ORIGINAL PAPER
Genetic transformation and pyramiding of aprotinin-expressing sugarcane with cry1Ab for shoot borer (Chilo infuscatellus )resistance
S.Arvinth •S.Arun •R.K.Selvakesavan •
J.Srikanth •N.Mukunthan •P.Ananda Kumar •
M.N.Premachandran •N.Subramonian
Received:5October 2009/Revised:1February 2010/Accepted:3February 2010/Published online:24February 2010ÓSpringer-Verlag 2010
Abstract We evaluated the insecticidal toxicity of Cry1Aa,Cry1Ab and Cry1Ac toxins against neonate larvae of sugarcane shoot borer Chilo infuscatellus Snellen (Lepidoptera:Crambidae)in vitro on diet surface.With the lowest LC 50value,Cry1Ab emerged as the most effective among the three toxins.Sugarcane culti-vars Co 86032and CoJ 64were transformed with cry1Ab gene driven by maize ubiquitin promoter through particle bombardment and Agrobacterium -mediated transforma-tion systems.Gene pyramiding was also attempted by retransforming sugarcane plants carrying bovine pancre-atic trypsin inhibitor (aprotinin)gene,with cry1Ab .Southern analysis confirmed multiple integration of the transgene in case of particle bombardment and single site integration in Agrobacterium -mediated transformants.The expression of cry1Ab was demonstrated through Western analysis and the toxin was quantified using ELISA.The amount of Cry1Ab protein in different events varied from 0.007to 1.73%of the total soluble leaf protein;the events transformed by Agrobacterium method showed significantly higher values.In in vivo bioassay with neonate larvae of shoot borer,transgenics produced considerably lower percentage of deadhearts despite
suffering feeding damage by the borer compared with the untransformed control plants.Expressed Cry1Ab content was negatively related to deadheart damage.Aprotinin-expressing sugarcane pyramided with cry1Ab also showed reduction in damage.The potential of producing sugar-cane transgenics with cry1Ab and aprotinin genes resis-tant to early shoot borer was discussed in the light of the results obtained.
Keywords Genetic transformation ÁSugarcane Á
cry1Ab ÁAprotinin ÁGene pyramiding ÁAgrobacterium ÁParticle bombardment ÁShoot borer ÁC hilo infuscatellus ÁInsect resistance
Introduction
Sugarcane (Saccharum spp.hybrid),grown mainly for sugar and ethanol in both tropical and subtropical parts of the world,plays a significant role in world economy.Insect pests constitute a major biotic stress in all sugar-cane growing countries including India,one of the leading producers of sugar in the world.Among insect pests,shoot borer Chilo infuscatellus Snellen (Lepidoptera:Crambidae)attacks the crop in the early stages leading to loss of population and ultimately reduction in yield.Generally,predominant in the hot and dry weather of summer (Avasthy and Tiwari 1986),the borer can survive throughout the year in mild climates reaching attack levels of 28.6%deadhearts (Srikanth et al.2009)in tropical India and higher levels (34.1%)in subtropical India (Kaur et al.2003).Despite a high economic threshold level of 23.4%(Sardana 1997)and compensa-tory tillering ability,the pest can inflict cultivar-dependent losses of up to 19.63%in cane weight and 1.72–8.02%in
Communicated by kshmanan.
S.Arvinth ÁS.Arun ÁR.K.Selvakesavan ÁJ.Srikanth Á
N.Mukunthan ÁM.N.Premachandran ÁN.Subramonian (&)Sugarcane Breeding Institute,Coimbatore 641007,India e-mail:monian_52@
P.Ananda Kumar
National Research Center on Plant Biotechnology,New Delhi 110012,India
Plant Cell Rep (2010)29:383–395DOI 10.1007/s00299-010-0829-5
