Hydrobiologia 398/399: 469–472, 1999. J.M. Kain (Jones), M.T. Brown & M. Lahaye (eds), Sixteenth International Seaweed Symposium, © 1999 Kluwer Academic Publishers. Printed in the Netherlands.
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Review of genetic engineering of Laminaria japonica (Laminariales, Phaeophyta) in China Song Qin, Guo-Qiong Sun, Peng Jiang, Li-Hong Zou, Yun Wu & Cheng-Kui Tseng Institute of Oceanology, the Chinese Academy of Sciences, Qingdao 266071, China E-mail:
[email protected] Key words: genetic transformation, Laminaria japonica Abstract Progress has been made in establishing a genetic transformation model for Laminaria japonica (Phaeophyta, Laminariales). The model includes introduction of foreign genes by biolistic bombardment, use of promoter SV40 to drive gene expression, algal regeneration by parthenogenesis and selection by chloramphenicol or hygromycin. Introduction Genetic transformation of seaweeds was first proposed by Saga (1991), who highlighted potential difficulties in introducing foreign DNA, the vector to express foreign genes, and the selectable marker to screen transgenic plants. Since then, efforts have been made towards establishing transformation models for seaweeds. For example, Cheney & Kurtzman (in litt.) bombarded uidA, the gene which encodes βglucuronidase (GUS), into Eucheuma (Rhodophyta) cells and succeeded in obtaining its transient expression by using CaMV35S and NOS promoters. Transient expression of uidA in protoplasts of Porphyra miniata (Rhodophyta) was carried out by Kuebler et al. (1994) using electroporation and CaMV35S as the promoter. Qin et al. (1994) transferred CaMV35SuidA into explants of Laminaria japonica and Undaria pinnatifida (Phaeophyta) using biolistic bombardment, and transient expression was observed in rhizoids of Laminaria japonica and blades of U. pinnatifida. Here we provide a review of the work carried out since then on the genetic transformation model for L. japonica.
Significance of transformation studies From the late 1950s to the early 1960s, Chinese scientists had worked on genetic breeding of kelp (L. japonica), and created a few highly productive strains. These new strains were partially responsible for the
increase in the annual production of kelp from about 10 t dry weight in 1952 to the current 350 000 t dry weight (Tseng & Qin, 1991). Kelp production, which can be as much as 40 t of dry material per hectare is presently consumed as a subsidiary food and used as a source of low value products, such as iodine, mannitol and alginate. Transgenic land plants are now being explored as a new source of proteins and drugs (Goddijn & Pen, 1995; Haq, 1995; Moffat, 1995). Genetic engineering is expected to be an effective means to develop kelp as a marine bioreactor to produce oral drugs such as vaccines. The basis for genetic engineering is genetic transformation studies.
Methods to introduce foreign genes Since there is very little understanding of the genomes of bacteria or viruses associated with seaweeds, only direct modes of transformation including electroporation and microparticle bombardment should be used. Protoplasts, from sporophytes and gametophytes, of Laminaria are unable to regenerate (Saga & Sakai, 1984; Qin et al., 1995), and so these cannot be used as hosts. Therefore, since only intact cells can be used as gene recipients, methods such as microparticle bombardment and ultrasonication have to be used to introduce foreign DNA through cell walls. It has been found that biolistic bombardment, using a Bio-Rad Biolistic PDS-1000/He Particle Delivery System (Bio-Rad Company, Hercules, CA,
470 U.S.A.), can effectively introduce foreign DNA through cell walls. No GUS or lacZ(β-galactosidase) background was detected by histochemical staining. A low background level (about 549.8 ng/mg total protein) of CAT (chloramphenicol acetyltransferase) was detected by ELISA (Qin et al., 1998b). Using this method sporophytes of L. japonica, which regenerated by parthenogenesis, exhibited CAT and lacZ activity, suggesting that random integration of foreign genes could occur in this process (Qin et al., 1998a, b). Ultrasonic treatment has been developed as a new method for introducing foreign genes into higher plant cells through their cell walls (Zhang et al., 1991). Results of our study revealed that ultrasonic treatment reduced the size of filamentous female gametophytes and could partially break down cell walls. Observations under a fluorescent microscope showed that cell walls of filamentous female gametophytes of L. japonica were partially removed. The positive aspects of this method are lower cost and saving of time, the negative aspects are the lethal effects on gametophyte cells, characterized by the appearance of empty cells, and a decrease in the number of regenerated parthenogenetic sporophytes (Wang et al., 1998a). A method using brown algal viruses for introducing foreign DNA into algal genomes proposed by Henry & Meints (1994) is now being assessed.
Promoter study A lack of knowledge on promoters which work effectively in macroalgae has affected transformation studies. Until powerful seaweed promoters can be isolated, promoters from land plants have to be used for establishing a model. Both CaMV35S and SV40 promoters have been tested and the former was found to work transiently in rhizoids, but the latter worked in an non-tissue-specific manner (Qin et al., 1998a, b). Results of our experiments showed that SV40 promoter induced stable expression of reporter genes after regeneration of sporophytes.
Regeneration of plants Protoplasts, single cells and tissues are the main gene recipients in plant genetic engineering. But neither protoplasts nor single cells from sporophytes of L. japonica can regenerate into new plants. Tissue culture in Laminariales has been studied extensively (e.g.
Notoya et al., 1992, 1994), but as yet no efficient regeneration system has been obtained. More than two-thirds of the tissues excised from blades, stipes and rhizoids could produce calli after 4 months of culturing in enriched MS medium, but callus only differentiates in ASP12-NTA medium; when PESI medium was used calli appeared within 24 days but no further differentiation was achieved (Wang et al., 1998b). Three types of differentiation from callus have been reported in the Laminariales: formation of sporophytes directly from callus, formation of male and female gametophytes, from which sporophytes are formed by fertilization, and differentiation directly from male and female gametophyte – like filaments without fertilization. From our observations, the regeneration of sporophytes was similar to the latter type of regeneration (Wang et al., 1998b). In genetic transformation of seaweeds, it is critical that attached bacteria do not produce any falsepositive results in the gene expression test. The use of bacteria-inhibited algal materials and the establishment of controls is necessary. In transformation and culture of kelp tissues, pretreatment with 1.5% (w/v) KI produced good sterilization results (Qin et al., 1998b; Wang et al., 1998b). Both blank controls (without bombardment) and negative controls (bombarded with gold particles without DNA) are required since gold particles may introduce bacteria if they are not properly sterilized. Fang et al. (1978) reported that female gametophytes can develop by parthenogenesis. They thought that after parthenogenesis, diploid sporophytes formed but Lewis et al. (1993) have shown that all parthenogenetic sporophytes were haploid. Female gametophytes could grow vegetatively to form a filamentous clone which can be maintained for prolonged periods in the laboratory and used to store valuable strains. Stimulated by certain conditions, these vegetative clones can develop into parthenogenetic sporophytes. This route can be used for regeneration. In our experiments, filamentous female gametophytes were first ground with glass, then captured onto a 0.2-µm membrane, and finally bombarded using the Bio-Rad Biolistic Particle Delivery System. After a few weeks new plants, with an average length of a few mm, were regenerated. Selection in an antibiotic solution could be done at this stage. Usually haploid sporophytes showed an abnormal form of thallus but, if put into specially designed containers and cultured in the sea, they matured and only female gametophytes
471 resulted. Diploid sporophytes generate as if female gametophytes are hybridized with male gametophytes.
Screening of transgenic plants Antibiotics and herbicides are used in genetic engineering of land crops as selectable markers. We tested the sensitivity of L. japonica to eight antibiotics, including lincomycin, ampicillin, streptomycin, kanamycin, neomycin, chloramphenicol (Qin et al., 1998b), hygromycin (Li, 1998), zeocin and G-418. Our result showed that L. japonica is sensitive only to chloramphenicol and hygromycin (Qin et al., 1998b; Li, 1998). The LD50 of hygromycin to parthenogenetic sporophytes was much lower than that of chloramphenicol, and was not correlated with algal length while that of chloramphenicol was (Li, 1998). Using the biolistic particle delivery system, the SV40 promoter as transcription initiator, and female gametophytes as the gene recipients, both cat and lacZ were expressed stably in regenerated parthenogenetic sporophytes (Qin et al., 1998a, b). These results suggest that random integration of foreign genes might occur, since 2 months after transformation with pCATcontrol, followed by selection in chloramphenicolcontaining medium, about 0.7% of the regenerated thalli survived, while all controls died (Qin et al., 1998b). Seven months after transformation, about 6% of regenerated plants showed lacZ expression (% area, 0.25–10%) (Qin et al., 1998a). In lacZ transformation, about 1 × 106 female gametophytes were bombarded with pSV-lacZ plasmid, and after about 80 days among the 435 regenerated plants (algal length including rhizoid, stipe and frond, 2–3 mm) tested for lacZ expression, seven plants exhibited blue spots. There were on average three to four blue spots in the meristematic zone or stipe of each plant. No blue spots were found in the 520 regenerated controls (Wu, 1996). In another experiment, the same (1 × 106 ) number of female gametophytes was bombarded with equivalent amounts of (pSV-lacZ) DNA. After 165 days of regeneration via parthenogenesis, 256 regenerated plants (algal length including rhizoid, stipe and frond, 1–3 mm) were tested and eight plants showed lacZ expression (% area, 0.25 – 1%). In 286 control plants no lacZ activity was observed. The increased number of transformants obtained in this latter experiment may be because egg-releasing gametophytes were used or because histochemical staining was carried out later, i.e. more expressing cells were
produced by cell division so that transformants could be more easily found and counted. By using eggreleasing materials rather than stimulating egg release after bombardment, more gametophytes bombarded with the foreign gene regenerated into sporophytes and thus transformation efficiency was increased. The remaining regenerated plants and controls were cultured for a further 45 days, including 1 month in the sea, 30 of the 496 plants exhibited lacZ expression (% area, 0.25–10%) while no lacZ activity was detected in the 500 controls (Qin et al., 1998a). The increase in both transformants and expressed area after culturing in the sea may be due to a more suitable environment for foreign gene expression and/or more expressing cells appearing after cell division. In a recent experiment, the same number of eggreleasing gametophytes was bombarded with the same amount of DNA. Three months later, 75.5% of regenerated parthenogenetic sporophytes exhibited lacZ expression and no lacZ activity was found in any control plant. The expression level is still low and may due to gene integration at a very late stage; the later the integration, the lower the ratio of transformed cells. In conclusion, by using biolistic bombardment as the method of introduction, the SV40 promoter as a transcription initiator, female gametophytes as gene hosts, parthenogenesis as regeneration route and chloramphenicol (or hygromycin) as a selectable reagent, a transformation model has been established.
Environmental release of transformed algae Environmental release of transgenic L. japonica should be given serious consideration. The vectors and genes used in transformation studies should be restricted in order to avoid any negative phenotype they may encode. In our opinion transgenic L. japonica should not cause any negative impact on natural populations or on seed stocks. Laminaria japonica was originally introduced from Japan and is now cultured extensively in China. In most parts of the China Sea, it cannot withstand the summer seawater temperature and plants die naturally; there are no ‘natural’ population of L. japonica. To prevent transgenic kelp being incorporated into the marine food chain or carried away by marine currents containers are needed to ensure that the kelp cannot escape or be eaten by marine animals when cultured in the sea. We have designed two types of containers to hold transformed algae in the sea. One type has a volume of about 3.6 l and is made of 0.2-
472 mm nylon membrane (Qin et al., 1998b); this type of container can be easily fouled but the membrane is solid enough not to be perforated. The second type is made by removing the base of a 1.25-l cola bottle, fixing a 0.2-mm nylon membrane to the bottom, and drilling many holes on the surface of the bottle. The membrane can be changed when it is fouled. To avoid the release of spores through the membrane or holes, it is necessary to harvest the kelp prior to the formation of sporangia and to collect the gametophytes indoors.
Acknowledgements This project was sponsored by the National Climbing Plan (B6-4-1) from the China Science and Technology Committee, and by National Natural Science Grants (No.39400076 and 39670367) from the National Natural Science Foundation of China.
References Fang, Z., J. Dai, J. Cui & Y. Ou, 1978. The first record of female sporophytes of Laminaria japonica. Chin. Sci. Bull. 23: 43–44. Goddijn, O. J. M. & J. Pen, 1995. Plants as bioreactors. Trends Biotechnol. 13: 379–387. Haq, T. A., 1995. Oral immunization with a recombinant bacterial antigen produced in transgenic plants. Science 268: 714–716. Henry, E. C. & R. H. Meints, 1994. Recombinant viruses as transformation vectors of marine macroalgae. J. appl. Phycol. 6: 247–253. Kuebler, J. E., S.C. Minocha & A. C. Mathieson, 1994. Transient expression of the GUS reporter gene in protoplasts of Porphyra miniata (Rhodophyta). J. mar. Biotechnol. 1: 165–169. Lewis, R. J., B. Y. Jiang, M. Neushul & X. G. Fei, 1993. Haploid parthenogenetic sporophytes of Laminaria japonica (Phaeophyceae). J. Phycol. 29: 363–369. Li, X., 1998. Studies on genetic transformation of Laminaria japonica (Phaeophyta) by using female gametophytes as gene recipients. Master Thesis, Institute of Oceanology, Chinese Academy of Sciences, 49 pp. Moffat, A. S., 1995. Exploring transgenic plants as a new vaccine source. Science 268: 658–660.
Notoya, M., M. Nagashima & Y. Aruga, 1992. Influence of light intensity and temperature on callus development in young sporophytes of four species of Laminariales (Phaeophyta). Kor. J. Phycol. 7: 101–107. Notoya, M., M. Nagashima & Y. Aruga, 1994. Influence of light intensity and temperature on callus development in young sporophytes of three species of Laminariales (Phaeophyta). J. mar. Biotechnol. 2: 15–18. Qin, S., P. Jiang, X. Li & C. K. Tseng, 1998a. The expression of lacZ in regenerated sporophytes of parthenogenetic Laminaria japonica. Proceedings of the 2nd Asia-Pacific Marine Biotechnology Conference and the 3rd Asia-Pacific Conference on Algal Biotechnology: 205–208. Qin, S., J. Wu, X. Wang, X. Li, P. Jiang & C. K. Tseng, 1998b. The expression of foreign genes in Laminaria japonica (Phaeophyta). In Morton, B. (ed.), The Marine Biology of South China Sea. Hong Kong University Press, Hong Kong: 209–217. Qin, S., J. Zhang, W. Li, X. Wang, S. Tong, Y. Sun & C. K. Tseng, 1994. Transient expression of GUS gene in phaeophytes using biolistic particle delivery system. Oceanol. Limnol. Sin. 25: 353– 356 (in Chinese with English abstract). Qin, S., X. Wang, S. Tong and C. K. Tseng, 1995. Isolation and culture of female haploid protoplasts from Laminaria japonica. Oceanol. Limnol. Sin. 26 (suppl.): 126–129 (in Chinese with English abstract). Saga, N., 1991. Protoplasts and somatic hybridization-controversal discussion ‘No’ side. Proceedings of a COST-48 Workshop, Spain: 25–30. Saga, N. & Y. Sakai, 1984. Isolation of protoplasts from Laminaria and Porphyra. Bull. jap. Soc. Sci. Fish. 50:1085. Tseng, C. K. & S. Qin, 1991. Marine algal biotechnology in China: present conditions and prospects. Proceeding of International Symposium on Biotechnology of Salt Ponds, Tianjin: 61–68. Wang, X., S. Qin, X. Li, P. Jiang, C. K.Tseng & M. Qin, 1998a. Effects of ultrasonic treatment on female gametophytes of Laminaria japonica (Phaeophyta). Chin. J. Oceanol. Limnol. 16 (Suppl.): 62–66. Wang, X., S. Qin, X. Li, P. Jiang, C. K.Tseng & M. Qin, 1998b. High efficient induction of callus and regeneration of sporophytes in Laminaria japonica (Phaeophyta). Chin. J. Oceanol. Limnol. 16 (Suppl.): 67–74. Wu, J., 1996. Study of selectable markers and expression of foreign genes in Laminaria japonica, Master Thesis, Institute of Oceanology, Chinese Academy of Sciences, 39 pp. (in Chinese with English abstract). Zhang, L., L. Cheng, J. Yuan , C. Li, S. Jia, N. Xu & N. Zhao, 1991. Ultrasonic direct gene transfer – the establishment of high efficiency genetic transformation system for tobacco. Sci. Agric. Sin. 24: 83–89 (in Chinese with English abstract).
