Long-term subculture randomly affects morphology and subsequent maturation of early somatic embryos in maritime pine

Abstract

Somatic embryogenesis is affected by highly variable maturation yields in Pinus pinaster. Origins of this variability were investigated by testing effects of spatial and temporal division of initial embryogenic tissue into independently cultivated pieces. One embryogenic cell-line was proliferated to obtain six embryonal-suspensor masses (ESM) treated as six sub-lines within a single dish. After proliferation to reach 2, 4 and 8 dishes (12, 24, 48 ESM), the 48 ESM (six sub-lines) were maintained by weekly subculture (34 weeks). ESM observations and maturation tests were regularly performed during the amplification and maintenance periods. Relations between maturation yields as well as cotylfedonary embryo length and immature embryo morphology were analyzed. ESM yielded variable results, independently from culture spatial (dish) and temporal (sub-line) subdivision. Maturation yields and length of regenerated embryos globally decreased as a function of subculture number. Concomitantly, morphological degradation of immature embryos occurred, indicating a global loss of embryogenic ability. Maturation variability probably results from immature embryos heterogeneity, which could be increased by manipulations during subculture. This is the first time that this evolution along time in culture is precisely described in conifers somatic embryos. Keywords Maturation AE Pinus pinaster AE Somatic embryogenesis AE Subculture AE Culture ageing Abbreviations ESM embryonal-suspensor masses f.wt fresh weight CE cotyledonary somatic embryos AE abnormally developed embryos PC precotyledonary and early cotyledonary embryos Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at http://dx.

Somatic Embryogenesis for More Effective Breeding and Deployment of Improved Varieties in Pinus spp.: Bottlenecks and Recent Advances

“…EM culture subdivision during proliferation at both spatial (Petri dish) and temporal levels (subline) did not affect the se production capacity of P. pinaster EM lines (Breton et al 2006), indicating that environmental conditions were homogeneous and standardized. In contrast, EM growth was strongly impacted by cell density on the filter paper (optimal in the range of 50-100 mg fresh mass for most embryogenic lines) and medium formulation.…”

“…Considering the ratio of fully developed cotyledonary se to precotyledonary and abnormal se at various sampling times during proliferation, Breton et al (2006) showed that the maturation of se was qualitatively unchanged, but that it quantitatively and progressively reduced as the number of consecutive subcultures and, hence, the chronological age of the cultures increased.…”

“…Cryopreservation of embryogenic lines shortly after initiation (usually within 2-4 months post-initiation) is necessary because se production capacity decreases rapidly during proliferation (between 6-10 months of subculture; Breton et al 2006;Trontin et al 2011). Considering the ratio of fully developed cotyledonary se to precotyledonary and abnormal se at various sampling times during proliferation, Breton et al (2006) showed that the maturation of se was qualitatively unchanged, but that it quantitatively and progressively reduced as the number of consecutive subcultures and, hence, the chronological age of the cultures increased.…”

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“…Since elite genotype www.intechopen.com selection is on the basis of both in vitro and ex vitro evaluations, the process from somatic embryo initiation to mass seedling production can be long. A consequence of this extended time period is that embryogenicity may diminish or even be irrevocably lost in a few weeks or months (Breton et al, 2006). A way around this problem is to cryopreserve tissues while they are at their peak productivity, shortly after embryogenic tissue induction and when enough tissue is available (Kong and Aderkas, 2011).…”

New Challenges and Opportunities of Protein Engineering in Plants

“…Maybe the main reason of this has been the difficulty to make transgenic crop plants, and the limited number of full sequenced genomes compared to microorganisms until now. Although at present, it is possible to transform almost all crop plants [3], including some trees like poplar [4] and pine [5,6], and the number of full sequenced genomes of plant species is highly increased in the last years; thanks to next generation sequencing techniques [7]. In the present moment, we have enough technology to attend the protein engineering in plants.…”