengths on Capsules Germination of S. while with sucrose and fructose at 20 g l-1 gave the highest leaves number (84.60 41. ‘ MS or  MS) on growth ability of stored encapsulated stevia buds.25 shoots). After storage. leaf number (85.60 6. leaves number and shoot length. MS medium was found to be superior to WP medium in terms of shoot numbers and shoot length of Dalbergia sissoo Roxb (Thirunavoukkarasu et al. rebaudiana This investigation was performed to study the effect of different salts strengths of MS medium (MS.40 11. The number of shoot obtained with glucose and fructose were 12. length and number of leaves was recorded on LS and DKW media (Fig.05 according to LSD test: F-test significant at 0.25 mg l-1) combination was superior for multiple shoot bud induction (4. Sucrose is the carbohydrate of choice as carbon source for in vitro plant culture.60 3. Thorpe 1982.33. LS. shoot length of Stevia (5.33 and 13.36 ± 0. 11. Lemos and Baker 1998. (2012) found that BAP (1 mg l-1) and IAA (0.05 level Fig.40).20 3.00 14. Among the cytokinins tested. Germplasm Conservation Table 2 Effect of different media on shoot proliferation of S. BAP was provided to be the most efficient cytokinin for multiple shoot regeneration (Taiyagarajan and Venkatachalam 2012). 2011).12 Shoot length (cm) 5. probably because it is the most common carbohydrate in the phloem sap of many plants.80 cm) than other media.40 26. 2000). Data of the main effect of different media type in Table 2. Effect of Media Type on Shoot Proliferation After 4 weeks of culture of Stevia on N6.80 8.80 21. 3). Fuentes et al. rebaudiana 123 . The results have shown that different sugar type and concentrations had a significant effect on the regeneration of plants.55) was also obtained on MS medium supplemented with 4 % fructose (Preethi et al.678 Leave no.4) and leaves number (68. The minimum number of shoots. respectively (Fig. 41. Laribi et al. Our results are in accordance with Hossain et al.4 ± 0. Increasing in sugar concentration resulted in decrease in shoot. (2008) who found that BA (1. BA was more effective than either KN or TDZ. ’ MS.20 7. NN. 2 Effect of different media on shoot proliferation of S.0 mg l-1) was superior to all other hormonal treatments for shoot proliferation.Sugar Tech other treatments. MS medium supplemented with sucrose gave the highest number of shoots (11.80 5.

00 3.42 3.33 2.33 9.30 0.8555 4.53 %) compared to quarter MS medium salt strength which had low growth of shoots.66 3.46 0.52 A = 0.16 3.33 41.20 4.00 68.08 10. Data on the main effect of the beads stored indicated that the growth of shoot was increased and observed in beads stored for 5 weeks (79.00 67.33 10.66 35.93 A 9 B = 1.33 8.33 26.83 2.66 26.33 3.33 %) than other treatments.66 13.913 11.9111 5.16 5.04 4.16 3.66 1.00 17.80 3.00 86.33 2.66 3.66 11.06 B = 0.00 3.16 3. the growth of beads was dependent on salt strength and storage time as shown in Table 4. rebaudiana Concentration (g l-1) 20 Shoot length (cm) Sucrose Glucose Fructose Sorbitol Mannitol Mean (B) LSD at 5 % level Number of shoot Sucrose Glucose Fructose Sorbitol Mannitol Mean (B) LSD at 5 % level Number of leaves Sucrose Glucose Fructose Mean values followed by the same letter are not significantly different at p \ 0.46 A 9 B = 0.22 2.66 34.46 B = 0.66 13.33 2.00 3.00 1.08 7.66 2.66 38.05 level Sorbitol Mannitol Mean (B) LSD at 5 % level 84. Generally. As for as the interaction is concerned.95 40 60 80 Mean (A) Sugar types (A) Fig.53 A = 0.08 5.33 21.Sugar Tech Table 3 Effect of different sugar types and concentrations on shoot proliferation of S.8555 31.00 2.41 7.04 B = 0.66 23.26 A 9 B = 1.33 2.9565 36.05 according to LSD test: F-test significant at 0.00 12.33 12. 3 Effect of sucrose concentrations on shoot proliferation of S.16 2.33 38.33 1.33 0.4075 3.4556 5.50 19.33 3.66 3.16 3.00 5.33 3.00 27.00 5.33 12. rebaudiana although they appeared to be healthy and still green.66 5.33 13.33 3.33 35.9565 6.913 68.83 4.66 3.19 1.66 6.66 7. our presented results concerning the effect of different MS salt strengths and storage time after 5 weeks appeared better for encapsulated buds when 123 . The effect of full MS medium salt strength significantly showed higher percentage of shoot growth (69.46 A = 0.33 3.43 3.00 1.33 3.41 3.

00 2.3 % in A3 (MS ? BAP ? Kinetin (1.00 0.077 6 Mean (B) Mannitol 0.00 0.06 55.05 1.00 3.00 0.00 0.00 0.33 55.66 60.00 0.66 78.00 0.00 61. rebaudiana Sugar alcohol (M) (A) Period after Capsules germination % culture 1 2 3 4 5 (week) 0.33 72.86 57.00 0.579 50.00 30.00 0. 2010). Nower et al.00 2.05 level Solanum germplasm storage.00 0.00 Mean (A) LSD at 5 % level A B A9B 0.00 1.00 0.00 0. (2007) showed that the frequency of conversion pear encapsulated buds into plantlets was affected by MS strengths.00 40.289 4.00 0.00 Sorbitol 0. Effect of Sugar Alcohol (Mannitol or Sorbitol) on Capsules Germination The addition of osmotic agent mannitol at 0.05 according to LSD test: F-test significant at 0. The most suitable to conversions of capsules was 0.00 0.66 0.00 0.73 Mean values followed by the same letter are not significantly different at p \ 0.00 69.05 1.00 0.00 3.00 79.00 0. A decline in survival and re-growth occurred on culture storage at higher concentrations of osmotic agents 3 M mannitol and 2 and 3 M sorbitol.048 2.441 4.00 0.00 0. followed by 90 % in A1 (MS medium) and 83.66 36. Hemant et al. however.66 72.0 mg l-1 BAP). The use of the osmotic agents was not an efficacious procedure.66 62.00 0.44 2. rebaudiana was observed (100 %) in A2 medium (liquid MS medium containing 1.00 0.22 0.00 44. Maximum conversion of shoot tips and nodal segments of S.00 0. 4).00 0.00 0.00 0.66 34. although with few applications on in vitro storage.00 26. with deterioration of cultures in terms of quality of growth.00 0.Sugar Tech Table 4 Effect of MS Salts Strengths on capsules germination of S.00 0.00 13. In this respect.00 13.33 64.00 76. rebaudiana cultured on MS full salt strength which gave the highest conversion percentage (100 %) compared with other treatments (Fig.00 0.00 25.83 Mean values followed by the same letter are not significantly different at p \ 0.66 58.00 0. rebaudiana after different culture period in vitro Culture Period (week) Capsules germination % 1 2 3 4 5 Mean (A) MS strengths MS ’ MS ‘ MS  MS Mean (B) LSD at 5 % A B A9B 2.05 according to LSD test: F-test significant at 0.00 0.00 40. These results corroborate with Westcott’s (1981) descriptions on the toxic effects of mannitol in Table 5 Effect of sugar alcohol (Mannitol or Sorbitol) on capsules germination of S.66 58.00 0.00 0.00 0.00 0.25 mg l-1) medium (Ali et al.33 0.00 0.00 55.00 0.53 61.00 0.05 M mannitol after 6 weeks from storage of synthetic seeds of Stevia (Table 5).00 0.91 62.00 60.00 9.44 5. The research in synthetic seed technology has been prolific.66 100.00 0.66 64.00 0.00 0.0 ? 0.00 0.66 0.05 level Fig.05 M to the medium increased culture survival.00 0.00 80. (2010) 123 .664 1.00 4.33 0.00 0.00 60.00 56.33 69. 4 Effect of MS strengths on germination of capsules of S.

or sorbitol to the media. (2012) adding sorbitol or mannitol to the media at 0.00 3.0 2.73 0. IBA 0. Results revealed that.00 23.33 2.33) while.46 1. Rizkalla et al. rebaudiana 123 .66 %) and number of root per shoot (12.0 2.33 10.0 NAA 0.66 11.00 22.00 12.67 18.33 4.33 5.00 13.67 14.00 8.1 0.73 0.057 14.66 2.66 1.33 8. NAA and IAA) on in vitro rooting of S. rebaudiana Auxin conc.33 4.66 0. The effect of auxin concentrations was observed where.00 2.33 0. even Fig.1 0. the greatest length of plant was obtained on MS medium free auxin (14.50 m7 l-1). mannitol.00 3.66 7.67 12.66 6.0 2. NAA and IAA) efficiently stopped shoot multiplication and supported nearly 100 % rooting.33 18.30 2.53 0.33 3.00 14.33 23.0 and 2.66 6.1 mg l-1).5 1. IBA at 1. The greatest number of leaves observed with IAA at 0.00). IBA at 1 or 2 mg l-1 resulted in the tallest root as shown in Table 6. stevia shoots cultured on MS medium supplemented with IAA.088 2.43 0. (2007) who found that the highest rooting percentage (97.2 mg l-1 (26.2 mg l-1 resulted in the greatest number of roots/explant (10.33 26.00 12.67 3.33 13.0 IAA 0.0 mg l-1 resulted in longest root/shoot and gave the same length of root (Fig. it could be concluded that. Laribi et al.5 1.251 18.33 4.33 who showed that in vitro storage of shoot cultures was also evaluated by supplementing osmotic agents.33 12.43 0.33 4.00 11.33 cm) than the other treatments.00 10.67 12.2 0.333 7.33 2.1 0.2 0.00 15.00 15.5 1.66 1. at 0.0 0. 5). These results are in harmony with those of Ahmed et al.543 2. Such treatment had a negative impact on post-storage re-growth (at 25 °C).67 7.33 9.67 13.67 14.10 root/shoot) in Stevia rebaudiana were noted on MS medium fortified with IAA but with less low concentration (0. In general.0 lsd AT 5 % level 12. 5 Development and root induction of S.56 0.33 5.2 0.70 cm) were observed on MS medium fortified with IAA (0. (mg l-1) Root formation Plant length (cm) No of leaves No of roots Root length (cm) though the inclusion of 2 % w/v sorbitol and mannitol each to the media increased plantlet survival during 10 °C storage treatment.33 12. (2012) showed that shoots produced roots within 2 weeks of culture.05 M increased plantlet survival of sugar beet synthetic seeds (Francesca and Toro).Sugar Tech Table 6 Effect of different concentrations (mg l-1) IBA. Root Induction Auxins (IBA.00 3.90 root/shoot) as well as the root length (1.53 1. the highest percent (97 %) of root induction and maximum number of roots (8.00 4.93 0.00 11.

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