An efficient shoot regeneration system andAgrobacterium-mediated transformation withcodAgene in a doubled haploid line of broccoli

Abstract

Broccoli is an important vegetable crop belonging to the genus of Brassica. However, it is often affected by biotic and abiotic stresses that result in large losses in yield and quality. Genetic manipulation has opened the opportunity for germplasm improvement of broccoli. In this study, an efficient shoot regeneration and Agrobacterium-mediated transformation system was established. The optimum medium for shoot induction was selected for three types of explant including hypocotyl, petiole, and peduncle, and up to 90% regeneration frequency was obtained. The transformation procedure was developed with the EHA105 strain, harboring the PUC19 vector, along with the target gene of codA and a hygromycin-resistance gene. Several factors were optimized, including hygromycin concentration for selection and the Agrobacterium density yielding the highest transformation frequency. Among the three types of explants, peduncle explants showed the highest response to this procedure, and the highest frequency of transformation (3.4%) was obtained depending on the analysis of polymerase chain reaction and Southern blot. In conclusion, the present study introduced a reliable system for plant regeneration and genetic transformation with three types of explants for a DH line of broccoli, and peduncle explant resulted in the highest transformation frequency. Résumé : Le brocoli est une importante culture maraîchère appartenant au genre Brassica. Cette culture est souvent affligée par des stress biotiques et abiotiques à l'origine de fortes baisses de rendement et de qualité. La manipulation génétique laisse entrevoir la possibilité d'une amélioration de l'espèce. Dans le cadre de cette étude, les auteurs ont mis au point un système efficace pour obtenir des pousses et les modifier génétiquement avec Agrobacterium. Ils ont sélectionné le support idéal pour l'induction des pousses parmi trois types d'explant (hypocotyle, pétiole et pédoncule) et obtenu un taux de régénération allant jusqu'à 90 %. La technique de manipulation génétique a été élaborée avec la souche EHA105, renfermant le vecteur PUC19 et le gène désiré codA, ainsi qu'un gène codant la résistance à l'hygromycine. Plusieurs paramètres ont été optimisés, notamment la concentration d'hygromycine, pour la sélection, et la densité d'Agrobacterium donnant la plus forte fréquence de modifications. Des trois types d'explant, ceux du pédoncule réagissent le mieux à la méthode employée et donnent le plus fort taux de transformation (3,4 %), après analyse par la technique de réaction en chaîne de la polymérase combinée à celle de Southern. En conclusion, cette étude propose un système fiable pour obtenir des plants et les modifier génétiquement à partir des explants de trois sortes venant d'une lignée haploïde double de brocoli. Les explants du pédoncule sont ceux qui donnent le taux le plus élevé de transformations. [Traduit par la Rédaction]

High frequency in vitro regeneration of adventitious shoots in daylilies (Hemerocallis sp) stem tissue using thidiazuron

“…Growth regulators' signal conversions into metabolic activities and developmental morphologies in cells or organisms have been established in biology [28][29][30][31][32][33]. The concept is broadly utilized for downstream applications particularly in cell, tissue, and organ cultures as well as plant transformation across plant species [24,[34][35][36][37]. Generally, TDZ is used at lower concentrations to effect plant micropropagation [23,26,27,38].…”

CRISPR/Cas9-mediated mutagenesis of homologous genes in Chinese kale

“…The transformation of Chinese kale was conducted as previously described, with modifications 53 , 54 . The explants pre-cultured for 3 d in Murashige and Skoog (MS) medium with 0.5 mg/L 2,4-D and 0.8% phytagar were infected with the Agrobacterium strain GV3101 (optical density at 600 nm between 0.6 and 0.8) by immersion for 1–2 min.…”

Optimization of Protoplast Preparation System from Leaves and Establishment of a Transient Transformation System in Apium graveolens

“…Additionally, PEG-mediated transformation allows the direct targeting of individual cells, enabling a more direct investigation of transient gene expression and study of exogenous substances. As a result, it is extensively utilized in plant molecular biology research [14,15]. At present, protoplast isolation and the preparation of a transient transformation system using protoplasts have been successfully conducted on various plant species.…”