Callogenesis and Shoot Organogenesis from Flowers of Stevia rebaudiana (Bert

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The callogenic and shoot organogenic potential of the medicinaly important plant, S. rebaudiana were investigated. Callus induction and shoot regeneration were induced from flower explants of greenhouse plants incubated on Murashige and Skoog
  SHORT COMMUNICATION Callogenesis and Shoot Organogenesis from Flowersof   Stevia rebaudiana  (Bert.) Nisar Ahmad  • Hina Fazal  • Roshan Zamir  • Shahid Akbar Khalil  • Bilal Haider Abbasi Received: 8 January 2011/Accepted: 17 June 2011   Society for Sugar Research & Promotion 2011 Abstract  The callogenic and shoot organogenic potentialof the medicinaly important plant,  S. rebaudiana  wereinvestigated. Callus induction and shoot regeneration wereinducedfromflowerexplantsofgreenhouseplantsincubatedon Murashige and Skoog (MS)-medium supplemented withdifferent plant growth regulators (PGRs). The best callo-genic response was observed on explants incubated on MS-medium supplemented with 2.0 mg/l 6-benzyladenine (BA)along with 2.0 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D)after 30-days of culture. Subsequent transfer of callogenicexplants onto MS-medium supplemented with 2.0 mg/l BAgave 85.2% shoot organogenesis after 30-days followingculture. Maximum number (21.6) of shoots/explant wasrecorded for 2.0 mg/l BA. Subsequent sub culturing of shoots after 3 weeks of culture, on medium with similarcompositions of PGRs gave 5.1 cm longest shoot. The pal-ynology of   S. rebaudiana  has also been investigated. Keywords  Stevia rebaudiana    Callogenesis   Shoot organogenesis    Flowers    Palynology Abbreviations BA 6-BenzyladenineGA 3  Gibberellic acidNAA  a -Naphthaleneacetic acid2,4-D 2,4-dichlorophenoxyacetic acidPGRs Plant growth regulatorsMS0 MS medium without plant growth regulatorsSEM Scanning Electron Microscopy Introduction Stevia rebaudiana  ( S. rebaudiana ) is one of the potentmembers of family  Asteraceae  and cultivated in tropi-cal and sub-tropical countries (Sreedhar et al. 2008). S. rebaudiana  leaves contain steviol glycosides. The puri-fied steviosides imparts about 300 times sweetness thansucrose and is an important sugar substitute (Hwang 2006). For centuries, the Guarani tribes of Paraguay and Brazilused Stevia species, primarily  S. rebaudiana  as a sweetenerin yerba mate and medicinal teas for treating heartburn andother ailments (Singh and Rao 2005). The World Health Organization (WHO) performed a thorough evaluation of recent experimental studies of stevioside and steviolsconducted on animals and humans, and concluded thatstevioside and rebaudioside are not genotoxic in vitro or invivo and that the genotoxicity of steviol and some of itsoxidative derivatives in vitro is not expressed in vivo(Benford et al. 2006). The plants are traditionally raised from cutting becauseseeds of   Stevia  show a very low germination rate (Sivaramand Mukundan 2003). In vitro propagation methods offerpowerful tools for the mass-multiplication and germplasm N. Ahmad ( & )    B. H. AbbasiDepartment of Biotechnology, Faculty of Biological Sciences,Quaid-i-Azam University, Islamabad 45320, Pakistane-mail: nisarbiotech@gmail.comN. Ahmad    R. Zamir    S. A. KhalilNuclear Institute for Food and Agriculture,Tarnab Peshawar 25000, PakistanH. FazalPakistan Council of Scientific and Industrial Research (PCSIR)Laboratories Complex, Peshawar 25000, Pakistan  1 3 Sugar TechDOI 10.1007/s12355-011-0083-3  conservation of this medicinally important species. So far,there are no reports on successful in vitro regeneration of  S. rebaudiana  from flower explants of greenhouse plants.However, several protocols for regeneration and somaticembryogenesis in  S. rebaudiana  from leaves, shoot tips andnodal explants have been reported (Sreedhar et al. 2008; Hwang 2006; Ibrahim et al. 2008a, b; Sivaram and Mukundan 2003).In the literature cited, there is no evidence of palynologyof   S. rebaudiana , however the palynology of other mem-bers of family Asteraceae has been reported (Hayat et al.2010; Qureshi et al. 2009). Pollen grains of Asteraceae family are helianthoid, spherical, tricolporate, echinatewith abnormalities in size and colpus (Meo and Khan 2006).Hayat et al. (2010) also reported that pollen grains with tiny spinules and globular morphology markedly representAsteraceae family. The present study also included anaccount of the palynological observations of   S. rebaudiana .Leaves were collected from potted plants of   S. rebau-diana  maintained inside the greenhouse. Leaves weresterilized according to the method of Abbasi et al. (2010).Floral parts were immersed in 70% (v/v) ethanol for 60 s,0.1% (w/v) mercuric chloride (HgCl 2 ) solution for * 2 min, and rinsed three times with sterile distilled water.These surface sterilized explants were placed onto aMurashige and Skoog (1962) medium containing 30 g/lsucrose, and solidified with 8 g/l agar (Agar TechnicalLP0013, Oxoid, Hampshire, England). Different plantgrowth regulators (PGRs) were added to the medium, andthe pH was adjusted to 5.8. All media were autoclaved at121  C for 20 min. All cultures were maintained in agrowth room at temperature of 25  ±  1  C under a 16 hphotoperiod with a light intensity ranging from  * 40 to50  l mol m - 2 s - 1 provided by cool-white fluorescent tubelights (Ahmad et al. 2010). Pollen grains of   S. rebaudiana  were collected fromflowering plants from green house of NIFA, Peshawar. Thepollens were prepared for Scanning Electron Microscopy(SEM) by standard method described by Erdtman (1952).The pollen grains, suspended in acetic anhydride wereplaced on slide and crushed with the help of glass rod and Table 1  Effects of various concentrations of 2,4-D, BA, GA 3  andIAA (mg/l) on percent callus induction in  Stevia rebaudiana BA (mg/l) 2,4-D (mg/l) GA 3  (mg/l) IAA (mg/l) % Callusing0.5 0.5 – – 33.8  ±  6.30c1.0 1.0 – – 56.6  ±  10.11bc1.5 1.5 – – 81.7  ±  10.26ab2.0 2.0 – – 93.6  ±  7.12a0.5 2.0 0.5 0.5 85.0  ±  5.10ab0.5 1.5 0.5 0.5 80.2  ±  9.90ab0.5 1.0 0.5 0.5 58.3  ±  18.93bc0.5 0.5 0.5 0.5 35.8  ±  14.36cMeans with the same letter in the same column are not significantlydifferent (Duncan’s Multiple Range Test) Fig. 1  Callogenesis and organogenesis in  Stevia rebaudiana .  A  Flowers with callus,  B  Petals closed and callus increases,  C  Yellowish greencallus,  D–E  Shoot regeneration,  F  Shoot multiplication. (Color figure online)Sugar Tech  1 3  poured on the metallic stub, placed for drying for 30 minand then coated with gold in a sputter chamber (Ion-sputterJFC-1100) with coating restricted to 150  A on Jet micro-scope JSM-T200. The measurements were based on 10–15readings, characters like shape, class, size aperture andexine ornamentations of pollen grains were recorded.Micro photographs were taken at the Centralized ResourceLaboratories, University of Peshawar.The effects of various PGRs such as BA in combinationwith 2,4-D (0.5, 1.0, 1.5 and 2.0 mg/l) or 0.5 mg/l IAA or0.5 mg/l GA 3  on indirect organogenesis were evaluated(Table 1). Flower explants of   S. rebaudiana  used in presentstudy responded to all PGRs used (Fig. 1A). Best callusinduction was recorded on MS medium supplemented with2.0 mg/l BA (93.6%) along with 2.0 mg/l 2,4-D and0.5 mg/l BA with 2.0 mg/l 2,4-D, 0.5 mg/l GA 3  and0.5 mg/l IAA (85.0%). Callus induction recorded for BA athigher concentration 2.0 mg/l was significantly higher thanother PGRs, and no callus was observed on MS0 medium.The callus was initially white in color turning yellow andthen green after about 20 days (Fig. 1A–C). We noted incurrent report that addition of 2 mg/l BA in medium con-taining Auxins enhanced callus production in  S. rebaudi-ana  flower explants, however lower callus was observedwhen MS medium was supplemented with different con-centration of 2,4-D. In previous reports, most of theresearch work was on direct regeneration from bud, leaf,shoot apex and nodal explants but no reports on successfulregeneration from flower explants were observed (Sivaramand Mukundan 2003; Sreedhar et al. 2008). Findings of  Ahmad et al. (2010) about shoot tip explant of   P. nigrum are in agreement with our data. BA has been shown toinduce efficient callusing in several elite plant species(Abbasi et al. 2010).Callogenesis is considered as a significant feature of indirect organogenesis and for research on biologicallyactive molecules in medicinal species (Abbasi et al. 2010).Data on organogenesis was determined after 5 weeks of sub-culture and the best % shooting was recorded for2.0 mg/l of BA (85.2%) (Table 2). However, 2.0 mg/l BAwith 2.0 mg/l 2,4-D produced callus but inhibit % shoot-ing. Nonetheless, BA was more effective than other PGRsused in current report. With similar concentration of BA,Sreedhar et al. (2008) found maximum number of shoots (93%) from bud explants of   S. rebaudiana  and maximumshoots/explant (4.93). In another study highest number of shoots and shoots/explant (6.63) was recorded when the Table 2  Effects of various concentrations of BA on shoot length,shoot number and percent shooting in  Stevia rebaudiana BA (mg/l) Shoot length (cm) Shoot/explant % shooting0.5 3.2  ±  0.45b 3.1  ±  0.85c 71.2  ±  10.22b1.0 3.2  ±  0.77b 10.4  ±  2.52b 79.9  ±  4.05ab1.5 4.3  ±  0.62ab 15.2  ±  5.23ab 82.7  ±  7.66a2.0 5.1  ±  0.75a 21.6  ±  1.86a 85.2  ±  9.70aMeans with the same letter in the same column are not significantlydifferent (Duncan’s Multiple Range Test) Fig. 2  Microphotographs of pollen grains of   S. rebaudiana .( A – C ) Exine view of pollengrains. ( D ) Polar view of pollengrainsSugar Tech  1 3  MS medium was incorporated with 2 mg/l BA (Ibrahimet al. 2008a). The finding of Sivaram and Mukundan (2003), are in agreement with our data that BA induce 70%organogenic response from shoot apex, nodal and leaf explants. Shooting was recorded on all BA concentrationtested (Fig. 1F). Data on mean shoot length showed thatincorporation of BA into medium produced best shoots of 5.1 cm length. Highest number (21.6) of shoots/explantwas recorded for 2.0 mg/l of BA (Fig. 1F) and lowestnumber (3.1) of shoots/explant was recorded for 0.5 mg/lBA. During shoot organogenesis it was observed thataddition of IAA or 2,4-D in medium incorporated with BAsignificantly inhibited number of shoots/explant.Contrarily to data on shoots/explant, addition of BA inmedium increased means shoot length. Similar findingswere made by Makunga et al. (2003) for  Thapsia  spp.However, Abbasi et al. (2007) and Lucchesinia et al.(2009) reported optimum shoot length for  Echinacea spe-cie  on medium containing BA. Magyar-Tabori et al. (2010) also concluded from their work that type and concentrationof cytokinins significantly influence shoot organogenesis indifferent apple genotypes. Stevia rebaudiana  flowers was investigated for polyno-morph features, is generally radially symmetrical, usuallyprolate-spheroidal and trizonocolporate to tetrazonocolpo-rate (Fig. 2). The polar and equatorial views can be easilyrecognized, Fig. 2A–D shows the sculpture patterns of exine surface of   S. rebaudiana  pollens represented bymarked tiny spinules. These spinules were prominent inoverall surface of pollen grains and such homogeneityverified its monophyly. In flora of Pakistan only three tribesof Family Asteraceae have been published, while others areyet to be documented and  S. rebaudiana  is one amongstthose unreported species. However palynology of othermembers of Asteraceae family has been reported by severalworkers (Meo and Khan 2006; Mbagwu et al. 2007; Hayat et al. 2010; Qureshi et al. 2009; Zafar et al. 2007). References Abbasi, B.H., P.K. Saxena, S.J. Murch, and C.-Z. Liu. 2007.Echinacea biotechnology: challenges and opportunities.  In vitroCellular & Developmental Biology - Plant   43: 481–492.Abbasi, B.H., M.A. Khan, T. Mahmood, M. Ahmad, M.F. Chaudhary,and M.A. Khan. 2010. Shoot regeneration and free-radicalscavenging activity in  Silybum marianum  L.  Plant Cell, Tissueand Organ Culture  101: 371–376.Ahmad, N., H. Fazal, B.H. Abbasi, M. Rashid, T. Mahmood, and N.Fatima. 2010. Efficient regeneration and antioxidant potential inregenerated-tissues of   Piper nigrum  L.  Plant Cell, Tissue and Organ Culture  102: 129–134.Benford, D.J., M. DiNovi, and J. Schlatter. 2006. Safety evaluation of certain food additives: steviol Glycosides.  WHO Food AdditivesSeries  5: 117–144.Erdtman, G. 1952.  Pollen morphology and plant taxonomy: (Angio-sperm. An introduction to palynology-I) . Stockholm: Almqvistand Wiksell.Hayat, M.Q., M. Ashraf, M.A. Khan, G. Yasmin, N. Shaheen, and J.Jabeen. 2010. Palynological study of the Genus  Artemisia (  Asteraceae ) and its systematic implications.  Pakistan Journalof Botany  42: 751–763.Hwang, S.J. 2006. Rapid in vitro propagation and enhanced steviosideaccumulation in  Stevia rebaudiana  Bert.  Journal of Plant  Biology  49: 267–270.Ibrahim, I.A., M.I. Nasr, B.R. Mohammed, and M.M. EI-Zefzafi.2008a. Nutrient factors affecting in vitro cultivation of   Steviarebaudiana .  Sugar Tech  10: 148–253.Ibrahim, I.A., M.I. Nasr, B.R. Mohammed, and M.M. EI-Zefzafi.2008b. Plant growth regulators affecting in vitro cultivation of  Stevia rebaudiana .  Sugar Tech  10: 254–259.Lucchesinia, M., A. Bertolib, A. Mensuali-Sodic, and L. Pistelli.2009. Establishment of in vitro tissue cultures from  Echinaceaangustifolia  D.C. adult plants for the production of phytochem-ical compounds.  Scientia Horticulturae  122: 484–490.Magyar-Tabori, K., J. Dobranszki, J.A.T. da Silva, S.M. Belley, and I.Hudak. 2010. The role of cytokinins in shoot organogenesis inapple.  Plant Cell, Tissue and Organ Culture  101: 251–267.Makunga, N.P., A.K. Jager, and J. van Staden. 2003. Microprogationof   Thapsia garganica —a medicinal plant.  Plant Cell Report   21:967–973.Mbagwu, F.N., E.G. Chime, and C.I.N. Unamba. 2007. Palynologicalstudies on five species of   Asteraceae .  Life Science Journal  5:73–76.Meo, A.A., and M. Khan. 2006. Palynogical observations on the Genus Calendula  ( Calenduleae - Compositeae ) from Pakistan. Pakistan Journal of Botany  38: 511–520.Murashige, T., and F. Skoog. 1962. A revised medium for rapidgrowth and bioassays with tobacco tissue cultures.  PhysiologiaPlantarum  15: 473–497.Qureshi, S.J., M.A. Khan, M. Arshad, Rashid, A. Ahmad, and M.Zafar. 2009. Morphological, palynological, and anatomicalinvestigations of   Hieracium  L. (Lactuceae, Asteraceae) inPakistan. Me´todos en Ecologı´a y Sistema´tica 4: 1–22.Singh, S.D., and G.P. Rao. 2005. Stevia: the herbal sugar of 21stcentury.  Sugar Tech  7: 17–24.Sivaram, L., and U. Mukundan. 2003. In vitro culture studies on Stevia rebaudiana .  In vitro Cellular & Developmental BiologyPlant   39: 520–523.Sreedhar, R.V., L. Venkatachalam, R. Thimmaraju, N. Bhagya-lakshmi, M.S. Narayan, and G.A. Ravishankar. 2008. Directorganogenesis from leaf explants of   Stevia rebaudiana  andcultivation in bioreactor.  Biologia Plantarum  52: 355–360.Zafar, M.Z., M. Ahmad, and M.A. Khan. 2007. Palynology of familyAsteraceae from flora of Rawalpindi-Pakistan.  International Journal of Agriculture and Biology  9: 156–161.Sugar Tech  1 3
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