Tomato Transformation

S. Kimura; N. Sinha

doi:10.1101/pdb.prot5084

Gene editing in tomatoes

Emerging Topics in Life Sciences ◽

10.1042/etls20170056 ◽

2017 ◽

Vol 1 (2) ◽

pp. 183-191 ◽

Cited By ~ 2

Author(s):

Joyce Van Eck

Keyword(s):

Gene Editing ◽

Crop Improvement ◽

Site Directed Mutagenesis ◽

Dna Uptake ◽

Food Crop ◽

Transcription Activator ◽

Base Editing ◽

Crop Models ◽

Functional Studies ◽

Tomato Transformation

Tomato is an effective model plant species because it possesses the qualities necessary for genetic and functional studies, but is also a food crop making what is learned more translatable for crop improvement when compared with other non-food crop models. The availability of genome sequences for many genotypes and amenability to transformation methodologies (Agrobacterium-mediated, direct DNA uptake via protoplasts, biolistics) make tomato the perfect platform to study the application of gene-editing technologies. This review includes information related to tomato transformation methodology, one of the necessary requirements for gene editing, along with the status of site-directed mutagenesis by TALENs (transcription activator-like effector nucleases) and CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated Proteins). In addition to the reports on proof-of-concept experiments to demonstrate the feasibility of gene editing in tomato, there are many reports that show the power of these technologies for modification of traits, such as fruit characteristics (ripening, size, and parthenocarpy), pathogen susceptibility, architecture (plant and inflorescence), and metabolic engineering. Also highlighted in this review are reports on the application of a recent CRISPR technology called base editing that allows the modification of one base pair in a gene sequence and a strategy that takes advantage of a geminivirus replicon for delivery of DNA repair template.

Download Full-text

Approaches for Studying the Interaction of Geminiviruses with Their Whitefly Vector Bemisia tabaci

10.32747/1995.7604928.bard ◽

1995 ◽

Author(s):

Yehezkiel Antignus ◽

Ernest Hiebert ◽

Shlomo Cohen ◽

Susan Webb

Keyword(s):

Amino Acid ◽

Coat Protein ◽

Bemisia Tabaci ◽

Trialeurodes Vaporariorum ◽

Open Reading Frames ◽

Single Amino Acid ◽

Tomato Transformation ◽

Amino Acid Mutations ◽

Transmission Studies ◽

Whitefly Vector

The DNA of tomato yellow leaf curl virus (TYLCB) was detected in its whitefly vector, Bemisia tabaci, by dot spot hybridization as early as 1 h after acquisition access. The retention of the virus nucleic acid in the vector was at least 23 days after a 48 h acquisition access. However, the retention of TYLCV coat protein did not exceed 10 days. No replicative forms of TYLCV could be detected in B. tabaci, indicating a non-propagative relationship with the vector. Whiteflies were not able to accumulate naked virion ssDNA, virus cloned dsDNA, or virions with impaired coat protein. Deletion, frameshift, and single amino acid mutations were inserted into open reading frames (ORFs) V1 and V2 (Coat protein) of TYLCV. The ability of these mutants to replicate, to spread and to induce symptoms was tested both in leaf disks and in intact plants. No replication was found in tissues that were infected with a deletion mutant that lacked the carboxy half of the coat protein gene. Residual amounts of ssDNA and dsDNA were detected i tissues infected with a frameshift mutant in which an early termination at the extreme part of the protein. Two other mutants in which a single amino acid was changed in the overlapping part of V1 and V2 were able to spread systemically but infections remained symptomless and the production of ssDNA and dsDNA were significantly lower. These mutants were acquired and transmitted by Bemisia tabaci. Procedures for the the dissection, fixation and embedding of whiteflies were developed. The anatomy and ultrastructure of the salivary gland and the midgut of Bemisia tabaci and Trialeurodes vaporariorum (a vector and non-vector of geminiviruses respectively) was studied and described. Monoclonal antibodies against bean golden mosaic virus (BGMV) with narrow and broad spectrum were prepared. Transmission studies of tomato mottle geminivirus (TMoV) by B. tabaci were carried out. These studies were essential for a further work aimed to understand the interaction of geminiviruses with the insect and their localization in its tissues. To enable the production of transgenic plants procedures were developed for tomato transformation with both Agrobacterium and microparticle bombardment.

Download Full-text