Regeneration of sugarcane elite breeding lines and engineering of stem borer resistance

Abstract

Five elite sugarcane breeding lines were tested for efficiency in embryogenesis and plant regeneration. All of them produced regenerative embryogenic calli but with varied efficiencies. To engineer strongly insect-resistant sugarcanes, the GC content of a truncated cry1Ac gene, which encodes the active region of Cry1Ac insecticidal delta-endotoxin, was increased from the original 37.4 to 47.5% following the sugarcane codon usage pattern. The synthetic cry1Ac gene (s-cry1Ac) was placed under the control of maize ubiquitin promoter and introduced by microprojectile bombardment into the embryogenic calli of sugarcane lines YT79-177 and ROC16. Southern blotting analysis showed that multicopies of s-cry1Ac were integrated into the genomes of transgenic sugarcane lines. Immunoblotting analysis identified 18 transgenic lines expressing detectable levels of s-Cry1Ac, which were estimated in the range of 1.8-10.0 ng mg(-1) total soluble proteins. Four transgenic and two parental lines were assayed for sugarcane stem borer resistance in leaf tissue feeding trials and greenhouse plant assays. The results showed that, while the untransformed control lines were severely damaged in both leaves and stems, the transgenic sugarcane lines expressing high levels of s-Cry1Ac proteins were highly resistant to sugarcane stem borer attack, resulting in complete mortality of the inoculated insects within 1 week after inoculation.

Herbicide-tolerant sugarcane (Saccharum officinarum L.) plants: an unconventional method of weed removal

“…In our study, friable and embryogenic calli of all 4 cultivars were obtained on media containing 2,4-D; casein was added to enhance embryogenic potential of the callus. Similar studies were reported by Weng et al (2006) and Joyce et al (2010). In several other studies, 2,4-D was found to be mandatory for production of friable and embryogenic calli in sugarcane (Snyman, 2004;Ramanand et al, 2005;Franklin et al, 2006;Basnayake et al, 2011;Nawaz et al, 2013).…”

Genetic Engineering of Saccharum

“…(Falco et al 2000 ) • bar , CaMV 35S , Agrobacterium , Phosphinothricin (5 mg/l), No fi eld trial, Co 92061 and Co 671 (Manickavasagam et al 2004 ) • pat , Maize Ubi-1 , Biolistic, Glufosinate ammonium, No fi eld trial, NCo 310 (Leibbrandt and Snyman 2003 ) Biotic stress tolerance Insect • Sugarcane stem borer: -cry1A(b) , CaMV 35S / nos , Electroporation, GUS, Field trial, Ja60-5 (Arencibia et al 1997(Arencibia et al , 1999 , maize phopshoenolpyruvate carboxylase promoter and pith promoter, Biolistic, Neomycin, Field trial, SP80-1842 and SP80-3280 (Braga et al 2003 ) -cry1A(c) , Maize Ubi-1 / nos , Biolistic, Geneticin (50 mg/l), No fi eld trial, YT79-177 and ROC16 (Weng et al 2006 ) • Sugarcane canegrub, gna or pinII , Maize Ubi-1 / nos , Biolistic, Geneticin, No fi eld trial, Q117 (Nutt et al 1999 Metabolic engineering for other economically important traits such as enhancing sucrose content and using transgenic sugarcane to produce novel and value-added products represent a more recent era of genetic improvement (Table 11.2 ).…”

“…This marked a major milestone in sugarcane transformation. Further re fi nements in tissue culture media and explant preparation and improvements in biolistic transfer protocols allowed transformation of sugarcane to become fairly routine in many laboratories (Tables 11.1 and 11.2 ), using easily established target tissues, including embryogenic callus (Bower et al 1996 ;Falco et al 2000 ;Gallo-Meagher and Irvine 1996 ;Gilbert et al 2005 ;Ingelbrecht et al 1999 ;Joyce et al 1998 ;Snyman et al 1996 ;Weng et al 2006 ;Zhang et al 1999 ) and immature leaf roll disk explants (Snyman et al 2000(Snyman et al , 2006 . Agrobacterium -mediated gene transfer was successful in sugarcane for a limited number of varieties and target tissues such as embryogenic calli (Arencibia et al 1998 ;Elliott et al 1998 ) , immature leaf sections (Enríquez-Obregón et al 1997 , and axillary meristems (Manickavasagam et al 2004 ) .…”

“…Insect tolerant transgenic sugarcane engineered with the Bacillus thuringiensis ( Bt ) toxin genes cry1A(b) and cry1A(c) (Arencibia et al 1997(Arencibia et al , 1999Arvinth et al 2010 ;Braga et al 2003 ;Weng et al 2006 ) , the carbohydrate binding lectin gene gna and the proteinase inhibitor genes pinII , SKTI , SBBI , and aprotinin (Christy et al 2009 ;Legaspi and Mirkov 2000 ;Nutt et al 1999 ;Sétamou et al 2002 ) were also generated (Table 11.1 ), and fi eld or greenhouse trials have demonstrated encouraging levels of insect tolerance.…”

Caracterização de promotores de expressão especifica de cana-de-açucar (Saccharum ssp.) em sistema modelo Micro-Tom (Solanum lycopersicum L)

“…A expressão heteróloga de inibidores de proteinase em plantas transgênicas apresenta potencial para o controle de pragas (AUGUSTYNIAK et al, 1997;FALCO et al, 2003;WENG et al, 2006 As informações obtidas constituem uma importante base molecular para o desenvolvimento biotecnológico da cultura, uma vez que esforços direcionados ao melhoramento genético dos híbridos de cana-de-açúcar invariavelmente esbarram na complexidade do seu genoma (VETTORE et al, 2003).…”