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Effect of light quality and culture medium on somatic embryogenesis of Agave tequilana Weber var. Azul

Somatic embryogenesis in Agave tequilana Weber var. Azul was affected by the interaction between the light regimes applied during the induction phase and the expression phase. When embryogenic calli was exposed to white or red light during the
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  RESEARCH NOTE Effect of light quality and culture medium on somaticembryogenesis of   Agave tequilana  Weber var. Azul Araceli Rodrı´guez-Sahagu´n  • Gustavo Acevedo-Herna´ndez  • Jose´ M. Rodrı´guez-Domı´nguez  • Benjamı´n Rodrı´guez-Garay  • Jesu´s Cervantes-Martı´nez  • Osvaldo A. Castellanos-Herna´ndez Received: 13 February 2010/Accepted: 2 August 2010/Published online: 25 August 2010   Springer Science+Business Media B.V. 2010 Abstract  Somatic embryogenesis in  Agave tequilana Weber var. Azul was affected by the interaction betweenthe light regimes applied during the induction phase andthe expression phase. When embryogenic calli wasexposed to white or red light during the expression phase,an average of two germinated embryos per explant wasobtained regardless of the light treatment used for callusinduction. Conversely, the highest number of germinatedembryos, an average of 18 per explant, was obtained whenapplying either white or red light during the inductionphase and then wide-spectrum light during the expressionphase. Culture medium had also a great influence in thisprocess, with embryo germination being reduced by up to70%, depending on the light treatment, when using Schenk and Hildebrandt (SH) medium instead of Murashige andSkoog (MS) medium. Keywords  Embryogenic calli    Visible spectrum   Embryo germination  Agave tequilana  Weber var. Azul, commonly known asblue agave, is the only species allowed by law to be used asraw material for the production of tequila, an alcoholicbeverage recognized worldwide as a distinctive product of Mexico (Diario Oficial et al. 1993). Recently, this plant hasalso been proposed as a promising source of dietary fruc-tans with nutritional and health-promoting applications(Urı´as-Silvas et al. 2008). Consequently, cultivation of thiscrop holds remarkable cultural and economic importance.  A. tequilana  has a long life span (6–12 years), and sexualreproduction is very rare, and not very efficient when itoccurs. Traditionally, the species is propagated throughnatural offsets (asexual rhizomatous shoots), favoringpreservation of homogeneous and high-quality populations.However, use of this propagation method has raised con-cerns about the vulnerability of agave plantations to path-ogen attacks or to adverse environmental conditions as aresult of its narrow genetic base (Portillo et al. 2007).Somatic embryogenesis in  A. tequilana  has already beenreported and proposed not only as a valuable tool for thein vitro mass propagation of this species but also as apotential alternative source of genetic variability whichmay be suitable for use in breeding programs (Portillo et al.2007). However, there are many factors that need to betaken into account to optimize this protocol for practicaluse. Such factors, which include plant growth regulators,light regime, culture medium composition, and theirinteractions, play an important role in influencing somaticembryogenesis and can be manipulated to improve theefficiency of embryo production and germination (Aslamet al. 2008; Gow et al. 2009). Light conditions are partic- ularly important, and there are several reports on theinfluence of light quality on somatic embryogenesis; itseffects may vary depending on the species studied and thedevelopmental stage (Kaldenhoff et al. 1994; D’Onofrio A. Rodrı´guez-Sahagu´n ( & )    O. A. Castellanos-Herna´ndezDepartamento de Ciencias Ba´sicas, Centro Universitario de laCie´nega, Universidad de Guadalajara (CUCI-UdeG), Av.Universidad 1115, 47820 Ocotla´n, Jalisco, Mexicoe-mail: aracelicrs@gmail.comJ. M. Rodrı´guez-Domı´nguez    B. Rodrı´guez-Garay   J. Cervantes-Martı´nezUnidad de Biotecnologı´a Vegetal, Centro de Investigacio´n yAsistencia en Tecnologı´a y Disen˜o del Estado de Jalisco, A.C.(CIATEJ), Normalistas 800 Colinas de la Normal, 44270Guadalajara, Jalisco, MexicoG. Acevedo-Herna´ndezDepartment of Biology, University of Western Ontario, London,ON N6A 5B7, Canada  1 3 Plant Cell Tiss Organ Cult (2011) 104:271–275DOI 10.1007/s11240-010-9815-4  et al. 1998; Torne´ et al. 2001). Thus, the objective of thiswork is to determine the influence of medium composi-tion and light quality on the somatic embryogenesis of   A. tequilana  during both the callus induction and embryodevelopment processes.With this aim, in vitro cultivated  A. tequilana  plants(genotype L) were obtained and propagated via axillarybud proliferation according to Santacruz-Ruvalcaba et al.(1999), using Murashige and Skoog (MS, 1962) basal medium supplemented with 30 g L - 1 sucrose, 0.025mg L - 1 2,4-dichlorophenoxyacetic acid (2,4-D), and10 mg L - 1 6-benzyladenine (BA). Agar (8 g L - 1 ) wasadded to the medium, and pH was adjusted to 5.8. Thisplant material was maintained for 30 days in a growthchamber at 27  C under a 16/8 h photoperiod with lightintensity of 32  l mol m - 2 s - 1 .Leaves (3 cm long) were taken from the in vitro prop-agated plants and cut transversely into pieces about 1 cmlong. Two basal culture media were used for induction of embryogenic callus (induction phase): MS and Schenk andHildebrandt (SH, 1972), both supplemented with 30 g L - 1 sucrose, 3 mg L - 1 2,4-D, 0.3 mg L - 1 BA, and 8 g L - 1 agar. Leaf segments were cultured under the same condi-tions as described above, and medium was refreshed at2-week intervals. Calli produced after 40 days weretransferred to the media for production of somatic embryos(expression phase). These media consisted of SH basal saltmixture and a modified MS medium reported by Portilloet al. (2007), where the ammonium nitrate content wasreduced to 5 mM. Both media were supplemented with250 mg L - 1 casein hydrolysate, 500 mg L - 1 glutamine,30 g L - 1 sucrose, and 6 g L - 1 Phytagel  . The calli weresubcultured every 2 weeks, and the number of producedembryos was recorded after 60 days. The somatic embryoswere considered as germinated when the emergence of thecotyledon was evident. Germinated embryos could berecovered and developed full plants (Fig. 1).The in vitro cultures were exposed to different lightqualities (Fig. 2); white light (W, 32  l mol m - 2 s - 1 ), bluelight (B, 54  l mol m - 2 s - 1 ), and red light (R, 65  l molm - 2 s - 1 ) treatments were obtained using fluorescent lamps(Osram  F20T12/CW, F20T12/B, and F20T12/R, respec-tively). Wide-spectrum light (WS, 49  l mol m - 2 s - 1 ) wasobtainedbyusingaF20T12-PL/AQlamp(GeneralElectric  ). Fig. 1  Somatic embryogenesis in  Agave tequilana .  a , b  Callusproduced from leaf explant;  c  development of somatic embryos; d , e  somatic embryos produced from callus;  f   whole plant regeneratedthrough somatic embryogenesis Fig. 2  Irradiance spectra of thelight treatments obtained with aspectrophotometer (SF2000;Ocean Optics, Inc., Dunedin,FL)272 Plant Cell Tiss Organ Cult (2011) 104:271–275  1 3  Culture plates were positioned at a distance of 9 cm fromthe lamps, and maintained at 27  C under a 16/8 h pho-toperiod. The white light treatment was considered as thecontrol.Two multifactorial experiments were designed toinvestigate the effect of culture medium and light qualityon induction of embryogenic callus and production andgermination of somatic embryos. Each experiment con-sisted of eight treatments, with six replicates per treatment,and each replicate consisting of three explants per Petridish. Data were analyzed using analysis of variance(ANOVA), and means were compared by the least signif-icant difference (LSD) range test with a family error rate of 0.05. The software package Statgraphics (StatisticalGraphics Co.) was used for the analyses.The use of light from different sources during theinduction phase did not show a significant effect onthe efficiency of callus production, since almost all of the explants cultured in MS medium (95% in average)produced callus when exposed to different light treatments(data not shown). On the other hand, composition of cul-ture medium can exert a considerable influence on theefficiency of callus induction. On average, only about 75%of the leaf explants cultivated in SH medium were able toproduce callus, without any apparent effect of the lightregime (data not shown). Light conditions, however, seem Fig. 3  Effect of light regimeand culture medium on embryoproduction and germination.Mean of the number of embryosproduced or germinated perexplant for six replicates andstandard error are shown.  Different letters  on the  bars indicate significant differences( a  =  0.05). See text forabbreviationsPlant Cell Tiss Organ Cult (2011) 104:271–275 273  1 3  to have a greater effect on the production, maturation, andgermination of agave somatic embryos. As can be seenfrom Fig. 3, different light qualities produced a ratherdiverse response pattern in terms of the total number of somatic embryos produced and the germination rates.Moreover, in some cases it was observed that embryogeniccalli subjected to different light treatment during theinduction phase exhibited distinct responses even if theywere exposed to the same light condition during theexpression phase. In this particular case, when using thesame light source during both phases, blue light treatmentproduced the highest number of embryos (20 per explant,on average), but this number was significantly increasedwhen applying either white or red light during the induc-tion phase and then wide-spectrum light during theexpression phase (average of 31 and 28 embryos perexplant, respectively). This result not only shows that lightfrom different sources influences the production and ger-mination of somatic embryos, but also suggests thatresponsiveness of the embryogenic callus to the lightstimulus is somehow determined by the light regime duringthe induction phase.To better understand this relationship, we used the samedata to generate interaction plots (Fig. 4), which allowvisualization of the presence of interactions among severalfactors. The more the lines in the graph deviate from beingparallel, the more significant the interaction effect. Weevaluated the interaction of the light regimes applied dur-ing the induction and expression phases on the number of produced and germinated somatic embryos. The analysisshows that, when blue or wide-spectrum light is appliedduring the expression phase, their positive effect onembryo germination depends on the light treatment towhich the callus was previously subjected. It is also note-worthy that interaction plots show that the two lighttreatments display opposite trends. On the other hand, forwhite and red light sources, the effect on embryo germi-nation (lower, compared with the other light sources) isindependent of the srcin of the embryogenic callus. Theoverall interaction of all light treatments is greatly reducedwhen using SH medium, probably due to the rather lownumber of embryos produced.The effect of light quality on somatic embryogenesis hasbeen previously reported, with different effects of lighttreatments depending on the species analyzed (D’Onofrioet al. 1998; Kaldenhoff et al. 1994; Michler and Lineberger 1987; Torne´ et al. 2001; Ascencio-Cabral et al. 2008). In some cases, the rate of somatic embryo differentiationhas been correlated with the ratio of the far-red- and red-absorbing forms of the phytochrome (Pfr/Pr ratio), report-ing promotive and inhibitory effects depending on theamount of activated phytochrome (D’Onofrio et al. 1998;Torne´ et al. 2001). Nevertheless, those studies have usuallybeen performed using the same light source along thewhole process of somatic embryogenesis, and the effectthat use of different light treatments during the inductionand expression phases may have on this photoequilibriumhas not been investigated. Mancinelli et al. (1992) reportedthat, in vivo, different pre-irradiation treatments affect theinitial Pfr/Pr ratio, which in turn determines the photo-conversion phytochrome rate under the subsequent lightsource. However, further experiments using more con-trolled light treatments and including biochemical analyseswould be required to confirm the role of phytochrome orany other photoreceptor in controlling agave somaticembryogenesis, and to fully understand the interactionsbetween different light treatments during the induction andexpression phases.Regarding the differences observed for different culturemedia, previous studies have demonstrated the effect of  Fig. 4  Interaction plots for the light conditions during both theinduction phase and the expression phase for two culture media. Dataused are the same as for Fig. 3. See text for abbreviations274 Plant Cell Tiss Organ Cult (2011) 104:271–275  1 3  mineral nutrients on plant morphogenesis in vitro, andsome of these reports have called attention to the differ-ences between MS and SH basal media (Gomez and Segura1995). These media differ basically in their total ionicstrength, nitrogen level, ammonium concentration, andammonium-to-nitrate ratio, all of which are higher in theformer. It is possible that one of these nutritional param-eters could account for the observed differences, thoughfurther study would be necessary to establish which factoris critical in determining the efficiency of the callusinduction and embryo production processes in agave.In conclusion, in this work we observed the importanceof medium composition in the efficiency of embryo pro-duction, and identified the impact of the interactionbetween the light conditions during the induction andexpression phases of somatic embryogenesis in  A. tequil-ana . This interaction plays an important role and should beconsidered when optimizing protocols for mass productionof somatic embryos in this species. References Ascencio-Cabral A, Gutie´rrez-Pulido H, Rodrı´guez-Garay B,Gutie´rrez-Mora A (2008) Plant regeneration of   Carica papaya L. through somatic embryogenesis in response to light quality,gelling agent and phloridzin. Sci Hortic 118:155–160. doi:10.1016/j.scienta.2008.06.014Aslam J, Mujib A, Fatima S, Sharma MP (2008) Cultural conditionsaffect somatic embryogenesis in  Catharanthus roseus  L. (G.)Don. Plant Biotechnol Rep 2:179–189. doi:10.1007/s11816-008-0060-9D’Onofrio C, Morini S, Bellocchi G (1998) Effect of light quality onsomatic embryogenesis of quince leaves. Plant Cell TissueOrgan Cult 53:91–98. doi:10.1023/A:1006059615088Diario Oficial, Secretarı´a de Comercio y Fomento Industrial, NormaOficial Mexicana NOM-006-SCFI-1993 (1993) BebidasAlcoho´licas-Tequila-Especificaciones, Me´xico, D.F. Octubre13, pp 48–52Gomez MP, Segura J (1995) In vitro control of adventitious buddifferentiation by inorganic media components in leaves of mature  Juniperus oxycedrus . In Vitro Cell Dev Biol Plant31:179–182Gow W-P, Chen J-T, Chang W-C (2009) Effects of genotype, lightregime, explants position and orientation on indirect somaticembryogenesis from leaf explants of   Phalaenopsis orchids . ActaPhysiol Plant 31:363–369. doi:10.1007/s11738-008-0243-6Kaldenhoff R, Henningsen U, Richter G (1994) Gene activation insuspension-cultured cells of   Arabidopsis thaliana  during blue-light-dependent plantlet regeneration. Planta 195:182–187. doi:10.1007/BF00199677Mancinelli AL, Rossi F, Moroni A (1992) Phytochrome photocon-version in vivo. Effect of the initial Pfr/Ptot ratio. PhotochemPhotobiol 56:593–598Michler CH, Lineberger D (1987) Effects of light on somatic embryodevelopment and abscisic levels in carrot suspension cultures.Plant Cell Tissue Organ Cult 11:189–207Murashige T, Skoog F (1962) A revised medium for rapid growth andbio assays with tobacco tissue cultures. Physiol Plant15:473–497. doi:10.1111/j.1399-3054.1962.tb08052.xPortillo L, Santacruz-Ruvalcaba F, Gutie´rrez-Mora A, Rodrı´guez-Garay B (2007) Somatic embryogenesis in  Agave tequilana Weber cultivar azul. In Vitro Cell Dev Biol Plant 43:569–575.doi:10.1007/s1162700790465Santacruz-Ruvalcaba F, Gutie´rrez-Pulido H, Rodrı´guez-Garay B(1999) Efficient in vitro propagation of   Agave parrasana  Berger.Plant Cell Tissue Organ Cult 56:163–167. doi:10.1023/A:1006232911778Schenk RV, Hildebrandt AC (1972) Medium and techniques forinduction and growth of monocotyledonous and dicotyledonousplant cell cultures. Can J Bot 50:199–204. doi:10.1139/b72-026Torne´ JM, Moysset L, Santos M, Simo´n E (2001) Effect of lightquality on somatic embryogenesis in  Arauija sericifera . PhysiolPlant 111:405–411. doi:10.1034/j.1399-3054.2001.1110319.xUrı´as-Silvas JE, Cani PD, Delme´e E, Neyrinck A, Lo´pez MG,Delzenne NM (2008) Physiological effects of dietary fructansextracted from  Agave tequilana  Gto and Dasylirion spp. Br JNutr 99:254–261. doi:10.1017/S0007114507795338Plant Cell Tiss Organ Cult (2011) 104:271–275 275  1 3
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