An experimental comparison of different transformation procedures assessed in tomato cv Rio Grande with yeast HAL 1 gene

Abstract

• Transformation through tissue culturing.• In planta transformation using in vitro fruit injection method.• In planta transformation using in vivo fruit and flower injection. Materials and Methods Bacterial strain and plasmid constructionThe Agrobacterium tumefaciens strain LBA4404 containing pPM7 was used for transformation experiment. The HAL1 expression cassette was constructed from the plasmid pMOG180 as described by Gisbert et al [14]. Transformation through tissue-culturingTissue-culture transformation of tomato was based on the protocol derived from Khoudi et al. [15] with some modifications. The cotyledon, apical meristems and hypocotyl explants cut from one week old seedlings were dipped in A. tumefacians culture for 10 minutes and shifted to cocultivation medium. After 2 days, explants washed with diluted cefotaxime (50 mg/l) were shifted to regeneration MS [16] selection medium (MS containing 0.5 mg/1 Zeatin and 0.5 mg/l IAA (SM1) or MS containing 1 mg/1 Zeatin and 0.5 mg/1 IAA (SM2) supplemented with 50 µg/ml kanamycin sulphate and 100 µg/ml cefotaxime. Rooting of the transgenic shoot was obtained on half MS medium. In planta transformation using in vitro fruit injection methodFresh, healthy and mature Rio Grande fruits were purchased from the local market, rinsed thoroughly with tap water and dried with blotting paper. Agrobacterium was grown in Luria Broth medium supplemented with rifampicin 40 mg/l and kanamycin 50 mg/l until an O.D.= 0.8 was achieved. Surface sterilization of fruits was done by washing with 0.1% aqueous mercuric chloride solution for 1 minute followed by rinsing thrice with autoclaved distilled water. One ml sterile hypodermic syringes were used to evenly inject the Agrobacterium culture throughout the fruit. Fruits were incubated at 28°C for 6, 10, 24 and 48 hours incubation periods. For each incubation period, three replications with total of 12 fruits were selected and placed in already cleaned and autoclaved 500 ml beaker inside the laminar flow hood.In order to observe the effect of Agrobacterium treatment on the germination of seeds compared to control (untreated), seeds were collected from Agrobacterium injected fruits and were inoculated on selection medium (half MS medium containing 100 mg/l kanamycin) under sterile conditions. Seeds from each incubation period were inoculated in three replications. Each replication contained approximately 40 seeds per fruit per flask. Germination

Tomato tolerance to abiotic stress: a review of most often engineered target sequences

“…So far, this methodology has not been broadly employed in tomato transformation. However, Safdar and Mirza (2014) performed a comparison of transformation through tissue culture and in planta transformation using an in vitro fruit injection method and in vivo fruit and flower injection.…”