Establishment of a highly efficient transformation system for pepper (Capsicum annuum L.)

Application of modern genetic manipulation has been limited in pepper ( Capsicum annuum L.) due to the lack of an efficient transformation system. Following the development of an efficient protocol for in vitro regeneration of pepper cotyledons, we investigated the key factors affecting transformation and established a highly efficient genetic transformation system using the pepper cotyledon as starting material. In this system, cotyledon explants are preconditioned for 2 days on kanamycin (km)-free DM1 medium [Murashige and Skoog (MS) salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients and a hormone combination of 1.0 mg/l indoleacetic acid (IAA) and 5.0 mg/l 6-benzyladenine (BA) solidified with 0.7% agar, pH 5.8], followed by co-cultivation with Agrobacterium tumefaciens on DM1 for 2 days and delay selection on DM1 with 500 mg/l carbenicillin (carb) for 2 days. The explants are then placed on DM1 containing 10 mg/l AgNO(3), 50 mg/l km-sulfate and 500 mg/l carb. After 4-5 weeks, the explants with buds are transferred to EM1 medium (MS salts/Gamborg B5 vitamins basal medium supplemented with 20 g/l sucrose, 5,000 mg/l DJ nutrients, 10 mg/l AgNO(3) and a hormone combination of 1.0 mg/l IAA, 3.0 mg/l BA and 2.0 mg/l gibberellic acid, solidified with 0.7% agar, pH 5.8) with 50 mg/l kanamycin and 500 mg/l carbenicillin for the elongation of buds. After 3-6 weeks, 1- to 2-cm-long elongated shoots are excised and planted on RM1 medium (MS basal medium supplemented with a hormone combination of 0.2 mg/l NAA and 0.1 mg/l IAA, solidified with 0.8% agar, pH 5.8) with 25 mg/l km and 200 mg/l carb for rooting. We tested four genotypes of pepper, and all presented a high differentiation efficiency (81.3% on average), elongation rate (61.5%) and rooting efficiency (89.5%). Polymerase chain reaction analysis results showed that 40.8% of the regenerated plantlets were transgenic plants.