Effect of ABA, the auxin antagonist PCIB and partial desiccation on stone pine somatic embryo maturation
- 298 Downloads
Plant regeneration by somatic embryogenesis (SE) was achieved in Pinus pinea L., a forest species of economic importance for its edible seeds, but improvements in the SE protocol are needed to make this technology feasible for breeding programs. In the present study, different maturation treatments on medium with high concentration of sucrose and gelling agent were tested. The effects of abscisic acid (ABA) concentration and culture procedure, the presence of the auxin antagonist 2-(4-chlorophenoxy)2-methylpropionic acid (PCIB), and the partial desiccation of embryonal masses before maturation on the reduction of proliferation and promotion of maturation in six embryogenic lines were evaluated. Increasing ABA concentration neither reduced proliferation nor improved maturation. The highest number of mature embryos was produced with 121 μM ABA in line 1F11 or 161 μM ABA in line 2F47. The culture procedure did not affect growth rate, but monthly subcultures onto maturation medium increased the normal embryo production 13-fold by compared with no subculturing. PCIB decreased proliferation only when it was included during the 12 weeks of the maturation period, and did not improve somatic embryo production. Partial desiccation of embryonal masses between 5 to 26% water loss did not reduce proliferation but enhanced maturation by 1.7 to 4.7-fold compared with the control, depending on the embryogenic line. Up to 256 normal cotyledonary embryos per gram fresh weight from the best line and culture condition were obtained. Somatic embryos germinated and converted to plants at over 70%. Although improvements in maturation are provided, problems such as growth arrest of somatic seedlings and low rates of acclimatization still remain to be solved before SE can be used for large scale plant production in stone pine.
KeywordsConifers Forest biotechnology Pinus pinea L. Somatic embryogenesis Vegetative propagation
The authors gratefully thank N. Cleto and Y. Vinuesa for their technical assistance. The suggestions of two anonymous reviewers that largely improved the manuscript are highly appreciated. Funds were provided by the Spanish National R + D Program (Projects AGL2007-66345-CO2-01 and AGL2010-22292-C03-01) and IMIDRA and INIA grants to E. Carneros. We wish to thank the National Forest Breeding Centre “Puerta de Hierro” (Madrid) of the Spanish Ministry of Environment and Dr. Mutke for all their help in collecting plant material.
EC, MT and CC conceived and designed research. EC and CC conducted experiments. EC, CC and MT analyzed results. MT wrote the manuscript with assistance from CC and EC. All authors read and approved the final manuscript.
- Klimaszewska K, Cyr DR (2002) Conifer somatic embryogenesis: I. development. Dendrobiology 48:31–39Google Scholar
- Klimaszewska K, Hargreaves C, Lelu-Walter M-A, Trontin J-F (2016) Advances in conifer somatic embryogenesis since year 2000. In: Germanà MA, Lambardi M (eds) In vitro embryogenesis in higher plants. Methods in molecular biology vol 1359. Springer Science + Business Media, New York, pp 131–166. doi: 10.1007/978-1-4939-3061-6_7 CrossRefGoogle Scholar
- Kong L, von Aderkas P (2007) Genotype effects on ABA consumption and somatic embryo maturation in interior spruce (Picea glauca x engelmanni). J Exp Bot 58:1525–1531. doi: https://doi.org/10.1093/jxb/erm019
- Lelu-Walter MA, Klimaszewska K, Miguel C, Aronen T, Hargreaves C, Teyssier C, Trontin JF (2016) Somatic embryogenesis for more effective breeding and deployment of improved varieties in Pinus spp.: Bottlenecks and recent advances. In: Loyola-Vargas V, Ochoa-Alejo N (eds) Somatic embryogenesis: fundamental aspects and applications. Springer, Cham, pp 319–365. doi: 10.1007/978-3-319-33705-0_19 CrossRefGoogle Scholar
- Othmani A, Bayoudh C, Drira N, Marrakchi M, Trifi M (2009) Somatic embryogenesis and plant regeneration in date palm Phoenix dactylifera L., cv. Boufeggous is significantly improved by fine chopping and partial desiccation of embryogenic callus. Plant Cell Tissue Organ Cult 97:71–79. doi: 10.1007/s11240-009-9500-7 CrossRefGoogle Scholar
- Park Y-S, Lelu-Walter MA, Harvengt L, Trontin JF, MacEacheron I, Klimaszewska K, Bonga JM (2006) Initiation of somatic embryogenesis in Pinus banksiana. P. strobus. P. pinaster and P. sylvestris at three laboratories in Canada and France. Plant Cell Tissue Organ Cult 86:87–101. doi: 10.1007/s11240-006-9101-7 CrossRefGoogle Scholar
- StatSoft. Inc (1996) STATISTICA for Windows. Tulsa. OKGoogle Scholar