Submitted to: Aflatoxin Elimination Workshop Proceedings
Publication Type: Abstract only
Publication Acceptance Date: 10/1/2002
Publication Date: 12/1/2002
Citation: Ozias-Akins, P., C. Niu, M. Joshi, Y. Deng, C.C. Holbrook Jr, R.E. Lynch. 2002. Genetic engineering of peanut for reduction of aflatoxin contamination. Proc. Aflatoxin Elimination Workshop p. 71. Interpretive Summary: not required
Technical Abstract: Through genetic engineering of peanut, we have focused mainly on two levels of protection against aflatoxin contamination: the entry of spores through insect-damaged tissues and the growth of the fungus after entry, although interfering with the aflatoxin biosynthetic pathway also is of interest. The insecticidal crystalline protein from the bacterium Bacillus thuringiensis (Bt cryIA(c)) was introduced into peanut (Arachis hypogaea L.) several years ago. Field tests have been conducted for five years to assess the level of insect resistance and more recently the level of aflatoxin reduction associated with insect resistance. In 2001 and 2002, both lesser cornstalk borer (LCB) infestation and Aspergillus inoculation were carried out in these field tests. Leaves from Bt-expressing plants contained toxin levels that were comparable between the two years (15-20 ng cryIA(c) per ml extract or ~0.03% of total protein). Pods were separated into four categories for aflatoxin analysis - damaged, non-Bt; undamaged, non-Bt; damaged, Bt; undamaged, Bt. There was a significant difference for aflatoxin levels between damaged pods vs. undamaged pods. There also was a highly significant difference in aflatoxin levels between non-transgenic, non-Bt and transgenic, Bt pods when the data were log transformed. The field experiment has been repeated in 2002 with the same experimental design and pods have been harvested. LCB damage and aflatoxin levels will again be quantified. A bacterial chloroperoxidase (CPO) gene, was obtained from Kanniah Rajasekaran and Jeff Cary (USDA-SRRC, New Orleans). This gene is under the control of the CaMV35S promoter and has been introduced into peanut. Fifty-seven hygromycin resistant lines have been recovered and 24 have been tested by PCR for the presence of the CPO transgene. All are positive, again indicating that hygromycin selection is very effective in peanut. Southern blot analysis of two independent lines demonstrated integration of the transgene with a relatively simple integration pattern. Out of 20 hygromycin-resistant lines analyzed by Northerns, 18 showed expression at the RNA level. Tissue extracts have been tested for activity against Aspergillus flavus in in vitro assays. For multiple lines, there is a significant reduction in the number of colony forming units recovered after incubation of pregerminated spores in tissue extract. All cell lines are in some phase of regeneration and several have reached the whole plant stage. Additional molecular analysis will be conducted on whole plants. A green fluorescent protein (GFP) gene driven by an enhanced double CaMV35S promoter has been introduced into peanut either alone or co-bombarded with CPO and the hygromycin resistance gene. Visual selection has allowed the recovery of several cell lines expressing GFP, but these lines appear to be mixtures of transgenic (GFP-expressing) and non-transgenic cells. More effective recovery of homogeneous GFP-expressing tissues was accomplished in the co-bombardment experiments after selection on hygromycin. The frequency of co-transformation is high enough to be of practical use. GFP allows the early detection of a transgenic line and would be particularly useful in future studies with novel selectable markers.