Phenotypic and Molecular Characterization of Phaseolus vulgaris Plants from Non-Cryopreserved and Cryopre-served Seeds

Inaudis Cejas, Roberto Méndez, Ariel Villalobos, Felix Palau, Carlos Aragón, Florent Engelmann, Domenico Carputo, Riccardo Aversano, Marcos Edel Martínez, José Carlos Lorenzo

Department of Soil, Plant, Environmental and Animal Production Sciences, University of Naples Federico II, Naples, Italy.
Faculty of Agronomy, Universidad de Ciego de ávila, Ciego de ávila, Cuba.
Institut de Recherche pour le Développement, UMR Diversité Adaptation et Developpement des Plantes, Montpellier, France.
Laboratory for Plant Breeding, Centro de Bioplantas, Universidad de Ciego de ávila, Ciego de ávila, Cuba.

DOI: 10.4236/ajps.2013.44103                

Abstract

The objective of this work was to evaluate if cryostorage of Phaseolus vulgaris L. seeds induced variations in regenerated plants at the phenotypic and molecular levels. A series of agricultural traits was measured on plants grown from control, non-cryopreserved and cryopreserved seeds, and the genetic stability of plants of the second generation was analysed at selected microsatellite loci. The phenotype of the second generation plants was evaluated as well. No statistically significant phenotypic differences were observed for the parameters measured, neither in the first nor in the second generations. Averaging both treatments, about 76% of the seeds had germinated 10 days after sowing. At harvest we recorded plants with about 73 cm in height, 13 stem internodes, 25 fruits, 103 grains and 4 grains per fruit. One hundred seeds weighted about 26 g. The genetic analyses performed on the second generation plants using six nuclear Simple Sequences Repeats (SSR) markers revealed no changes in microsatellite length between control and cryopreserved samples, implying that there was no effect of seed liquid nitrogen exposure on genome integrity. The phenotypic and molecular results reported here confirm that cryostorage is an efficient and reliable technique to conserve P. vulgaris seeds and regenerate true-to-type plants.

Keywords

Common Bean; Cryostorage; Genotype Variation; Phenotype Variation

Conflicts of Interest

The authors declare no conflicts of interest.

References

[1]	FAOSTAT, 2010. http://faostat.fao.org/
[2]	R. M. Welch, W. A. House, S. Beebe and Z. Cheng, “Genetic Selection for Enhanced Bioavailable Levels of Iron in Bean (Phaseolus vulgaris L.) Seeds,” Journal of Agricultural and Food Chemistry, Vol. 48, No. 8, 2000, pp. 3576-3580. doi:10.1021/jf0000981
[3]	P. B. Geil and J. W. Anderson, “Nutrition and Health Implications of Dry Beans: A Review,” Journal of the American College of Nutrition, Vol. 13, No. 6, 1994, pp. 549-558.
[4]	F. Engelmann and V. R. Rao, “Major Research Challenges and Directions for Future Research,” In: M. N. Normah, H. F. Chin and B. M. Reed, Eds., Conservation of Tropical Plant Species, Springer Verlag, Berlin, 2012.
[5]	K. Harding, “Genetic Integrity of Cryopreserved Plant Cells: A Review,” Cryoletters, Vol. 25, No. 1, 2004, pp. 3-22.
[6]	P. Revilla, A. Butrón, M. E. Cartea, R. A. Malvar and A. Ordás, “Breeding for Cold Tolerante,” In: M. Ashraf and P. J. C. Harris, Ed., Abiotic Stresses: Plant Resistance through Breeding and Molecular Approaches, Food Products Press, New York, 2005, pp. 301-400.
[7]	K. J. Dietz, “Plant Peroxidoxins,” Annual Review of Plant Biology, Vol. 54, 2003, pp. 93-107. doi:10.1146/annurev.arplant.54.031902.134934
[8]	P. Berjak, P. Bartels, E. Benson, K. Harding, D. J. Mycock, N. W. Pammenter and J. Wesley-Smith, “Cryoconservation of South African Plant Genetic Diversity,” In Vitro Cellular & Developmental Biology, Vol. 47, No. 1, 2012, pp. 65-81.
[9]	L. M. Díaz and M. W. Blair, “Race Structure within the Mesoamerican Gene Pool of Common Bean (Phaseolus vulgaris L.) as Determined by Microsatellite Markers,” Theoretical and Applied Genetics, Vol. 114, No. 1, 2006, pp. 143-154. doi:10.1007/s00122-006-0417-9
[10]	L. R. Hanai, T. Campos, L. E. A. Camargo, L. L. Benchimol, A. P. Souza, M. Melotto, S. A. M. Carbonell, A. F. Chioratto, L. Consoli, E. F. Formighieri, M. V. B. M. Siqueira, S. M. Tsai and M. L. C. Vieira, “Development, Characterization, and Comparative Analysis of Polymorphism at Common Bean SSR Loci Isolated from Genic and Genomic Sources,” Genome, Vol. 50, No. 3, 2007, pp. 266-277. doi:10.1139/G07-007
[11]	M. W. Blair, L. M. Díaz, H. F. Buendía and M. C. Duque, “Genetic Diversity, Seed Size Associations and Population Structure of a Core Collection of Common Beans (Phaseolus vulgaris L.),” Theoretical and Applied Genetics, Vol. 119, No. 6, 2009, pp. 955-972. doi:10.1007/s00122-009-1064-8
[12]	T. Campos, P. R. Oblessuc, D. A. Sforça, J. M. K. Cardoso, R. M. Baroni, A. C. B. Sousa, S. A. M. Carbonell, A. F. Chioratto, A. A. F. Garcia, L. B. Rubiano and A. P. Souza, “Inheritance of Growth Habit Detected by Genetic Linkage Analysis Using Microsatellites in the Common Bean (Phaseolus vulgaris L.),” Molecular Breeding, Vol. 27, No. 4, 2011, pp. 549-560. doi:10.1007/s11032-010-9453-x
[13]	F. Engelmann, “Use of Biotechnologies for Conserving Plant Biodiversity,” Acta Horticulturae, Vol. 812, 2009, pp. 63-82.
[14]	I. Cejas, K. Viveas, T. Laudat, J. González-Olmedo, F. Engelmann, M. E. Martínez-Montero and J. C. Lorenzo, “Effects of Cryopreservation of Phaseolus vulgaris L. Seeds on Early Stages of Germination,” Plant Cell Reports, Vol. 31, No. 11, 2012, pp. 2065-2073. doi:10.1007/s00299-012-1317-x
[15]	K. Yu, S. J. Park, V. Poysa and P. Gepts, “Integration of Simple Sequence Repeat (SSR) Markers into a Molecular Linkage Map of Common Bean (Phaseolus vulgaris L.),” Journal of Heredity, Vol. 91, No. 6, 2000, pp. 429-434. doi:10.1093/jhered/91.6.429
[16]	D. Sicard, L. Nanni, O. Porfiri, D. Bulfon and R. Papa, “Genetic Diversity of Phaseolus vulgaris L. and P. coccineus L. Landraces in Central Italy,” Plant Breeding, Vol. 124, No. 5, 2005, pp. 464-472. doi:10.1111/j.1439-0523.2005.01137.x
[17]	R. K. Kalia, M. K. Rai, S. Kalia, R. Singh and A. K. Dhawan, “Microsatellite Markers: An Overview of the Recent Progress in Plants,” Euphytica, Vol. 177, No. 3, 2011, pp. 309-333. doi:10.1007/s10681-010-0286-9
[18]	K. Harding and E. E. Benson, “The Use of Microsatellite Analysis in Solanum tuberosum L. in Vitro Plantlets Derived from Cryopreserved Germplasm,” Cryo Letters, Vol. 22, No. 3, 2001, pp. 199-208.
[19]	N. R. F. Castillo, N. V. Bassil, S. Wada and B. M. Reed, “Genetic Stability of Cryopreserved Shoot Tips of Rubus germplasm,” In Vitro Cellular & Developmental Biology-Plant, Vol. 46, No. 3, 2010, pp. 246-256.
[20]	E. Benson, “Cryopreservation of Phytodiversity: A Critical Appraisal of Theory & Practice,” Critical Reviews in Plant Sciences, Vol. 27, No. 3, 2008, pp. 141-219. doi:10.1080/07352680802202034
[21]	F. Engelmann, “Use of Biotechnologies for the Conservation of Plant Biodiversity,” In Vitro Cellular and Developmental Biology-Plant, Vol. 41, No. 7, 2011, pp. 5-16.
[22]	S. Fukai, M. Goi and M. Tanaka, “The Chimeric Structure of the Apical Dome of Chrysanthemum (Dendranthema grandiflorum (Ramat.) Kitamura) Is Affected by Cryopreservation,” Scientia Horticulturae, Vol. 57, No. 4, 1994, pp. 347-351. doi:10.1016/0304-4238(94)90117-1
[23]	J. J. Medina, I. Clavero-Ramírez, M. E. González-Benito, J. Gálvez-Farfán, J. M. López-Aranda and C. Soria, “Field Performance Characterization of Strawberry (Fragaria × ananassa Duch.) Plants Derived from Cryopreserved Apices,” Scientia Horticulturae, Vol. 113, No. 1, 2007, pp. 28-32. doi:10.1016/j.scienta.2007.01.030
[24]	E. K. Konan, T. Durand-Gasselin, Y. J. Koadio, A. C. Niamké, D. Dumet, Y. Duval, A. Rival and F. Engelmann, “Field Development of Oil Palms (Elaeis guineensis Jacq.) Originating from Cryopreserved Stabilized Polyembryonic Cultures (SPCs),” Cryo Letters, Vol. 28, No. 5, 2007, pp. 377-386.
[25]	G. Mix-Wagner, H. M. Schumacher and R. J. Cross, “Recovery of Potato Apices after Several Years of Storage in Liquid Nitrogen,” Cryo Letters, Vol. 24, No. 1, 2003, pp. 33-41.
[26]	F. X. Côte, O. Goue, R. Domergue, B. Panis and C. Jenny, “In-Field Behavior of Banana Plants (Musa AA sp.) Obtained after Regeneration of Cryopreserved Embryogenic Cell Suspensions,” Cryo Letters, Vol. 21, No. 1, 2000, pp. 19-24.
[27]	M. E. Martínez-Montero, M. T. González-Arnao, C. Borroto-Nordelo, C. Puentes-Diaz and F. Engelmann, “Cryopreservation of Sugarcane Embryogenic Callus Using a Simplified Freezing Process,” Cryo Letters, Vol. 19, No. 3, 1998, pp. 171-176.
[28]	M. E. Martínez-Montero, E. Ojeda, A. Espinosa, M. Sánchez, R. Castillo, M. T. González-Arnao, F. Engelmann and J. C. Lorenzo, “Field Performance of Cryopreserved Callus-Derived Sugarcane Plants,” Cryo Letters, Vol. 23, No. 1, 2002, pp. 21-26.
[29]	C. Walters, L. J. Wheeler and P. C. Stanwood, “Longevity of Cryogenically Stored Seeds,” Cryobiology, Vol. 48, No. 3, 2004, pp. 229-244. doi:10.1016/j.cryobiol.2004.01.007
[30]	C. W. Vertucci and E. E. Roos, “Theoretical Basis of Protocols for Seed Storage II. The Influence of Temperature on Optimal Moisture Levels,” Seed Science Research, Vol. 3, No. 3, 1993, pp. 201-213. doi:10.1017/S0960258500001793