Sugar Tech DOI 10.

1007/s12355-013-0228-7

RESEARCH ARTICLE

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

Received: 13 March 2013 / Accepted: 7 June 2013 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 Multiplication Rooting Stevia rebaudiana Bertoni 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 200300 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, Tofer 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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shoots within 2 weeks (Laribi et al. 2012). Synthetic seed technology is an advancement in micropropagation (Naik and Chand 2006). Encapsulation of germplasm along with micropropagation can be used for in vitro conservation of endangered species (West et al. 2006). Synthetic seed consists of encapsulation matrix such as sodium alginate and vegetative part of plant like shoot tips, axillary buds and somatic embryos. However, axillary buds and shoot tips can also be used for synthetic seeds (Sarka and Naik 1998). Synthetic seed technology is most effective alternation for the conservation of those plant species which produce non viable seeds and difcult to propagate by other means (Daud et al. 2008). The potential advantages of this technology include genetically identical, virus free germplasm, ease in transportation, long term storage and low cost of production (Ghosh and Sen 1994). The synthetic seed technology was developed in different economically important plant species such as vegetable crops, forage legumes, industrially important crops, cereals, spices, plantation crops, fruit crops, ornamental plants, orchids, medicinal plants and wood yielding forest trees etc. (Chandrasekhara Reddy et al. 2012). 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. The main objective of this study was to develop efcient system for synthetic seed production and storage of Stevia because it is dont gave seeds under Egyption conditions and germination it into plants for further propagation.

and 121 C for 20 min. The cultures were incubated at a constant temperature of 25 1 C and 16 h photoperiod (2,000 lux). Subcultures were done every 21 days interval. Nodal segments from the proliferated shoots were subcultured again for further multiple shoot induction. Effect of Different Media on Shoot Regeneration Shoot tips about 24 mm in length were cultured on MS (Nitsch and Nitsch 1969), NN (Linsmier and Skoog 1965), LS (Driver and Kuniyuki 1984), DKW (Chu 1978), N6 or (Lloyd and McCown 1980) WPM supplemented with 1 mg/l BA and 30 g l-1 sucrose. The pH of the medium was adjusted to 5.7 before adding 7 g l-1 agar. The culture vessels (350 ml) were incubated at 25 1 C and 16 h photoperiod. The formation of shoots was determined after 4 weeks of culture. Effect of Sugar Types and Concentrations on Shoot Regeneration Shoot tips about 24 mm in length were cultured on MS media supplemented with 1 mg l-1 BA and different sugar types (sucrose, glucose, sorbitol or mannitol) and various concentrations (15, 30, 45 or 60 g l-1). The pH of the medium was adjusted to 5.7 before adding 7 g l-1 agar. The culture vessels (350 ml) were incubated at 25 1 C and 16 h photoperiod. The formation of shoots was determined after 4 weeks of culture. Storage of Synthetic Seeds

Materials and Methods The explant nodal segments were collected from the farm of GEBRI. The explants were cut into small pieces (about 0.1 m long) and then were treated with 1 % savlon for 56 min with constant shaking and washed thoroughly with distilled water. They were then surface sterilized with 0.1 % mercuric chloride for 5 min followed by rinsing them ve times with double distilled water inside the Laminar Air ow chamber. The explants were then inoculated aseptically into MS medium (Murashige and Skoog 1962). Effect of Some Plant Growth Regulators on Shoot Regeneration Shoot tips cultured in MS medium at concentrations of growth regulators benzyl adenine (BA), kinetin (KN) and Thidiazuron (N-pheny1-N0 -1,2,3-thiadiazol-5yl-urea (TDZ) (0.0, 0.25, 0.5, 1 and 1.5 mg l-1) adding 30 g l-1 sucrose and 0.7 % Difco Bacto-agar. The pH of the medium was adjusted to 5.7 with 0.1 NaOH before autoclaving at 1.06 kg cm G2

Articial Seeds Preparation Sodium alginate (4 %) was prepared using MS liquid medium and 100 mM of Ca (NO3)24H2O solution for complexation and autoclaved. Encapsulation was accomplished by mixing the shoot tip explants into the alginate and dropping these into the Ca (NO3)24H2O solution. The droplets containing the explants were held for at least 30 min to achieve polymerization of the sodium alginate, beads were collected and rinsed with sterile distilled water to wash away Ca (NO3)24H2O residues. Effect of Different MS Salt Strengths on Capsules Germination Finally, the articial seeds were cultivated in a germination medium in jars (250 ml) contained , , , and MS media supplemented with 0.25 mg l-1 BA and 7 g l-1 agar. They were then left in the growth chamber at a temperature of 20 C in complete darkness. Data were taken after 6 weeks.

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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. The growth limitation has been achieved by mannitol or sorbitol. Since the germplasm remains viable, it can be grown again into shoots. Capsules were cultured on MS medium supplemented with mannitol or sorbitol at 0.05, 0.1, 0.2, and 0.3 M. Finally, the articial seeds were cultivated in a germination medium in 250 ml jars on MS medium supplemented with

0.25 mg l-1 BA and 7 g l-1 agar. They were kept in the culture chamber at a temperature of 20 C in complete darkness for 6 weeks to slow growth. Root Induction Regenerated multiple shoots were cut and individual shoots were placed in 350 ml jars MS medium containing different concentrations 0.0, 0.1, 0.2, 0.5, 1.0 and 2 mg l-1 of IBA, NAA and IAA for root induction. Layout of the Experiments The all experimental design was completely randomized with 5 replicates in treatment each replicate had 5 explants (shoot tip or articial seeds). All experiments were designed in factorial design and data were compared according to method described by Snedecor and Cochran (1989).

Table 1 Effect of some plant growth regulators on shoot proliferation of S. rebaudiana Cytokinin conc. (mg l-1) Shoot no. Leave no. Shoot length (cm) BA 0.0 0.25 0.50 1.0 1.5 KN 0.25 0.50 1.0 1.5 TDZ 0.25 0.50 1.0 1.5 LSD at 5 % level 4.00 6.00 2.75 4.00 3.410 31.00 52.500 19.50 12.50 18.59 4.00 8.25 3.87 3.25 1.075 4.50 15.50 22.25 22.75 13.00 8.50 13.00 16.25 20.00 24.75 72.50 89.00 59.00 62.00 47.50 57.50 81.00 112.00 5.25 5.25 6.75 5.37 5.25 4.00 5.87 7.12 7.37

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. 1. In general, the higher percentage of cultures producing shoot number were achieved when placed on media with higher concentrations of BA at 0.5 or 1.0 mg l-1 (22.25 and 22.75 respectively). Maximum number of leaves obtained at the concentration of 1.0 mg l-1 KN (81.00) compared to the other concentrations. However, lower responses were obtained with TDZ at all concentrations. Surpassed shoot length on MS medium supplemented with 0.5 mg l-1 TDZ and 1.5 mg l-1 KN (8.25 and 7.37 cm respectively) than

Mean values followed by the same letter are not signicantly different at ** p \ 0.05 according to LSD test: F-test signicant at 0.05 level

Fig. 1 Effect of different concentrations of BA on shoot proliferation of S. rebaudiana

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other treatments. Among the cytokinins tested, BA was more effective than either KN or TDZ. Our results are in accordance with Hossain et al. (2008) who found that BA (1.0 mg l-1) was superior to all other hormonal treatments for shoot proliferation. Laribi et al. (2012) found that BAP (1 mg l-1) and IAA (0.25 mg l-1) combination was superior for multiple shoot bud induction (4.25 shoots). BAP was provided to be the most efcient cytokinin for multiple shoot regeneration (Taiyagarajan and Venkatachalam 2012). Effect of Media Type on Shoot Proliferation After 4 weeks of culture of Stevia on N6, NN, MS, LS, DKW and WPM solid medium containing 1 mg l-1 BA and 30 g sucrose were tested. Data of the main effect of different media type in Table 2, the result showed that NN medium was suitable for formation number of shoot (21.40), leaf number (85.80) and shoot length (7.80 cm) than other media. The minimum number of shoots, length and number of leaves was recorded on LS and DKW media (Fig. 2). 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. 2010). Effect of Different Sugar Types on Regeneration The effect of different types and concentrations of sugar on regeneration of Stevia, MS medium supplemented with sucrose gave the highest number of shoots (11.4) and leaves number (68.33) (Table 3). Increasing in sugar concentration resulted in decrease in shoot, leaves number and shoot length. Maximum shoot formation was obtained with fructose at 20 and 40 g l-1, while with sucrose and fructose at 20 g l-1 gave the highest leaves number (84.00 and 86.66 respectively). The number of shoot obtained with glucose and fructose were 12.33 and 13.33, respectively (Fig. 3). The results have shown that different sugar type and concentrations had a signicant effect on the regeneration of plants, which is in agreement with the nding of (Murashige and Skoog 1962; Thorpe 1982; Lemos and Baker 1998; Fuentes et al. 2000). Sucrose is the carbohydrate of choice as carbon source for in vitro plant culture, probably because it is the most common carbohydrate in the phloem sap of many plants. The highest number of shoots (21.4 0.80), shoot length of Stevia (5.36 0.55) was also obtained on MS medium supplemented with 4 % fructose (Preethi et al. 2011). Germplasm Conservation

Table 2 Effect of different media on shoot proliferation of S. rebaudiana Media type N6 NN MS LS DKW WPM LSD at 5 % Shoot no. 11.80 21.40 11.60 6.80 8.00 8.20 3.678 Leave no. 41.00 85.80 34.40 26.40 21.60 41.00 14.12 Shoot length (cm) 5.20 7.80 5.10 4.60 3.20 7.10 1.029

Effect of Different MS Salt Strengths on Capsules Germination of S. rebaudiana This investigation was performed to study the effect of different salts strengths of MS medium (MS, MS, MS or MS) on growth ability of stored encapsulated stevia buds. After storage, 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 signicantly different at p \ 0.05 according to LSD test: F-test signicant at 0.05 level

Fig. 2 Effect of different media on shoot proliferation of S. rebaudiana

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Sugar Tech Table 3 Effect of different sugar types and concentrations on shoot proliferation of S. 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 signicantly different at p \ 0.05 according to LSD test: F-test signicant at 0.05 level Sorbitol Mannitol Mean (B) LSD at 5 % level 84.00 68.00 86.66 34.66 23.33 26.53 A = 0.9565 36.66 38.00 67.33 12.66 13.66 26.46 B = 0.8555 31.00 27.00 3.00 3.00 3.66 35.33 12.33 9.66 11.00 17.33 38.33 35.93 A 9 B = 1.913 68.33 41.33 21.08 5.50 19.08 10.00 12.33 13.33 10.00 2.33 3.46 A = 0.9565 6.33 8.66 13.33 2.66 2.66 5.06 B = 0.8555 4.33 3.00 1.00 1.66 1.66 6.20 4.33 1.33 2.33 3.00 5.66 7.26 A 9 B = 1.913 11.41 7.66 3.16 2.16 3.08 7.00 5.83 4.16 3.16 5.33 3.52 A = 0.4556 5.33 3.16 3.83 2.66 3.33 3.42 3.43 3.33 0.30 0.46 0.80 3.04 B = 0.4075 3.33 2.33 2.16 3.66 3.66 3.46 A 9 B = 0.9111 5.04 4.41 3.19 1.22 2.95 40 60 80 Mean (A)

Sugar types (A)

Fig. 3 Effect of sucrose concentrations on shoot proliferation of S. rebaudiana

although they appeared to be healthy and still green. 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 (79.33 %) than other treatments. The effect of full MS medium salt strength signicantly showed higher percentage of shoot growth (69.53 %) compared to quarter MS

medium salt strength which had low growth of shoots. As for as the interaction is concerned, the growth of beads was dependent on salt strength and storage time as shown in Table 4. Generally, our presented results concerning the effect of different MS salt strengths and storage time after 5 weeks appeared better for encapsulated buds when

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Sugar Tech Table 4 Effect of MS Salts Strengths on capsules germination of S. 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 2.048 2.289 4.579 50.66 34.66 36.00 40.00 40.33 55.66 64.00 60.00 44.00 55.91 62.66 62.66 58.66 58.66 60.66 78.66 72.00 61.33 64.00 60.66 100.00 76.00 69.33 72.00 79.33 69.53 61.86 57.06 55.73

Mean values followed by the same letter are not signicantly different at p \ 0.05 according to LSD test: F-test signicant at 0.05 level

Fig. 4 Effect of MS strengths on germination of capsules of S. rebaudiana

cultured on MS full salt strength which gave the highest conversion percentage (100 %) compared with other treatments (Fig. 4). In this respect, Nower et al. (2007) showed that the frequency of conversion pear encapsulated buds into plantlets was affected by MS strengths. Maximum conversion of shoot tips and nodal segments of S. rebaudiana was observed (100 %) in A2 medium (liquid MS medium containing 1.0 mg l-1 BAP), followed by 90 % in A1 (MS medium) and 83.3 % in A3 (MS ? BAP ? Kinetin (1.0 ? 0.25 mg l-1) medium (Ali et al. 2010). Effect of Sugar Alcohol (Mannitol or Sorbitol) on Capsules Germination The addition of osmotic agent mannitol at 0.05 M to the medium increased culture survival, however, with deterioration of cultures in terms of quality of growth. 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. The use of the osmotic agents was not an efcacious procedure. The most suitable to conversions of capsules was 0.05 M mannitol after 6 weeks from storage of synthetic seeds of Stevia (Table 5). These results corroborate with Westcotts (1981) descriptions on the toxic effects of mannitol in

Table 5 Effect of sugar alcohol (Mannitol or Sorbitol) on capsules germination of S. rebaudiana Sugar alcohol (M) (A) Period after Capsules germination % culture 1 2 3 4 5 (week) 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 1.664 1.441 4.077

Mean (B)

Mannitol 0.05 1.00 2.00 3.00 Sorbitol 0.05 1.00 2.00 3.00 Mean (A) LSD at 5 % level A B A9B

0.00 0.00 0.00 0.00 80.00 13.33 0.00 0.00 0.00 0.00 56.66 0.00 0.00 0.00 0.00 30.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 26.66 0.00 0.00 0.00 0.00 13.33 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 0.00 9.44 5.00 0.00 4.44 2.22 0.00 0.00

0.00 0.00 0.00 0.00 25.83

Mean values followed by the same letter are not signicantly different at p \ 0.05 according to LSD test: F-test signicant at 0.05 level

Solanum germplasm storage. The research in synthetic seed technology has been prolic, although with few applications on in vitro storage. Hemant et al. (2010)

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Sugar Tech Table 6 Effect of different concentrations (mg l-1) IBA, NAA and IAA) on in vitro rooting of S. rebaudiana Auxin conc. (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. Rizkalla et al. (2012) adding sorbitol or mannitol to the media at 0.05 M increased plantlet survival of sugar beet synthetic seeds (Francesca and Toro). Root Induction Auxins (IBA, NAA and IAA) efciently stopped shoot multiplication and supported nearly 100 % rooting. The effect of auxin concentrations was observed where, IBA at 1 or 2 mg l-1 resulted in the tallest root as shown in Table 6. Results revealed that, the greatest length of plant was obtained on MS medium free auxin (14.33 cm) than the other treatments. The greatest number of leaves observed with IAA at 0.2 mg l-1 (26.00). In general, it could be concluded that, stevia shoots cultured on MS medium supplemented with IAA, at 0.2 mg l-1 resulted in the greatest number of roots/explant (10.33) while, IBA at 1.0 and 2.0 mg l-1 resulted in longest root/shoot and gave the same length of root (Fig. 5). These results are in harmony with those of Ahmed et al. (2007) who found that the highest rooting percentage (97.66 %) and number of root per shoot (12.10 root/shoot) in Stevia rebaudiana were noted on MS medium fortied with IAA but with less low concentration (0.1 mg l-1). Laribi et al. (2012) showed that shoots produced roots within 2 weeks of culture, the highest percent (97 %) of root induction and maximum number of roots (8.90 root/shoot) as well as the root length (1.70 cm) were observed on MS medium fortied with IAA (0.50 m7 l-1).

IBA 0.0 0.1 0.2 0.5 1.0 2.0 NAA 0.1 0.2 0.5 1.0 2.0 IAA 0.1 0.2 0.5 1.0 2.0 lsd AT 5 % level 12.00 12.67 7.333 7.66 7.66 11.00 11.33 9.33 8.00 8.66 1.251 18.00 14.67 13.00 15.33 18.67 18.33 26.00 22.00 23.33 23.33 4.088 2.66 2.33 4.33 2.00 3.00 11.00 10.33 4.66 6.00 3.00 2.543 2.93 0.43 0.43 0.53 0.73 0.56 0.33 0.46 1.53 1.66 1.057 14.33 12.33 10.67 12.00 12.33 12.67 14.33 13.67 12.00 13.00 15.67 14.67 3.66 6.33 5.33 5.00 4.33 4.66 0.73 0.30 2.33 2.00 3.33 3.33

who showed that in vitro storage of shoot cultures was also evaluated by supplementing osmotic agents, mannitol, or sorbitol to the media. Such treatment had a negative impact on post-storage re-growth (at 25 C), even

Fig. 5 Development and root induction of S. rebaudiana

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