ZEMUN-BELGRADE, YUGOSLAVIA

Maize Research Institute

Allozyme polymorphism of maize plants transformed by bacterial gene integration

--Kosana Konstantinov, Snezana Mladenovic, D.Kovacevic, J.Dumanovic and Bojana Tadic

Transformation of the inbred lines B73 and Mo17 was performed by pollen tube pathway and dry seed incubation in plasmid DNA solution. Plants, self-pollinated by pollen grains pasted both in plasmid pRT100neo (Topfer et al., Nuclei Res. 15(14):5990, 1987) and total DNA of A. tumefaciens strain B6S3, were labelled as T0 plants. Also, plants derived from the seeds germinated and grown for 5 days on medium containing plasmid pRT100neo DNA (designated as cocultivated seeds) were labelled as T0 plants. In these plants, obtained from cocultivated seeds, neomycinphosphotransferase activity was determined, and plants with the enzyme in active form propagated to obtain T1 and T2 plants (Mladenovic, S et al., Genetika 23(3), 1991). Dot-blot and Southern blot analysis, by plant DNA hybridization with a specific probe, showed both NPTII gene integration and transmission through plant generations (Konstantinov, K et al., Genetika 23(2), 1991).

Bacterial DNA integration induced several phenotypic changes, such as barren stalk, curled (rolled) leaves, dwarfs, anthocyanin synthesis, that were almost eliminated in the next generation. Plant height alteration and reduction of the period from seed sowing to flowering (days to flowering) were inheritable and no segregation was obtained in the T2 generation.

To test the level of genetic divergence of "early" flowering transformed plants from the original inbred lines, allozyme polymorphism was determined in typical T1 plants. Starch gel electrophoretic systems were employed to investigate several allozyme genotypes from coleoptile tissue (according to Stuber, CW et al., N.C. State Exp. Res. Bull. No 286, 1988): acid phosphatase (Acp1), isocitrate dehydrogenase (Idh1,Idh2), malate dehydrogenase (Mdh1, Mdh2, Mdh3, Mdh4, Mdh5), phosphoglucomutase (Pgm1, Pgm2), 6-phosphogluconate dehydrogenase (Pgd1, Pgd2), phosphohexose isomerase (Phi1), betaglucosidase (Glu1) and arginine aminopeptidase (Amp1, Amp3). Distinct alterations were found in six of the 16 analysed loci. The results are presented in Table 1. The pattern of two enzyme systems, Acp1 and Mdh, was changed in plants belonging to both B73 and Mo17 genotypes, transformed either by plasmid pRT100neo or A. tumefaciens B6S3 DNA. Characteristic electrophoregrams are presented in Figs. 1 and 2. Experiments, including bacterial gene localization in maize chromosomes and studies on allozyme polymorphism in T2 plants are in progress.

Table 1. Allozyme genotypes of the transformed plants. Numbers and letters refer to allelic variants present in 5-day old coleoptile tissue.
 
Inbred/genotype   Alleles with genotype alteration
T1 plants lane1 Acp1 Mdh1 Mdh2 Mdh5 Idh2 Phi1
B73-control   2/2 6/6 3.5/3.5 12/12 4/4  
B73-cocultivated plants              
plant 1 1 2/4 10.5/10.5 3.5/6 15/N 4/6  
plant 2 2 2/4 10.5/10.5 6/6 15/N 6/6  
B73 - pollen pasted in pRT100 DNA               
plant 1 3 2/4 * 3.5/6 * 4/6  
plant 2 4 * * 3.5/6 * 4/6  
plant 3 7 * * 3.5/6 * 4/6  
plant 4 8 * * 3.5/6 * 4/6  
plant 5 9 4/4 * 6/6 * 6/6  
plant 6 10 4/4 * 6/6 * 6/6  
plant 7 11 * * 6/6 * 6/6  
plant 8 12 * * 3.5/6 * 4/6  
B73 - pollen pasted in B6S3 DNA              
plant 1 5 2/4 * 6/6 * 6/6  
plant 2 6 2/4 * 6/6 * 6/6  
Mo17 - control   2/2 6/6 6/6 12/12 4/4 4/4
Mo17 - pollen pasted in PRT100neo DNA              
plant 1 13 2/4 * * * *  
plant 2 14 4/4 * * * *  
Mo17-pollen pasted in B6S3 DNA              
plant 1 15 2/4 * 3.5/3.5 * * 4/5
plant 2 16 2/4 * 3.5/6 * * 5/5
*no changes

1number corresponds to the lanes on electrophoregrams (Fig. 1 and Fig. 2)

Figure 1.

Figure 2.


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