The effect of cocultivation treatments on transformation efficiency in pea (<Emphasis Type="Italic">Pisum sativum</Emphasis> L.)

The effect of chemical additives (acetosyringone, AS; L-cysteine, CYS; dithiothreitol, DTT; glutathione, GSH; cellulase, CEL; pectinase, PEC) and light regimes (16/8 light/dark photoperiod, 16L/8D; continuous light, 24L; continuous dark, 24D) applied during cocultivation procedure of pea explants with Agrobacterium tumefaciens on transformation efficiency was studied. A hypervirulent strain of A. tumefaciens EHA 105 with two plasmids, namely pGT89 and pBIN19, both carrying reporter gus-int gene, and bar or nptII selectable marker gene, respectively, was used for genetic transformation of cotyledonary node explants of three dry seed pea cultivars Adept, Komet and Menhir. The focus was laid on cocultivation step (48 h) of transformation protocol. After chemical or physical treatments, transient GUS expression was recorded 20 days after cocultivation as a measure of successful transformation, using a four category scale (0 – without GUS expression, 1 – weak, 2 – medium and 3 – strong GUS expression) for calculation of IGE (Intensity of GUS Expression). Of the tested chemical cocultivation additives, 100 μM AS and 50 mg CYS significantly improved GUS expression (IGE value), while DTT, GSH and both macerating enzymes (CEL, PEC used either separately or in combination) either had no positive effect or were even negative. There were no statistically significant differences between the light regimes tested. Nevertheless, cocultivation in 24L, without chemical additives, reproducibly resulted in the highest frequency of explants scored in category 3 of GUS expression (followed by 24D and 16L/8D treatment). However, application of 100 μM AS reverted this trend. Cv. Adept yielded higher transformation frequencies than cvs. Menhir and Komet. Plasmid pGT89 produced a higher IGE value than pBIN19. Based on our results, the improved cocultivation step for pea consists of 48 h cocultivation at 20 ± 2°C, with 50 mg l⁻¹ CYS and 100 μM AS, 16L/8D photoperiod (or without AS in continuous light).     

Efficient Agrobacterium tumefaciens-mediated transformation of embryogenic calli and regeneration of Hevea brasiliensis Müll Arg. plants

An efficient procedure for producing transgenic Hevea brasiliensis callus and plant lines from clone PB 260 was established with Agrobacterium tumefaciens using strain EHA105 harbouring the vector pCAMBIA2301. Transformation capacity and competence of the embryogenic calli were improved after two cycles of cryopreservation. When the cocultivation temperature was reduced from 27 to 20°C, the duration of this phase could be increased up to 7 days, promoting an increase in GUS activity. These transformation conditions led to the isolation of 24 callus lines resistant to paromomycin, which is used as a selection agent. Nineteen of these lines revealed the existence of one to four copies of T-DNA by Southern-blot analysis. Nine of them were transferred for regeneration by somatic embryogenesis. Three hundred seventy-four transgenic plants have thus been generated from six independent lines bearing 1, 2 or 3 copies of T-DNA. The efficiency and reproducibility of this method means that functional characterization of genes involved in natural rubber production can be envisaged.     

Cell-surface modification of non-GMO without chemical treatment by novel GMO-coupled and -separated cocultivation method

We developed a novel method to coat living non-genetically modified (GM) cells with functional recombinant proteins. First, we prepared GM yeast to secrete constructed proteins that have two domains: a functional domain and a binding domain that recognizes other cells. Second, we cocultivated GM and non-GM yeasts that share and coutilize the medium containing recombinant proteins produced by GM yeasts using a filter-membrane-separated cultivation reactor. We confirmed that GM yeast secreted enhanced green fluorescent protein (EGFP) fusion proteins to culture medium. After cocultivation, EGFP fusion proteins produced by GM yeast were targeted to non-GM yeast (Saccharomyces cerevisiae BY4741ΔCYC8 strain) cell surface. Yeast cell-surface engineering is a useful method that enables the coating of GM yeast cell surface with recombinant proteins to produce highly stable and accumulated protein particles. The results of this study suggest that development of cell-surface engineering from GM organisms (GMOs) to living non-GMOs by our novel cocultivation method is possible.     

Evidence for an interaction effect during <Emphasis Type="Italic">in vitro</Emphasis> rooting of oil palm (<Emphasis Type="Italic">Elaeis guineensis</Emphasis> Jacq.) somatic embryo-derived plantlets

In vitro rooting of oil palm shoots derived from somatic embryos was achieved through a single-phase protocol in which three shoots are cultured in the same culture tube on an α-naphtaleacetic acid-enriched culture medium. Rooting performance was dependent on both the genetic origin and initial size of the shoot explants. All shoots from a given tube showed a tendency to give roots of the same type, independent of the original size of the explant. Whatever the clonal line, longer-size shoots (L-type: >9 cm) showed higher rooting rates than medium-size (M-type: 7–9 cm) and short-size ones (S-type: 5–7 cm). When groups of three shoots from the same clonal line were rooted together in the same culture tube, the combination of plant size within the group impacted overall quality of rooting. Within triplets of shoots containing more than one short individual, the probability of obtaining adequate rooting was low. Similarly, when more than one long shoot was included in the triplet rooting, quality was also poor. By avoiding such combinations, the rate of well-rooted plantlets increased by 25%, with a maximum of 66% when triplets of S/M/L combination were used. Smaller shoots, which usually showed poor rooting performance, were therefore found to benefit from the presence of their neighbors. This interaction between the sizes of individuals in a given tube was found to be associated with a within-tube correlation effect, a phenomenon previously described as “event coupling,” which was estimated using a distorted binomial-type distribution of probabilities. The resulting calculation of a coupling factor (average r = 0.60) explains the behavior of shoots within the same culture tube and their average rooting performance. Modeling of the interactions that occurred during in vitro rooting is described here and is recommended for improvement of this critical step in micropropagation.     

An improved protocol for Agrobacterium-mediated transformation of grapevine (Vitis vinifera L.)

An improved protocol for efficient Agrobacterium-mediated transformation of grapevine (Vitis sp.) was developed through modification of cocultivation and subsequent washing procedures. It was determined that Agrobacterium-infected somatic embryos (SE) cocultivated on filter paper exhibited less browning and significantly higher transient GFP and GUS expression than those cultured on agar-solidified medium. Furthermore, such SE, when subjected to a prolonged washing period in liquid medium containing cefotaxime and carbenicillin, followed by another wash in similar medium with kanamycin added, exhibited significantly higher rates of stable transformation compared to previously-described procedures. Transgenic plant recovery was increased 3.5–6 Xs by careful excision of leafy cotyledons from SE that had been induced to germinate on MS medium containing 1 μM of BA. Southern blot analysis revealed the low copy number integration of transgenes in transgenic plants recovered using the improved protocol. These improved cocultivation and plant recovery procedures have been demonstrated to facilitate production of large populations of transgenic plants from V. vinifera ‘Merlot’, ‘Shiraz’ and ‘Thompson Seedless’ as well as Vitis hybrid ‘Seyval Blanc’.