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Effect of ABA, the auxin antagonist PCIB and partial desiccation on stone pine somatic embryo maturation

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Abstract

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.

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References

  • Abrahamsson M, Valladares S, Larsson E, Clapham D, von Arnold S (2012) Patterning during somatic embryogenesis in Scots pine in relation to polar auxin transport and programmed cell death. Plant Cell Tissue Organ Cult 109:391–400. doi: 10.1007/s11240-011-0103-8

    Article  CAS  Google Scholar 

  • Alonso P, Moncaleán P, Fernández B, Rodríguez A, Centeno ML, Ordás RJ (2006) An improved micropropagation protocol for stone pine (Pinus pinea L.). Ann For Sci 63:879–885. doi: 10.1051/forest:2006071

    Article  CAS  Google Scholar 

  • Álvarez JM, Bueno N, Cortizo M, Ordás RJ (2013) Improving plantlet yield in Pinus pinaster somatic embryogenesis. Scand J For Res 28:613–620. doi: 10.1080/02827581.2013.821516

    Article  Google Scholar 

  • Aronen T, Pehkonen T, Ryynänen L (2009) Enhancement of somatic embryogenesis from immature zygotic embryos of Pinus sylvestris. Scand J For Res 24:372–383. doi: 10.1080/02827580903228862

    Article  Google Scholar 

  • Barthélémy D, Caraglio Y (2007) Plant architecture: a dynamic, multilevel and comprehensive approach to plant form, structure and ontogeny. Ann Bot 99:375–407. doi:10.1093/aob/mcl260

    Article  PubMed  PubMed Central  Google Scholar 

  • Belz RG, Piepho H-P (2014) Interspecies variability of plant hormesis by the antiauxin PCIB in a laboratory bioassay. J Plant Growth Regul 33:499–512. doi: 10.1007/s00344-013-9400-2

    Article  CAS  Google Scholar 

  • Bonga JM (2004) The effect of various culture media on the formation of embryo-like structures in cultures derived from explants taken from mature Larix decidua. Plant Cell Tissue Organ Cult 77:43–48. doi: 10.1023/B:TICU.0000016488.79965.b7

    Article  Google Scholar 

  • Breton D, Harvengt L, Trontin JF, Bouvet A, Favre JM (2005) High subculture frequency, maltose-based and hormone-free medium sustained early development of somatic embryos in maritime pine. In Vitro Cell Dev Biol—Plant 41:494–504. doi: 10.1079/IVP2005671

    Article  CAS  Google Scholar 

  • Breton D, Harvengt L, Trontin JF, Bouvet A, Favre JM (2006) Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine. Plant Cell Tissue Organ Cult 87:95–108. doi: 10.1007/s11240-006-9144-9

    Article  Google Scholar 

  • Carneros E, Celestino C, Klimaszewska K, Park Y-S, Toribio M, Bonga JM (2009) Plant regeneration in Stone pine (Pinus pinea L.) by somatic embryogenesis. Plant Cell Tissue Organ Cult 98:165–178. doi: 10.1007/s11240-009-9549-3

    Article  CAS  Google Scholar 

  • Carrasquinho I, Freire J, Rodrigues A, Tomé M (2010) Selection of Pinus pinea L. plus tree candidates for cone production. Ann For Sci 67:814. doi: 10.1051/forest/2010050

    Article  Google Scholar 

  • Chand S, Sahrawat AK (2001) Stimulatory effect of partial desiccation on plant regeneration in indica rice (Oryza sativa L). J Plant Biochem Biotechnol 10:43–47. doi:10.1007/BF03263105

    Article  CAS  Google Scholar 

  • Claeys H, Van Landeghem S, Dubois M, Maleux K, Inzé D (2014) What is stress? Dose-response effects in commonly used in vitro stress assays. Plant Physiol 165:519–527. doi: 10.1104/pp.113.234641

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Drew AP, Ledig FT (1980) Episodic growth and relative shoot: root balance in loblolly pine seedlings. Ann Bot 45:143–148. doi: 10.1093/oxfordjournals.aob.a085805

    Article  Google Scholar 

  • Dyachok JV, Wiweger M, Kenne L, von Arnold S (2002). Endogenous Nod-factor-like signal molecules promote early somatic embryo development in Norway spruce. Plant Physiol 128:523–533. doi: 10.1104/pp.010547

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Egertsdotter U, von Arnold S (1998) Development of somatic embryos in Norway spruce. J Exp Bot 49:155–162. doi: 10.1093/jxb/49.319.155

    Article  CAS  Google Scholar 

  • Find J, Grace L, Krogstrup P (2002) Effect of anti-auxins on maturation of embryogenic tissue cultures of Nordmanns fir (Abies nordmanniana). Physiol Plant 116:231–237. doi: 10.1034/j.1399-3054.2002.1160213.x

    Article  CAS  PubMed  Google Scholar 

  • Humánez A, Blasco M, Brisa C, Segura J, Arrillaga I (2012) Somatic embryogenesis from different tissues of Spanish populations of maritime pine. Plant Cell Tissue Organ Cult 111:373–383. doi: 10.1007/s11240-012-0203-0

    Article  Google Scholar 

  • Jalonen P, von Arnold S (1991) Characterization of embryogenic cell lines of Picea abies in relation to their competence for maturation. Plant Cell Rep 10:384–387. doi: 10.1007/BF00232606

    Article  CAS  PubMed  Google Scholar 

  • Kim YW, Moon HK (2007) Regeneration of plant by somatic embryogenesis in Pinus rigida x P. taeda. In Vitro Cell Dev Biol—Plant 43:335–342. doi: 10.1007/s11627-007-9045-6

    Article  CAS  Google Scholar 

  • Klimaszewska K, Cyr DR (2002) Conifer somatic embryogenesis: I. development. Dendrobiology 48:31–39

    Google Scholar 

  • Klimaszewska K, Park Y-S, Overton C, MacEacheron I, Bonga JM (2001) Optimized somatic embryogenesis in Pinus strobus L. In Vitro Cell Dev Biol—Plant 37:392–399. doi: 10.1007/s11627-001-0069-z

    Article  Google 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

    Chapter  Google 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

  • Konrádová H, Gricová M, Lipavská H (2003) Cold-induced accumulation of raffinose family oligosaccharides in somatic embryos of Norway spruce (Picea abies). In Vitro Cell Dev Biol—Plant 39:425–427. doi: 10.1079/IVP2003426

    Article  Google Scholar 

  • Krajňáková J, Häggman H, Gömöry D (2009) Effect of sucrose concentration, polyethylene glycol and activated charcoal on maturation and regeneration of Abies cephalonica somatic embryos. Plant Cell Tissue Organ Cult 96:251–262. doi: 10.1007/s11240-008-9482-x

    Article  Google Scholar 

  • Kumria R, Sunnichan VG, Das DK, Gupta SK, Reddy VS, Bhatnagar RK, Leelavathi S (2003) High-frequency somatic embryo production and maturation into normal plants in cotton (Gossypium hirsutum) through metabolic stress. Plant Cell Rep 21:635–639. doi: 10.1007/s00299-002-0554-9

    CAS  PubMed  Google Scholar 

  • Lelu-Walter MA, Pâques LE (2009) Simplified and improved somatic embryogenesis of hybrid larches (Larix x eurolepis and Larix × marschlinsii). Perspectives for breeding. Ann For Sci 66:104. doi: 10.1051/forest/2008079

    Article  Google Scholar 

  • Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2006) Simplified and improved somatic embryogenesis for clonal propagation of Pinus pinaster (Ait.). Plant Cell Rep 25:767–776. doi: 10.1007/s00299-006-0115-8

    Article  CAS  PubMed  Google Scholar 

  • Lelu-Walter MA, Bernier-Cardou M, Klimaszewska K (2008) Clonal plant production from self- and cross-pollinated seed families of Pinus sylvestris (L.) through somatic embryogenesis. Plant Cell Tissue Organ Cult 92:31–45. doi: 10.1007/s11240-007-9300-x

    Article  Google Scholar 

  • Lelu-Walter MA, Thompson D, Harvengt L, Sanchez L, Toribio M, Pâques LE (2013) Somatic embryogenesis in forestry with a focus on Europe: state-of-the-art, benefits, challenges and future direction. Tree Genet Genomes 9:883–899. doi: 10.1007/s11295-013-0620-1

    Article  Google Scholar 

  • 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

    Chapter  Google Scholar 

  • Liao YK, Juan I-P (2015) Improving the germination of somatic embryos of Picea morrisonicola Hayata: effects of cold storage and partial drying. J For Res 20:114–124. doi: 10.1007/s10310-014-0445-2

    Article  CAS  Google Scholar 

  • Liao YK, Liao CK, Ho YL (2008) Maturation of somatic embryos in two embryogenic cultures of Picea morrisonicola Hayata as affected by alternation of endogenous IAA content. Plant Cell Tissue Organ Cult 93:257–268. doi: 10.1007/s11240-008-9371-3

    Article  CAS  Google Scholar 

  • Litvay JD, Verma DC, Johnson MA (1985) Influence of a loblolly pine (Pinus taeda L.). Culture medium and its components on growth and somatic embryogenesis of the wild carrot (Daucus carota L.). Plant Cell Rep 4:325–328. doi: 10.1007/BF00269890

    Article  CAS  PubMed  Google Scholar 

  • Lu J, Vahala J, Pappinen A (2011) Involvement of ethylene in somatic embryogenesis in Scots pine (Pinus sylvestris L.). Plant Cell Tissue Organ Cult 107:25–33. doi: 10.1007/s11240-011-9952-4

    Article  CAS  Google Scholar 

  • Malabadi RB, van Staden J (2005) Somatic embryogenesis from vegetative shoot apices of mature trees of Pinus patula. Tree Physiol 25:11–16. doi: 10.1093/treephys/25.1.11

    Article  PubMed  Google Scholar 

  • Malabadi RB, Choudhury H, Tandon P (2004) Initiation, maintenance and maturation of somatic embryos from thin apical dome sections in Pinus kesiya (Royle ex. Gord) promoted by partial desiccation and gellan gum. Sci Hort 102:449–459. doi: 10.1016/j.scienta.2004.06.001

    Article  Google Scholar 

  • Maruyama TE, Hosoi Y (2012) Post-maturation treatment improves and synchronizes somatic embryo germination of three species of Japanese pines. Plant Cell Tissue Organ Cult 110:45–52. doi: 10.1007/s11240-012-0128-7

    Article  Google Scholar 

  • Miguel C, Gonçalves S, Tereso S, Marum L, Maroco J, Oliveira M (2004) Somatic embryogenesis from 20 open-pollinated families of Portuguese plus trees of maritime pine. Plant Cell Tissue Organ Cult 76:121–130. doi: 10.1023/B:TICU.0000007253.91771.e3

    Article  CAS  Google Scholar 

  • Montalbán IA, De Diego N, Moncaleán P (2010) Bottlenecks in Pinus radiata somatic embryogenesis: improving maturation and germination. Trees 24:1061–1071. doi: 10.1007/s00468-010-0477-y

    Article  Google Scholar 

  • Montalbán IA, De Diego N, Aguirre Igartua E, Setién A, Moncaleán P (2011) A combined pathway of somatic embryogenesis and organogenesis to regenerate radiata pine plants. Plant Biotechnol Rep 5:177–186. doi: 10.1007/s11816-011-0171-6

    Article  Google Scholar 

  • Montalbán IA, Setién-Olarra A, Hargreaves CL, Moncaleán P (2013) Somatic embryogenesis in Pinus halepensis Mill.: an important ecological species from the Mediterranean forest. Trees 27:1339–1351. doi: 10.1007/s00468-013-0882-0

    Article  Google Scholar 

  • Mutke S, Gordo J, Gil L (2005) Cone yield characterization of a Stone pine (Pinus pinea L.) clone bank. Silvae Genetica 54:189–197

    Article  Google Scholar 

  • Oono Y, Ooura C, Rahman A, Aspuria ET, Hayashi KI, Tanaka A, Uchimiya H (2003) p-Chlorophenoxyisobutyric acid impairs auxin response in Arabidopsis root. Plant Physiol 133:1135–1147. doi: 10.1104/pp.103.027847

    Article  CAS  PubMed  PubMed Central  Google 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

    Article  Google Scholar 

  • Paquette A, Messier C (2010) The role of plantations in managing the world’s forests in the Anthropocene. Front Ecol Environ 8:27–34. doi: 10.1890/080116

    Article  Google Scholar 

  • Park Y-S (2014) Conifer somatic embryogenesis and multi-varietal forestry. In: Fenning T (ed) Challenges and Opportunities for the World’s Forests in the 21st Century. Forestry Sciences vol 81. Springer, Dordercht, pp 425–439. doi: 10.1007/978-94-007-7076-8_17

    Chapter  Google 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

    Article  Google Scholar 

  • Ramarosandratana A, Harvengt L, Bouvet A, Calvayrac R, Pâques M (2001) Influence of the embryonal-suspensor mass (ESM) sampling on development and proliferation of maritime pine somatic embryos. Plant Sci 160:473–479. doi: 10.1016/S0168-9452(00)00410-6

    Article  CAS  PubMed  Google Scholar 

  • Schenk RU, Hildebrandt AC (1972) Medium and techniques for induction and growth of monocotyledonous and dicotyledonous plant cell cultures. Can J Bot 50:199–204. doi: 10.1139/b72-026

    Article  CAS  Google Scholar 

  • Shimizu M, Miyazawa Y, Fujii N, Takahashi H (2008) p-Chlorophenoxyisobutyric acid impairs auxin response for gravity-regulated peg formation in cucumber (Cucumis sativus) seedlings. J Plant Res 121:107–114. doi: 10.1007/s10265-007-0121-0

    Article  CAS  PubMed  Google Scholar 

  • Stasolla C, Yeung EC (2003) Recent advances in conifer somatic embryogenesis: improving somatic embryo quality. Plant Cell Tissue Organ Cult 74:15–35. doi: 10.1023/A:1023345803336

    Article  CAS  Google Scholar 

  • Stasolla C, Kong L, Yeung EC, Thorpe TA (2002) Maturation of somatic embryos in conifers: morphogenesis, physiology, biochemistry, and molecular biology. In Vitro Cell Dev Biol—Plant 38:93–105. doi: 10.1079/IVP2001262

    Article  CAS  Google Scholar 

  • StatSoft. Inc (1996) STATISTICA for Windows. Tulsa. OK

  • Suprasanna P, Rupali C, Desai NS, Bapat VA (2008) Partial desiccation augments plant regeneration from irradiated embryogenic cultures of sugarcane. Plant Cell Tissue Organ Cult 92:101–105. doi: 10.1007/s11240-007-9299-z

    Article  Google Scholar 

  • von Arnold S, Sabala I, Bozhkov P, Dyachok J, Filonova L (2002) Developmental pathways of somatic embryogenesis. Plant Cell Tissue Organ Cult 69:233–249. doi: 10.1023/A:1015673200621

    Article  Google Scholar 

  • von Aderkas P, Pattanavibool R, Hristoforoglu K, Ma Y (2003) Embryogenesis and genetic stability in long term megagametophyte-derived cultures of larch. Plant Cell Tissue Organ Cult 75:27–34. doi: 10.1023/A:1024614209524

    Article  Google Scholar 

  • Vondráková Z, Eliášová K, Fischerová L, Vágner M (2011) The role of auxins in somatic embryogenesis of Abies alba. Cent Eur J Biol 6:587–596. doi: 10.2478/s11535-011-0035-7

    Google Scholar 

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Acknowledgements

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.

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  1. Research Institute of Madrid for Food, Agriculture and Rural Development (IMIDRA), Finca El Encín, Apdo 127, 28800, Alcalá de Henares, Madrid, Spain

    Elena Carneros, Mariano Toribio & Cristina Celestino

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  1. Elena Carneros
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  2. Mariano Toribio
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  3. Cristina Celestino
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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.

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Correspondence to Cristina Celestino.

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Communicated by Sergio J. Ochatt.

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Carneros, E., Toribio, M. & Celestino, C. Effect of ABA, the auxin antagonist PCIB and partial desiccation on stone pine somatic embryo maturation. Plant Cell Tiss Organ Cult 131, 445–458 (2017). https://doi.org/10.1007/s11240-017-1296-2

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