Abstract
Stone pine (Pinus pinea L.) is a Mediterranean forest species used for the commercial harvesting of its edible seeds, known as pine nuts. Plant regeneration by somatic embryogenesis on semi-solid medium has been achieved in stone pine, but suspension culture systems for the large-scale production of somatic embryos need to be developed. Suspension cultures were established from several embryogenic lines. Growth parameters as settled cell volume (SCV) and dry weight (DW) in suspension cultures were influenced by genotype, initial inoculum density, orbiting speed and type of carbohydrate in the culture medium. A higher linear growth (DW) up to week four was obtained using 20 mg mL−1 inoculum. The orbiting speed affected the SCV as well as the morphology of embryogenic cultures. Growth parameters decreased when orbiting speed was increased from 50 rpm to 150 rpm. Cultures at 50 rpm most commonly grew as clusters of embryonal cell masses, whereas at 150 rpm there were large numbers of single cells. Cultures grew slowly in medium with maltose instead of sucrose, but growth was not affected when plant growth regulators (PGRs) concentration was decreased. A sustainable suspension culture could be maintained by transferring 250 mg of filtered cell mass to 25 mL of liquid medium at 3-week intervals. Culture scaling-up in liquid medium resulted in a 23-fold DW increase in 6 weeks, compared with 12 fold increase on semi-solid medium. Cotyledonary embryos were obtained on semi-solid maturation medium from lines proliferated in suspension cultures. Increased somatic embryo production was obtained when proliferation was performed at 100 rpm orbiting speed in liquid medium with low PGRs concentration and sucrose instead of maltose.
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Acknowledgements
Funds were provided by the Spanish Ministry of Education and Science (AGL2010-22292-C03-01); the Madrid Regional R&D program S2009AMB-1668 (REGENFOR-CM) and IMIDRA grant to N. Gonzalez Cabrero. The participation of Dr. D. López-Vela and Dr. N. Alonso-Blázquez in some experiments is highly appreciated. Authors thank the comments and suggestions of two anonymous reviewers, which served to improve the manuscript. This work is part of the requirements to fulfill the N. Gonzalez-Cabrero Ph.D. degree.
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NG-C and MR-G participated in the experimental design, conducted experiments and helped to draft the manuscript. JA performed the statistical analyses and helped to draft the manuscript. MT wrote the final version of the manuscript. CC conceived the study, designed the experiments and drafted the manuscript. All authors read and approved the final manuscript.
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Communicated by Maurizio Lambardi.
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Supplementary material 1—Fig s1 Maturation and conversion into plants of somatic embryos from suspension cultures of Pinus pinea. a Maturing somatic embryos of 2F47 line cultured on 121 µM ABA-containing medium. Bar 1 mm. b Cotyledonary embryo after 12 weeks of culture. Bar 1 mm. c Germinating somatic embryos following 4 weeks on germination medium. Bar 1 cm. d Somatic seedlings growing in forest containers after 4 months of acclimatization in a mist chamber. Bar 5 cm (PDF 113 KB)
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González-Cabrero, N., Ruiz-Galea, M., Alegre, J. et al. Growth, morphology and maturation ability of Pinus pinea embryogenic suspension cultures. Plant Cell Tiss Organ Cult 135, 331–346 (2018). https://doi.org/10.1007/s11240-018-1467-9
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DOI: https://doi.org/10.1007/s11240-018-1467-9