A major objective of the present study was to test the method of electroporation for transformation (Objective 1), and also, to test the feasibility of using biolistic bombardment for transformation of Ulva lactuca and several native species of Porphyra (Objective 2).
Transformation by Electroporation
Following limited guidelines from the literature, we developed protocols for regeneration of whole plants from protoplasts of Ulva lactuca, and several native species of Porphyra. For different species of Porphyra, regeneration from protoplasts varied from 10-20% to as much as50-60% of the protoplasts depending upon the day of collection, the time of storage before making protoplasts, the temperature of growth, and the enzyme treatments. In the case of Ulva lactuca more than 60% of the protoplasts could routinely grow into germlings. Regeneration from swarmers was greater than 70%.
Electroporation was used for transformation with protoplasts and partially digested thallus fragments of Ulva lactuca, and protoplasts of six species of Porphyra. Detailed analysis of various parameters, such as pulse strength, voltage, time of pulse, etc. were tested for survival and transformation of these cells. The genes that were tested include neomycin phosphotransferase (NPTII) and β-glucuronidase (GUS). For Ulva, protocols for regeneration from both swarmers and protoplasts, before and after electroporation, were optimized. More than 50% of the protoplasts regenerated into normal plants (Huang et al. 1996). We tested field strengths ranging from 200-1,000 V/cm and found that 2-3 consecutive pulses of 300-400 V/cm were quite effective in causing transient expression of the GUS gene. Data from transformation studies indicate that the 35S CaMV promoters is quite active in Ulva cells in transient expression assays of the GUS gene.
Reliable protocols have been developed for the formation of protoplasts and their regeneration into whole plants, in Porphyra miniata, P. purpurea, P. amplissima, P. linearis, P. diocia, P. leucosticta and P. umbilicalis. The results on protoplast regeneration in different species of Porphyra (with the exception of P. linearis) are quite similar. The two most significant improvements that we made were (1) the use of commercially available crude enzyme preparations, and (2) the omission of the predigestion incubation with papain. The yield of protoplasts was typically 106- 107 protoplasts per g chopped tissue, with 70-80% of the protoplasts being viable as tested by staining. Protoplasts settled and adhered to cover slips within 24 h. Cell wall material was deposited quickly and the first cell division occurred around 7-10 days. Thereafter, cell divisions occurred more frequently, forming multicellular plantlets.
Another significant modification in the protocols that we have made is that we grow our cultures under nonaxenic conditions. This has resulted in a tremendous improvement in the regeneration frequency of the protoplasts.
The plasmids pB1121 (Clontech, Palo Alto, CA) was initially used for the expression of the GUS gene. This plasmid has a 35S CaMV promoter which has been quite successful with a large number of higher plants. A number of variations of this promoter have been suggested that are supposedly more effective in both transient and stable expression assays. We obtained one such variation (pCW122) where a 2x 35S promoter was attached to the GUS gene. The most effective electroporation conditions were as follows: 1-3 pulses at 100-300 V, from a 330 amp capacitor, exponentially decaying across a 5 mm pathlength. The conditions were optimized to produce 40-50% viability of electroporated cells as determined by vital staining with erythrocine B and trypan blue 1-3 h after treatment. Two days after electroporation, transient expression of the GUS gene was observed at a frequency of 1-15 cells/10,000 cells. No transient expression was observed in the samples electroporated without plasmid DNA.
In summary, we feel that the 35S promoter of CaMV is quite effective for at least transient expression inthe algal cells, and that these algae do not have any residual GUS activity that could interfere with the GUS assays in the transgenic cells.
Means of selection of transformed cells
We conducted detailed toxicity analysis for growth of protoplasts of Ulva lactuca and several species of Porphyra on kanamycin, geneticin, chloramphenicol and hygromycin. The results show that for Ulva, lower concentrations of kanamycin or geneticin caused improvement in protoplast regeneration while higher concentrations (0.5 mg/mL or more of kanamycin; 0.05 mg/mL geneticin) caused significant inhibition of growth (Huang et al., 1996). Thus it should be possible to select transformed cells with NPTII gene using these antibiotics. Porphyra cells also showed no inherent resistance to kanamycin or hygromycin, therefore, either NPT or HPT gene could be used to select transgenic cells.
Several experiments were conducted with Porphyra thallus using the technique of biolistic bombardment using the “gene gun.” While there was some indication of the development of blue color in some cells, it was extremely difficult to visualize the transformed cells from the thallus surface due to the fact that the cells are very small and they have a very thick cell wall that reflects incident light. Thus it was decided that further experiments with bombardment should be done with conchocelis cells which are quite elongated and would be easy to visualize.