Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 13, Num. 2, 1996

Transgenic sugarcane (Saccharum officinarum L.) plants are tolerant to stem borer (Diatraea saccharalis F.) attack despite the low expression levels of cryIA(b) gene from B. thuringiensis var. kurstaki HD-1

Ariel Arencibia, Roberto Vazquez, Dmitri Prieto, Elva R. Carmona, Pilar Tellez, Alberto Coego, Lazaro Hernandez, Gustavo de la Riva and Guillermo Selman-Housein

Plant Division, Centre for Genetic Engineering and Biotechnology, P.O. Box 6162,C.P. 10600, Havana, Cuba.

Code Number: BA96057
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Introduction

Sugarcane (Saccharum officinarum L.) is a widely spread and economically important monocot plant. Sugarcane stem borer (Diatraea saccharalis F.) is the most important pest of this crop, causing agricultural and industrial yield losses estimated in more than 200 million dollars annually for the producers of American continent (1). Bacillus thuringiensis is a Gram-positive bacterium producing a parasporal body during sporulation mainly composed by delta-endotoxins, which are active against several insects. The truncated gene cryIA(b) encode for active region of CryIA(b) delta-endotoxin from B. thuringiensis var. kurstaki HD-1 was expressed under the control of CaMV 35S promoter (2) in transgenic sugarcane plants transformed by intact cell electroporation. The levels of recombinant toxin in transgenic plants were established and biological activity test were performed against neonate sugarcane stem borer larvae. Transgenic sugarcane plants showed remarkable larvicidal activity despite the low expression level of B. thuringiensis delta-endotoxin gene cryIA(b).

Materials and Methods

Sugarcane cell suspension from Cuban commercial variety Ja 60-5 were transformed by intact cell electroporation (3). The biological activity of putative transgenic plants was assayed in vitro. Two stem borer larvae (24 hours after hatch) were carefully feeded on 12 weeks old transgenic plants during 7 days. Infected plants were scored for plant damaged and larvae size. The determination of recombinant CryIA(b) toxin was established by immuno-radiometric assay (IRMA). Immunopurified anti-CryIA rabbit polyclonal antibodies were iodinated by the chloramine T method (4). Specific activity of iodinated antibodies were 8.3 mCi/mg determined in a Clini-Gamma-o-counter (LKB Bromma, Sweden). IRMA assay was performed in microtiter plates (Titertek, FlowLaboratories, The Netherlands). Wells were coated with 100 uL of a solution of 10mg/mL immunopurified antibodies in Carbonate-bicarbonate buffer pH 9.6 at 37 C for 2 hours. Then, the plates were washed two times with PBST buffer and stored to 4 C. In coated plates, 50 uL neutralized extract (25 mg total soluble proteins from standard and samples) and 5x10^6 cpm of iodinated polyclonal antibodies were mixed into each well and incubated at 4 C for 12 hours. Then, the plates were washed five times and radioactivity bound to each well was determined after cutting each well from the plate.

Results and Discussion

By intact cell electroporation sugarcane transgenic plant lines of commercial variety Ja 60-5 were regenerated from transformed histochemically stained calli. By biological activity test 22 putative transgenic lines showed no significative plant damage and remarkable larvicidal activity. The selected plant lines were adapted to natural soil conditions and firstly in green house and then in experimental field conditions. After three months the transgenic plant lines were evaluated for their resistance to a super-infestation borer attack: two larvae per plant. Four weeks later were evaluated the number of dead stem hearts, the total number of stem segments and the total number of stem damaged. The expression in transgenic sugarcane plants is dramatically low because of the use of both unmodified bacterial cryIA(b) gene and CaMV 35S promoter in a monocot plant. Despite this fact, selected transgenic sugarcane plant lines are tolerant to borer attack (Figure 1, Table 1). However, higher expression levels are required for the effective control of this agronomically important pest. Taking into account these facts elite transgenic sugarcane plant lines are in progress.

Figure 1. Genomic Southern blot of autopesticide sugarcane transgenic plants.

Table 1. Tolerance to borer attack and the level of recombinant toxin in transgenic plants.

 Clone   CryIA(b) toxin (ng/mg total protein)    Plant Damage
--------------------------------------------------------------
   4                    0.61                          +
  48                    1.59                          +
  56                    0.48                         + +
  65                    0.55                         + +
 209                    0.66                          + 
 (-)Control          not detected                   +++++
(+) each represents 20 % of the total damage observed in negative control plant
line.

1. Bennet F. et al., GEPLACEA 1977; 9:1-32.

2. Gutierrez C et al., Biotecnologia Habana'92. Book of Short Reports 1992;14.3

3. Arencibia, et al., Plant Cell Report 1995;14: 305-309.

4. Greenwood F. Nature 1962;194: 495-496.

Copyright 1996 Elfos Scientiae

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