A biolistic-based genetic transformation system applicable to a broad-range of sugarcane and energycane varieties

Abstract

Sugarcane and energycane (Saccharum spp. hybrids) are prominent sources of sugar, ethanol, as well as high-value bioproducts globally. Genetic analysis for trait improvement of sugarcane is greatly hindered by its complex genome, limited germplasm resources, long breeding cycle, as well as recalcitrance to genetic transformation. Here, we present a biolistic-based transformation and bioreactor-based micro-propagation system that has been utilized successfully to transform twelve elite cane genotypes, yielding transformation efficiencies of up to 39%. The system relies on the generation of embryogenic callus from sugarcane and energycane apical shoot tissue, followed by DNA bombardment of embryogenic leaf roll discs (approximately one week) or calli (approximately 4 weeks). We present optimal criteria and practices for selection and regeneration of independent transgenic lines, molecular characterization, as well as a bioreactor-based micro-propagation technique, which can aid in rapid multiplication and analysis of transgenic lines. The cane transformation and micro-propagation system described here, although built on our previous protocols, has significantly accelerated the process of producing and multiplying transgenic material, and it is applicable to other varieties. The system is highly reproducible and has been successfully used to engineer multiple commercial sugarcane and energycane varieties. It will benefit worldwide researchers interested in genomics and genetics of sugarcane photosynthesis, cell wall, and bioenergy related traits.

Keywords: spp. hybrids; biolistic transformation; bioreactors; direct and indirect embryogeneses; energycane; micro-propagation; sugarcane.

FIGURE 1.

Typical sugarcane transformation procedure and timeline using direct (DE) and indirect (IE) embryogeneses. (a-b) Embryogenic leaf roll disc or callus preparation; (c-d) transformation by microprojectile bombardment of embryogenic callus; (e) selection and shoot regeneration; (f) shoot elongation and root initiation (g-h); soil and greenhouse adaptation; (i) bioreactor-based clonal micro-propagation to bulk up engineered lines.

FIGURE 2.

Bulk micro-propagation of transformants. Selection of explants from shoots generated (a) from apical meristems of mature plants or (b) from transformants in selection media; (c) multiplication of shoots in glass jars; (d-f) culturing shoots in bioreactors for rooting; (g-h) rooted seedlings; (i) potted seedlings growing in a shaded greenhouse.

FIGURE 3.

Molecular characterization of transgenic sugarcane and energycane. Typical results of (a) Southern blot analysis to assess transgene integration; (b) RT-PCR analysis of bar transgene expression (upper gel) and of constitutively expressing endogenous APRT2 gene (lower gel); (c) PAT immunostrip assay. Lanes: NT-Non-transformed control; #1 to #5-Independent generated lines; NC-Negative control; PC-Positive control. Strips with two lines (shown by ‘**’) indicate positive transgenic lines with active bar expression and are indicative of non-silenced transgenic lines. Line#1 appears to be a low-expresser, based on RT-PCR results. Marker: 1 Kb Plus ladder (Thermo Scientific, Thermo Fisher Scientific, Waltham, MA). Note: The low-level background noise observed in both NT and transgenic lanes in the Southern blot (Fig. 3b) is a result of the sugarcane codon-optimized bar transgene probe hybridizing to the sugarcane genomic DNA. This is not uncommon when codon-optimizations are performed to enhance expression in plants.⁴⁰ The specific signal however from the transgene can be readily discerned above the background in such cases.^40.