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Selection of Suitable Propagation Method for Consistent Plantlets Production in Stevia Rebaudiana (Bertoni)

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Selection of Suitable Propagation Method for Consistent Plantlets Production in Stevia Rebaudiana (Bertoni) View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Saudi Journal of Biological Sciences (2014) 21, 566–573 King Saud University Saudi Journal of Biological Sciences www.ksu.edu.sa www.sciencedirect.com ORIGINAL ARTICLE Selection of suitable propagation method for consistent plantlets production in Stevia rebaudiana (Bertoni) Shahid Akbar Khalil a,*, Roshan Zamir a, Nisar Ahmad b a Nuclear Institute for Food and Agriculture (NIFA), Peshawar, Pakistan b Department of Biotechnology, Faculty of Biological Sciences, Quaid-i-Azam University, Islamabad 45320, Pakistan Received 11 December 2013; revised 10 February 2014; accepted 20 February 2014 Available online 5 March 2014 KEYWORDS Abstract Stevia rebaudiana (Bert.) is an emerging sugar alternative and anti-diabetic plant in Paki- Stevia rebaudiana; stan. That is why people did not know the exact time of propagation. The main objective of the Seed germination; present study was to establish feasible propagation methods for healthy biomass production. In Seed radiation; the present study, seed germination, stem cuttings and micropropagation were investigated for Stem cuttings; higher productivity. Fresh seeds showed better germination (25.51–40%) but lost viability after a Micropropagation few days of storage. In order to improve the germination percentage, seeds were irradiated with 2.5, 5.0, 7.5 and 10 Gy gamma doses. But gamma irradiation did not show any significant change in seed germination. A great variation in survival of stem cutting was observed in each month of 2012. October and November were found the most suitable months for stem cutting survival (60%). In order to enhance survival, stem cuttings were also dipped in different plant growth reg- ulators (PGRs) solution. Only indole butyric acid (IBA; 1000 ppm) treated cutting showed a higher survival (33%) than control (11.1%). Furthermore, simple and feasible indirect regeneration system was established from leaf explants. Best callus induction (84.6%) was observed on MS-medium aug- mented with 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid (2,4-D; 2.0 mg lÀ1). For the first time, we obtained the highest number of shoots (106) on a medium containing BA (1.5 mg lÀ1) À1 and gibberellic acid (GA3; 0.5 mg l ). Plantlets were successfully acclimatized in plastic pots. The current results preferred micropropagation (85%) over seed germination (25.51–40%) and stem cutting (60%). ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. * Corresponding author. Address: Nuclear Institute for Food and 1. Introduction Agriculture (NIFA), Peshawar-25120, Pakistan. Tel.: +92 300 5900250; fax: +92 91 2964059. E-mail address: [email protected] (S.A. Khalil). Stevia rebaudiana (S. rebaudiana) in the Asteraceae family is an Peer review under responsibility of King Saud University. emerging economical species throughout the world (Sreedhar et al., 2008). S. rebaudiana is considered as an important plant due to its active compound present in the leaves known as Production and hosting by Elsevier steviol glycosides. The purified form of steviol glycoside is 1319-562X ª 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University. http://dx.doi.org/10.1016/j.sjbs.2014.02.005 Selection of best method for consistent plantlets production in Stevia rebaudiana 567 known as Stevioside which is 300 times sweeter than commer- 2.2. Seed germination in petri plates, soil and on MS medium cially available sucrose (Hwang, 2006). Stevia species are specially used for the treatment of diabetic patients. The natu- Carefully 20 radiated seeds were placed in each petri plate ral Steviosides cannot enter into the blood stream due to the along with the control (without radiation). Petri plates con- absence of receptor for absorbance. Still today there are no taining filter paper and 1 L bottle containing distilled water reports that a single patient is completely recovered from dia- were autoclaved before seed inoculation. Seeds were surface betes after using different synthetic drugs (Thiyagarajan and sterilized with 0.2% mercuric chloride solution for 5 min and Venkatachalam, 2012). It is expected that 57 million people washed several times with sterile distilled water. Before inocu- would be affected by diabetes in the year of 2025. Stevia lation the seeds were dried with the help of autoclaved filter pa- extracts have no reported side effects and can be used as an per. Irradiated seeds were placed in triplicate on a wet filter alternative to sugar and other synthetic sweeteners paper and the mean data was collected after 25 days. For seed (Thiyagarajan and Venkatachalam, 2012). germination in soil, 100 pots were filled with a mixture of sand, This species originated from Paraguay and Brazil, and silt and clay. 20 pots were used for each dose (2.5, 5.0, 7.5 and currently considered as an alternate substitute of cane and beet 10 Gy) along with the control. Irradiated seeds were also sugar (Ahmad et al., 2011a,b). According to the literature placed on a solid MS-medium for germination without the Brazilian and Paraguay tribes used this valuable species for addition of plant growth regulators (PGRs). A total of 180 the treatment of heartburn in medicinal teas and Yerba mate seeds were inoculated on MS-medium along with the control. (Singh and Rao, 2005; Fazal et al., 2011). In vitro and in vivo For each treatment 36 seeds were cultured in test tubes in three experiments have been conducted on human beings and replicates. Untreated fresh seeds were also sown in the soil to animals under the control of the WHO which showed that test the best germination time for a period of 6 weeks. the intake of steviosides or rebaudiosides does not induce genotoxicity and the oxidative derivatives were not expressed 2.3. Propagation through stem cuttings in vivo (Ahmad et al., 2011a). S. rebaudiana plants are conventionally propagated Equal of Sixty stem cuttings were planted in each month of through cuttings, but this traditional method cannot produce 2012 to obtain the best month for survival. In another set of a large number of plants. The seeds of this species are smaller experiment, stem cuttings of suitable length with 3–4 leaves in size and the germination % age is very low (Singh and Rao, were dipped for 1 min in NAA (500, 1000 ppm) and IBA 2005). Therefore modern techniques of propagation such as (500, 1000 ppm) solutions respectively. Stem cuttings were in vitro regeneration or tissue culture are needed to enhance then planted in pots containing combinations of soil, sand the production of this important species. For these reasons tis- and manure in 2:1:1 ratio. Data on different parameters of sue culture techniques are widely used to produce maximum shoot (length of cuttings, No. of leaves per cutting, No. of mass from a single plant in a short span of time and also pro- branches per cutting and internodes length) and root (length vide opportunities for germplasm conservation of important of longest root, No. of roots per cutting, fresh weight of roots plants (Jagatheeswari and Ranganathan, 2012; Sivaram and and dry weight of roots) was collected after 90 days of culture. Mukundan, 2003; Anbajhagan et al., 2010; Taware et al., 2010; Sabah and Rasha, 2013). The main objective of the present study was to improve the 2.4. Micropropagation production of Stevia rebaudiana using different propagation methods. This is an emerging species in Pakistan and most 2.4.1. Collection and sterilization of leaf explants of the researchers are unaware regarding the exact time of seed Fresh leaf explants were taken from S. rebaudiana mother sowing and cutting. In this study seed germination (irradiated) plant grown in the Nuclear Institute for Food and Agriculture and stem cuttings were compared with micropropagation. So, (NIFA), Peshawar, Pakistan. The leaves were collected in a we established a simple micropropagation system via indirect container with tap water to maintain viability. The leaves were regeneration for healthy and consistent plantlets production then placed under running water for 15 min to remove dust from leaf explants. These results will provide an opportunity particles. Ethanol (70%) for 1 min and mercuric chloride for selection of the best method of propagation for future (0.2%) for 120 s were used for explant sterilization. The ex- studies. plants were then rinsed with double distilled water and dried with the help of filter paper (autoclaved). 2. Materials and methods 2.4.2. Media and growth conditions 2.1. Seed collection and irradiation Murashige and Skoog, 1962 basal medium was selected for in vitro regeneration augmented with sucrose (3%) and then À1 Fresh seeds were collected from field grown plants of S. rebau- solidified with agar (8 g l , Merk). Plant growth regulators diana at the Nuclear Institute for Food and Agriculture (PGRs) from stock solutions were added to the media and (NIFA), Peshawar. Healthy and black coated seeds were the pH (5.6–5.8) was adjusted through a pH meter (330i/ selected for gamma irradiation. Two thousand seeds were irra- SET, Germany). After pH adjustment the media were boiled diated with 2.5, 5.0, 7.5 and 10 Gy doses through 60Co gamma in a microwave oven and then autoclaved (Hirayama, Japan) source in the presence of air and room temperature. The same at 121 °C for 20 min. All prepared media were then placed in number of untreated seeds was used as control. growth room (25 ± 1 °C) under a 16 h photoperiod. 568 S.A. Khalil et al. 2.4.3. Plant growth regulators for micropropagation 50 For callus induction, leaf explants were placed on MS medium Petri plates incorporated with different concentrations (1.0–3.0 mg lÀ1)of Soil 40 6-benzyladenine (BA) and 2,4-dichlorophenoxyacetic acid MS-medium (2,4-D) alone (0.5–4.0 mg lÀ1) or in combination with BA À1 (0.5–3.0 mg l ).
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