Single-stranded DNA used as an efficient new vehicle for transformation of plant protoplasts.
In relation to the question which DNA form (single- or double-stranded) is transferred by Agrobacterium tumefaciens to plant cells, we studied the behaviour of single-stranded DNA, as compared to double-stranded DNA, when it is introduced into plant protoplasts by electroporation. To this end, we cloned a construct with a plant NPTII gene as well as a CAT gene in the M13 vectors tg130 and tg131. We found that both complementary single-stranded molecules gave rise to substantial CAT activity in plant protoplasts, suggesting that single-stranded DNA is converted into double-stranded DNA by the plant cell replication machinery. Unexpectedly, we found that single-stranded DNA leads to a 3-10-fold higher frequency of stable transformation (selection for kanamycin resistance) than double-stranded DNA. These results indicate that the use of single-stranded DNA might be considered in experiments in which optimal transformation frequencies are needed, e.g. with protoplasts from recalcitrant plant species.
Rodenburg
K W
KW
Department of Plant Molecular Biology, Leiden University, Netherlands.
de Groot
M J
MJ
Schilperoort
R A
RA
Hooykaas
P J
PJ
D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004277:DNA, Single-Stranded / Q000235:genetics*; D004560:Electricity; D005821:Genetic Techniques; D005822:Genetic Vectors; D010944:Plants / Q000235:genetics*; D011523:Protoplasts; D012231:Rhizobium / Q000235:genetics; D014170:Transformation, Genetic
D004277:DNA, Single-Stranded; D004247:DNA