Kn1 gene overexpression drastically improves genetic transformation efficiencies of citrus cultivars

Abstract

The effects of a maize knotted1 (kn1) gene under the control of the cauliflower mosaic virus 35S promoter on genetic transformation efficiencies of six citrus genotypes were tested. The kn1 gene construct was used to transform 'Pineapple', 'Hamlin', 'Sucarri' andOur results demonstrate that expression of the kn1 gene enhances transformation efficiencies from 3 to 15 fold compared to a control vector, 3-11 fold relative to the highest transformation efficiencies reported for these citrus genotypes. Stable incorporations of T-DNA into the citrus genome have been confirmed with both histochemical staining of GUS activity and molecular analyses. The majority of kn1 over-expressing citrus plants grow and develop normally at young seedling stages, similar to those of the wild type plants. With all six genotypes of citrus tested including Eureka lemon, a cultivar difficult to be transformed, our results demonstrate that the kn1 gene may provide an effective molecular tool to enhance genetic transformation efficiencies of various citrus varieties. High transformation efficiency of citrus is of great importance for large scale characterization of gene functions and also cultivar development via transgenic and genome editing technologies.

An Improved Procedure for Agrobacterium-Mediated Transformation of Citrus

“…Earlier studies used both etiolated seedlings and light-grown seedlings for citrus transformation [18][19][20][21][22][23]. Our study shows higher transformation efficiency can be achieved using explants derived from light-grown seedlings, consistent with the study in kumquat that a relatively higher transformation efficiency was obtained when light-grown seedlings were used as explant source [29].…”

Opportunities for Innovation in Genetic Transformation of Forest Trees

“…Expression of STM/Knotted-1 orthologs from apple or maize enhanced shoot regeneration from leaf explants in the absence of exogenous cytokinins in tobacco, though were not effective in plum under the conditions studied ( Srinivasan et al, 2011 ). In citrus, expression of maize Knotted-1 enhanced in vitro regeneration after transformation, with rates varying widely among varieties ( Hu et al, 2016 ). In the gymnosperm forest tree Picea abies (Norway spruce), overexpression of a KNOTTED-1 ortholog similarly promoted in vitro somatic embryogenesis ( Belmonte et al, 2007 ).…”

An Improved Procedure for Agrobacterium-Mediated Transformation of ‘Carrizo’ Citrange

“…Earlier studies used both etiolated seedlings and light-grown seedlings for citrus transformation [ 18 , 19 , 20 , 21 , 22 , 23 ]. Our study shows higher transformation efficiency can be achieved using explants derived from light-grown seedlings, consistent with the study in kumquat that a relatively higher transformation efficiency was obtained when light-grown seedlings were used as explant source [ 29 ].…”

“…Attempts have been made by Dutt and Grosser to improve the citrus transformation method through optimizing acetosyringone concentrations and co-cultivation time, resulting in a relatively high transformation efficiency in ‘Carrizo’ citrange [ 19 ]. Transformation efficiencies of ‘Carrizo’ citrange were improved by the use of isopentenyl transferase gene ( IPT ) and knotted1 gene ( KN1 ) [ 20 ]. In 2015, Orbovic and Grosser [ 21 ] reported 7.8% transformation efficiency for ‘Carrizo’ citrange, which appears to be an average transformation efficacy for ‘Carrizo’ citrange observed by many investigators [ 19 , 20 , 21 , 22 , 23 ].…”

“…Transformation efficiencies of ‘Carrizo’ citrange were improved by the use of isopentenyl transferase gene ( IPT ) and knotted1 gene ( KN1 ) [ 20 ]. In 2015, Orbovic and Grosser [ 21 ] reported 7.8% transformation efficiency for ‘Carrizo’ citrange, which appears to be an average transformation efficacy for ‘Carrizo’ citrange observed by many investigators [ 19 , 20 , 21 , 22 , 23 ].…”