Early antibiotic selection and efficient rooting and acclimatization improve the production of transgenic plum plants ( Prunus domestica L.)

Abstract

We describe here an improved system for routinely developing transgenic plum plants (Prunus domestica L.) through the use of Agrobacterium tumefaciens. The production of non-transformed "escapes" has been virtually eliminated, and rates of plant establishment in the greenhouse have been dramatically improved. The system is based on the regeneration of shoots from hypocotyls extracted from mature seed. The shoot regeneration medium is Murashige and Skoog (MS) salts and vitamins supplemented with 7.5 microM thidiazuron and 0.25 microM indole-butyric acid. Transferring the explants after co-cultivation to shoot regeneration medium containing 80 mg l(-1) of kanamycin and 300 mg l(-1) of Timentin reduced the total number of regenerated shoots without affecting the transformation rate. Transformation rates using the described system averaged 1.2% of the hypocotyl slices producing transgenic plants, with a range of 0-4.2%. The transgenic shoots rooted at a rate of 90% on half-strength MS salts and vitamins supplemented with 5 microM alpha-naphthaleneacetic acid and 0.01 microM kinetin. Plantlets were transferred to a greenhouse directly from culture tubes with a 90% average survival.

Breeding in peach, cherry and plum: from a tissue culture, genetic, transcriptomic and genomic perspective

“…Medium supplemented with thidiazuron (TDZ) 7.5 μM in combination with indole-3-butyric acid (IBA) 0.25 μM has been used for shoot regeneration, with a transformation effi ciency of 1.2% (Scorza et al, 1994;González-Padilla et al, 2003). Petri et al (2012) reported 42% transformation effi ciency increase using 9 μM 2,4-dichlorophenoxyacetic acid (2,4-D) for three days during co-cultivation, followed by a regeneration medium containing TDZ (7.5 μM) and kanamycin 80 mg L-1 as selective agent.…”

Regeneration of Prunus salicina Lindl (Japanese plum) from hypocotyls of mature seeds

“…Approximately 45 and 68% of shoots of 'Shiro' and 'Early Golden' produced roots, respectively ( Table 3). The rooting efficiency was relatively high compared to that of P. domestica (Gonzalen-Padilla et al 2003, Tian et al 2007 on the regular rooting medium. Shoots with developed roots were transferred to Magenta boxes containing the same medium, and plantlets were developed (Fig.…”

“…These include cotyledons, hypocotyls, and young leaves (Miguel and Oliveira 1999;Tang et al 2002;Gonzalen-Padilla et al 2003;Pérez-Clemente et al 2004). However, regeneration appeared to be explant specific for a Prunus species.…”

“…Stone fruits, belonging to the genus Prunus, are important fruit crops, which include peach (Prunus persica), European plum (P. domestica), Japanese plum (P. salicina), apricot (P. armeniaca), sweet cherry (P. avium), sour cherry (P. cerasus), and almond (P. dulcis). Plant regeneration as well as genetic transformation has been reported in most of these Prunus fruit plants (e.g., Mante et al 1989Mante et al , 1991Escalettes and Dosba 1993;Miguel and Oliveira 1999;Tang et al 2002;Gonzalen-Padilla et al 2003;Pérez-Clemente et al 2004). Moreover, genetic transformation research in some of these species has been greatly advancing.…”

Genetic Transformation Strategies in Fruit Crops

“…To get this procedure, 1-phenyl-3-(1,2,3-thidiazol-5-il) urea (thidiazuron, TDZ) at 7.5 µM and indole-3-butyric acid (IBA) at 0.25 µM were applied to the explants in order to regenerate Agrobacterium-mediated transformed hypocotyl segments. The hypocotyls based system was later improved by González-Padilla et al (2003); these authors described a reduction in the total number of regenerated shoots without affecting the transformation rate by application of early selection (80 mg/L of kanamycin and 300 mg/L of Timentin) during shoot regeneration in a medium consisting on half-strength MS salts and vitamins supplemented with 5 mM -naphthaleneacetic acid (NAA) and 0.01 mM kinetin. From these works, new improvement was made in the diploid species Prunus salicina.…”