GENTECH archive 8.96-97


Rapid Progress in DNA-Tampering Techniques

Introduction of foreign DNA molecules into plant cells is a pivotal
but routine technique in crop biotechnology. The present technology,
however, does not permit the introduction of more than three or four
genes at a time. A technique that enables scientists to produce
transgenic plants with large inserts of DNA can thus have an exciting
impact on both basic and applied biotechnology. Scientists at Cornell
University report in the Proceedings of the National Academy of
Sciences USA the development of exactly such a technique (1). Using a
newly constructed plasmid vector they transferred a 150 kilobase
human DNA fragment into plant cells - about ten times the normal DNA
size that could be transferred with conventional approaches.

The key to the success of the Cornell team, consisting of Carol
Hamilton, Steven Tanksley and colleagues, was the methodical
development of a unique vector called BIBAC (Binary Bacterial
Artificial Chromosome) (2). Libraries with large chromosomal inserts
are now increasingly made in the bacterial artificial chromosome
(BAC) vectors in E. coli, while the workhorse for transfer of genes
into plants is Agrobacterium, which transfers a piece of its DNA into
plant cells during infection. The BIBAC vector can multiply in both
E. coli and Agrobacterium, and also has additional copies of the virG
and virE genes whose products help in the efficient transfer of DNA
from Agrobacterium to plant cells. Most of the transgenic tobacco
plants developed with the BIBAC vector showed that the introduced
human DNA fragment (150 kb) was present in an intact form and the
fragment was passed on to subsequent progeny without any changes.

Now that the size barrier for plant DNA transfer is broken,
scientists can seek to alter quality and yield traits that are
controlled by multiple genes. Many agronomically important traits
such as seed weight in soybean or tuber dormancy in potato are
controlled by such quantitative trait loci. Additionally, many
disease resistance genes are known to occur in clusters spanning a
large chromosomal segment. The BIBAC vector may, for the first time,
facilitate engineering of such complex traits in plants. Tanksley's
group has recently isolated a chromosomal segment from tomato
containing a major fruit weight quantitative trait locus (3). Richard
Michelmore of the University of California, Davis, predicts that
BIBAC's most significant applications will be in the map-based
cloning of plant genes and a better understanding of how plant
genomes are organized (4). According to Hamilton, BIBAC vectors may
also be used in the future to reconstitute secondary product
pathways, create new pathways for the production of novel compounds
and reduce position-dependent expression of transgenes.

1. Hamilton, C. M., A. Frary, C. Lewis & S.D. Tanksley. 1996. Stable
transfer of intact high molecular weight DNA into plant chromosomes.
Proc. Natl. Acad. Sci. USA 93:9975-9979.

2. Hamilton, C.M. 1997. Binary-BAC system for plant transformation
with high molecular weight DNA. Nuc. Acid Res. (Submitted)

3. Alpert, K.B. & S.D. Tanksley. 1996. High-resolution mapping and
isolation of a yeast artificial chromosome contig containing tw2: A
major fruit weight quantitative trait locus in tomato. Proc. Natl.
Acad. Sci.USA 93:15503-15508.

4. Michelmore, R. 1996. Big news for plant transformation. Nature
Biotechnology 14:16553-1654.

C. S. Prakash
Center for Plant Biotechnology Research
Tuskegee University