Transgenosis in Higher Plant Cells — A Reevaluation

Summary Experiments were designed to investigate the possible transfer of bacterial lac gene into the haploid callus of Datura innoxia using bacteriophage lambda as a gene carrier. For detecting possible transgenosis, λplac phage treated calli were analyzed at short intervals for the enzyme β-galactosidase. Parallely, the phage-treated calli were also tested as such or after dissociation into single cells for their capability to grow on lactose medium. Approximately 60% of the calli showed higher levels of β-galactosidase over the controls. The average increase was 3.5 fold. Since, unexpectedly, the control calli also showed a basal level of β-galactosidase, it became essential to ascertain whether the induced enzyme was E. coli specific. Accordingly, the callus β-galactosidase and E. coli β-galactosidase were compared. The properties and characteristics taken into consideration were the effect of pH and temperature on enzyme activity, and the electrophoretic mobility of the two enzymes. These properties, however, failed to provide any definite evidence for the contention that the induced enzyme is E. coli specific. Unfortunately, in tissue cultures also we failed to isolate any transformant. Finally, in an attempt to verify whether the β-galactosidase activity that was being assayed indeed represented a lactase activity, in vitro hydrolysis of radioactive lactose was also carried out employing callus extracts. Possible hydrolytic products were separated by paper chromatography. These studies indicate that callus β-galactosidase assayed by the common colorimetric procedure of ONPG hydrolysis, actually lacks lactose hydrolysing ability, whereas E. coli β-galactosidase does have this ability. On the basis of the methods employed for detection we conclude that no gene has been incorporated into the plant cell genome and the increase in enzyme activity after λplac phage inoculation does not really represent an increase in E. coli specific β-galactosidase. These results cast serious doubt on the validity of transgenosis reported in recent years.