Biolistics transformation of callus and cell suspension cultures of Capsicum annuum L. ‘Serrano’ is useful for in vitro studies of the relative contents of secondary metabolites

Sara Cristina Brito-Sánchez, Fredy Alan Zaragoza-Pérez, Teresa de Jesús Olivera-Flores, José Fausto Rivero-Cruz, Jessica Amacosta, Francisca Morayna Gutiérrez-Luna, Lilián Gabriela Valencia-Turcotte, Rogelio Rodríguez-Sotres

Abstract


Capsicum annuum is a crop species of economic importance able to produce capsaicinoids, capsinoids, and pigments with nutritional and medicinal value. Methods to propagate and transform this species have been reported, but most are phenotype dependent, rely on Agrobacterium for transformation, and their success has been limited. This relates to only one commercial transgenic variety currently on trial. In the present work, we report the conditions to produce callus and cell suspension cultures of C. annuum ‘Serrano’ using commercial seeds. The culture could be induced to produce capsaicin and dihydrocapsaicin in detectable quantities and was amenable to transformation using biolistics. The expression of the Arabidopsis thaliana soluble inorganic pyrophosphatase 4 fused to a fluorescent protein was demonstrated using confocal microscopy. Evidence of the integrity of the fusion was obtained by immunoblot. The transformation induced a change in the ratio of capsaicin to dihydrocapsaicin measured using high resolution direct sample analysis-mass spectrometry (DSA-MS). The method is thus useful for the study of capsaicinoid production under controlled conditions for special purposes and metabolic studies.

Keywords


plant secondary metabolites; direct sample analysis-mass spectrometry; soluble inorganic pyrophosphatase; transgenic plant cells

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References


Paran I, van der Knaap E. Genetic and molecular regulation of fruit and plant domestication traits in tomato and pepper. J Exp Bot. 2007;58(14):3841–3852. https://doi.org/10.1093/jxb/erm257

Wahyuni Y, Ballester A, Sudarmonowati E, Bino RJ, Bovy AG. Metabolite biodiversity in pepper (Capsicum) fruits of thirty-two diverse accessions: variation in health-related compounds and implications for breeding. Phytochemistry. 2011;72(11–12):1358–1370. https://doi.org/10.1016/j.phytochem.2011.03.016

Wahyuni Y, Ballester A, Sudarmonowati E, Bino RJ, Bovy AG. Secondary metabolites of Capsicum species and their importance in the human diet. J Nat Prod. 2013;76(4):783–793. https://doi.org/10.1021/np300898z

Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N. Chilli peppers – a review on tissue culture and transgenesis. Biotechnol Adv. 2010;28(1):35–48. https://doi.org/10.1016/j.biotechadv.2009.08.005

Gammoudi N, Pedro TS, Ferchichi A, Gisbert C. Improvement of regeneration in pepper: a recalcitrant species. In Vitro Cell Dev Biol Plant. 2018;54:145–153. https://doi.org/10.1007/s11627-017-9838-1

Barchenger DW, Lamour KH, Bosland PW. Challenges and strategies for breeding resistance in Capsicum annuum to the multifarious pathogen, Phytophthora capsici. Front Plant Sci. 2018;9:628. https://doi.org/10.3389/fpls.2018.00628

Kehie M, Kumaria S, Tandon P, Ramchiary N. Biotechnological advances on in vitro capsaicinoids biosynthesis in Capsicum: a review. Phytochem Rev. 2014;14(2):189–201. https://doi.org/10.1007/s11101-014-9344-6

Ochoa-Alejo N, Ireta-Moreno L. Cultivar differences in shoot-forming capacity of hypocotyl tissues of chilli pepper (Capsicum annuum L.) cultured in vitro. Sci Hortic (Amsterdam). 1990;42(1–2):21–28. https://doi.org/10.1016/0304-4238(90)90144-4

Cheng Y, Ma R, Jiao Y, Qiao N, Li T. Impact of genotype, plant growth regulators and activated charcoal on embryogenesis induction in microspore culture of pepper (Capsicum annuum L.). S Afr J Bot. 2013;88:306–309. https://doi.org/10.1016/j.sajb.2013.08.012

Heidmann I, de Lange B, Lambalk J, Angenent GC, Boutilier K. Efficient sweet pepper transformation mediated by the BABY BOOM transcription factor. Plant Cell Rep. 2011;30(6):1107–1115. https://doi.org/10.1007/s00299-011-1018-x

Horstman A, Li M, Heidmann I, Weemen M, Chen B, Muino JM, et al. The BABY BOOM transcription factor activates the LEC1-ABI3-FUS3-LEC2 network to induce somatic embryogenesis. Plant Physiol. 2017;175:848–857. https://doi.org/10.1104/pp.17.00232

Gunay AL, Rao PS. In vitro plant regeneration from hypocotyl and cotyledon explants of red pepper (Capsicum). Plant Sci Lett. 1978;11(3–4):365–372. https://doi.org/10.1016/0304-4211(78)90024-X

Haque SM, Ghosh B. An improved micropropagation protocol for the recalcitrant plant Capsicum – a study with ten cultivars of Capsicum spp. (C. annuum, C. chinense, and C. frutescens) collected from diverse geographical regions of India and Mexico. J Hortic Sci Biotechnol. 2018;93(1):91–99. https://doi.org/10.1080/14620316.2017.1345331

Balázs E, Bukovinszki Á, Csányi M, Csilléry G, Divéki Z, Nagy I, et al. Evaluation of a wide range of pepper genotypes for regeneration and transformation with an Agrobacterium tumefaciens shooter strain. S Afr J Bot. 2008;74(4):720–725. https://doi.org/10.1016/j.sajb.2008.05.005

Liu J, Yu Y, Lei J, Chen G, Cao B. Study on Agrobacterium-mediated transformation of pepper with Barnase and Cre gene. Agric Sci China. 2009;8(8):947–955. https://doi.org/10.1016/S1671-2927(08)60299-0

Ko MK, Soh H, Kim K, Kim YS, Im K. Stable production of transgenic pepper plants mediated by Agrobacterium tumefaciens. HortScience. 2007;42(6):1425–1430. https://doi.org/10.21273/HORTSCI.42.6.1425

Kreuze JF, Valkonen JP. Utilization of engineered resistance to viruses in crops of the developing world, with emphasis on sub-Saharan Africa. Curr Opin Virol. 2017;26:90–97. https://doi.org/10.1016/j.coviro.2017.07.022

Bawa AS, Anilakumar KR. Genetically modified foods: safety, risks and public concerns-a review. J Food Sci Technol. 2013;50(6):1035–1046. https://doi.org/10.1007/s13197-012-0899-1

Timmons AM, Charters YM, Crawford JW, Burn D, Scott SE, Dubbels SJ, et al. Risks from transgenic crops. Nature. 1996;380(6574):487. https://doi.org/10.1038/380487a0

Ochoa-Alejo N, Ramirez-Malagon R. In vitro chili pepper biotechnology. In Vitro Cell Dev Biol Plant. 2001;37:701–729. https://doi.org/10.1007/s11627-001-0121-z

Nugroho LH. Red pepper (Capsicum spp.) fruit: a model for the study of secondary metabolite product distribution and its management. AIP Conf Proc. 2016;1744:020034. https://doi.org/10.1063/1.4953508

Phimchan P, Chanthai S, Bosland PW, Techawongstien S. Enzymatic changes in phenylalanine ammonia-lyase, cinnamic-4-hydroxylase, capsaicin synthase, and peroxidase activities in Capsicum under drought stress. J Agric Food Chem. 2014;62(29):7057–7062. https://doi.org/10.1021/jf4051717

Turner LB, Wellburn AR. Changes in adenylate nucleotide levels in the leaves of Capsicum annuum during water stress. J Plant Physiol. 1985;120(2):111–122. https://doi.org/10.1016/S0176-1617(85)80015-8

Gutiérrez-Luna FM, Hernández-Domínguez EE, Valencia-Turcotte LG, Rodríguez-Sotres R. Review: pyrophosphate and pyrophosphatases in plants, their involvement in stress responses and their possible relationship to secondary metabolism. Plant Sci. 2018;267:11–19. https://doi.org/10.1016/j.plantsci.2017.10.016

Kim S, Park M, Yeom S, Kim Y, Lee JM, Lee H. et al. Genome sequence of the hot pepper provides insights into the evolution of pungency in Capsicum species. Nat Genet. 2014;46(3):270–278. https://doi.org/10.1038/ng.2877

Murashige T, Skoog F. A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant. 1962;15(3):473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x

Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Exp Cell Res. 1968;50(1):151–158. https://doi.org/10.1016/0014-4827(68)90403-5

Gutiérrez-Luna FM, Navarro de la Sancha E, Valencia-Turcotte LG, Vázquez-Santana S, Rodríguez-Sotres R. Evidence for a non-overlapping subcellular localization of the family I isoforms of soluble inorganic pyrophosphatase in Arabidopsis thaliana. Plant Sci. 2016;253:229–242. https://doi.org/10.1016/j.plantsci.2016.10.005

Navarro de la Sancha E, Coello-Coutiño MP, Valencia-Turcotte LG, Hernández-Domínguez EE, Trejo-Yepes G, Rodríguez-Sotres R. Characterization of two soluble inorganic pyrophosphatases from Arabidopsis thaliana. Plant Sci. 2007;172(4):796–807. https://doi.org/10.1016/j.plantsci.2006.12.011

Zor T, Selinger Z. Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies. Anal Biochem. 1996;236(2):302–308. https://doi.org/10.1006/abio.1996.0171

Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970;227(5259):680–685. https://doi.org/10.1038/227680a0

R Core Team. R: a language and environment for statistical computing. Vienna: R Foundation for Statistical Computing; 2014.

Min J, Shin SH, Jeon EM, Park JM, Hyun JY, Harn CH. Pepper, chili (Capsicum annuum). In: Wang K, editor. Agrobacterium protocols. New York, NY: Springer; 2015. p. 311–320. (Methods in Molecular Biology; vol 1223). https://doi.org/10.1007/978-1-4939-1695-5_25

Kehie M, Kumaria S, Tandon P. Osmotic stress induced-capsaicin production in suspension cultures of Capsicum chinense Jacq. cv. Naga King Chili. Acta Physiol Plant. 2012;34(5):2039–2044. https://doi.org/10.1007/s11738-012-0991-1

Weathers PJ, Fadzillah NM, Cheetham RD. Light inhibits the formation of capsaicin from Capsicum callus. Planta Med. 1992;58(3):278–279. https://doi.org/10.1055/s-2006-961455




DOI: https://doi.org/10.5586/aa.1792

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