Study On Genetic Transformation Of Gametophytes Of Undaria Pinnatifida

Undaria pinnatifia is one of the three main seaweed species under commercial cultivation in China. In order to improve its' quantity and quality, developing new strains is needed. Much advanced technology was applied, including somatic hybridization, genetic transformation and so on. In this paper some experiments related to U. pinnatifia gene engineering are conducted as follows. The protoplasts of gametophytes were isolated and regenerated under axenic conditions. The materials were incubated in enzyme solution, which was extracted from the digestive organs of sea snail. Some of the protoplast isolation conditions, such as duration of incubation, pH, temperature, were studied. It's found that pH 6-7 gave higher protoplast yields, the optimum incubation period was 2-3h, and 20-25℃ was proven to be appropriate. Contrast between protoplast yields of male and female gametophytes was conducted in this paper too. Results show that there was no significant difference between them. Purified protoplasts were cultured in PES medium. After 2-day's culture new cell wall was formed. Then cells underwent division. At the 3rd or 4th day, cells divided into two cells. And about a week later, they regenerated to a filamentous form. Effects of ultrasonic treatment on gametophytes were studied. It was found that after ultrasonic treatment the filaments were shortened, and they grew faster than control group which was not treated by ultrasonic. Effect of ultrasonic treatment to plasmid DNA was also investigated. Results showed that treatment which was in the rang of output of 20W-50W didn't damage DNA, that is, DNA was integrate after the treatment. X-gal was applied to detect lacZ activity. Both control and treated groups were dyed blue. Experiment showed that the possible reason maybe the bacteria which attacted on the filaments produced pseudo-positive result. Gametophyte sensitivity to four antibiotics was tested. They are hygromycin(Hm), chloramphenicol(Cm), kanamycin(Km), and streptomycin(Str), all of which are frequently used in plant gene engineering. Results show that female and male gametophytes of U. pinnatifida were not sensitive to Km. and Str., but they were sensitive to both Hm. and Cm. Further test indicated that there was significant difference in the sensitivity to Cm. between female and male gametophytes.