Influence of the embryonal-suspensor mass (ESM) sampling on development and proliferation of maritime pine somatic embryos

Towards genetic engineering of maritime pine (Pinus pinaster Ait.)

“…ESM were initiated from immature zygotic embryos according to the method of Bercetche and Pâques [3] and weekly subcultured in the dark (25 ± 1 o C). ESM maintenance and plantlet regeneration were done according to Ramarosandratana et al [36,37]. Petri dishes are sealed with two rounds of cling film.…”

“…Using our improved protocols for maturation and germination of somatic embryos in maritime pine [3,36,37] somatic plants were recovered from hygromycin-resistant lines (figure 2) produced either by particle bombardment of genotype 1463-13 or Agrobacterium-mediated DNA transfer to genotype PN519. Selection and stabilisation of hygromycinresistant lines were achieved within one to four months, depending on their growth rate ( figure 2A).…”

“…At this stage, all transformed materials were cryopreserved using an efficient technique developed by AFOCEL in order to maintain juvenility and maturation ability. When transferred onto our improved maturation media [36] using adapted ESM sampling [37], hygromycin-resistant lines produced cotyledonary somatic embryos within 3 months (figure 2B). These embryos were able to germinate.…”

“…Compared to horticultural cuttings usually subjected to decreased rooting with ageing, in vitro biotechnology is considered to have greater potential for sustained clonal propagation at lower cost, especially the most powerful somatic embryogenesis technique coupled with long-term frozen storage to preserve juvenility [34,42,45]. Since our initial work [3], significant improvement of protocols from initiation of embryonal-suspensor masses (ESM) to maturation of somatic embryos have been obtained [28,36,37].…”

“…Based on the expertise of AFOCEL on somatic embryogenesis [3,36,37], we used ESM initiated from selected seeds as target tissues for transformation experiments to produce transgenic maritime pines. Two different methods commonly used for successful transformation of plants were independently evaluated: (i) the microprojectile bombardment technique (biolistic) using our affordable method previously developed for Picea abies [4] and (ii) a modification of the Agrobacterium tumefaciens-mediated protocol established by Levée et al [30] to transform Pinus strobus.…”