Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants

Abstract

1997. Transformation and regeneration of transgenic aspen plants via shoot formation from stem explants. -Physiol. Plant. 99: 554-561.An Agrobacterium-mtdmted transformation procedure for aspen (Populus tremula L.), involving the direct regeneration of shoot-buds from stem explants, is described. Disarmed Agrobacterium tumefaciens strain EHAlOl harboring the binary plasmid pKIWI105 (which carries the uidA and n pi 11 gen&s, coding for/?-glucuronidase [GUS] and neomycin phosphotransferase II, respectively) was used for the transformation of stem explants. An incubation period of 48 to 72 h was found to be most effective in terms of transient GUS expression on the cut surface of the stem explants. Adventitious shoots regenerated after 2-3 weeks of culture in a woody plant medium (WPM) supplemented with TDZ (l-phenyl-3-[l,2,3-thiadiazol-5-yl]-urea, Thidiazuron) and carbenicillin. Three different kanamycin-based selection schemes were evaluated for optimization of transformation efficiency: (1) Kanamycin was added only to the rooting medium (5 to 6 weeks post-inoculation), or (2) to the regeneration medium 10-14 days after inoculation, or (3) after 2 days of co-cultivation. The third selection scheme was found to be optimal for adventitious shoots with regard to both the time required and the transformation efficiency, the latter being much higher than with the other schemes. Leaf samples from kanamycin-resistant shoots and plantlets were tested for GUS expression, and subjected to polymerase chain reaction (PCR) analysis of uidA and nptll genes. A Southern blot of the corresponding PCR-amplified fragments confirmed their authenticity and Southern blots of total plant DNA confirmed integration of the nptll gene into the plant genome.

Aspen micropropagation: use for phytoremediation of soils

“…Several transformants were selected for further analysis, and all of them showed kanamycin resistance as reflected by the high ability of rooting in the presence of kanamycin. Similar results were reported by TZFIRA et al (1997). The transgenic origin of the plants obtained during this study will be confirmed by PCR analyses.…”

Efficient Agrobacterium-Mediated Transformation of Hybrid Poplar Populus davidiana Dode × Populus bollena Lauche

“…IBA was used to induce roots for leaf of aspen and zeatin was used for shoot regeneration [24]. Adventitious shoots regenerated in a woody plant medium (WPM) supplemented with TDZ (Thidiazuron) from aspen stem explants [16]. Ferreira et al [14] reported that adventitious shoot regeneration of P. euphratica from organogenic nodules of leaf explants was achieved within a range of concentrations of a-naphtalenacetic acid and 6-benzylaminopurine, at a fixed 2:1 ratio.…”

“…Growth and differentiation response is not only controlled by the cultural environment, but is also dependent upon the genotype [11]. In vitro regeneration has been attempted in several poplar species using different explants such as leaves [8,11–14], petioles [15], internodes [8,14], stems [5,16], roots [8,11], and shoot tips [17]. The leaf disc transformation method [18] has been widely used in plant genetic engineering.…”