Xiaoying XU, Wenkai SHI, Lei LI, Ya’nan CAO, Xiaoguang LU, Peng LIU

Yantai Marine Economic Research Institute, Yantai 264006, China

Abstract This paper discussed the technique of algal protoplast fusion, including the method of protoplast-induced fusion, the screening and identification of hybrid cells and so on. In this paper, the research progress of protoplast-induced fusion of microalgae was reviewed, and the problems existing in the process of protoplast-induced fusion were discussed in order to provide a theoretical basis for the development of algae genetics and breeding.

Key words Algae, Protoplast fusion, Research progress

1 Protoplast fusion technique

Protoplast fusion is a process in which protoplasts of different varieties are hybridized by artificial methods to form heterozygous cells, and then cultured in vitro into complete plants after induction and differentiation. In short, protoplast fusion makes it possible for cells of two different varieties to hybridize. This technique has the advantages of wide range of application and strong feasibility, and it opens up a new way for the inheritance of various organisms and the effective cultivation of new varieties with good characters.

2 Methods of protoplast-induced fusion

Spontaneous fusion between homologous or heterogeneous protoplasts can often be observed in the process of protoplast preparation or culture[1]. However, the spontaneous fusion rate is relatively low, so artificial methods can be used to induce fusion in order to improve the fusion rate, in which chemical fusion, electrofusion and laser fusion are commonly used to induce protoplast fusion.

2.1 Chemical fusion methodChemical fusion induces protoplast aggregation, adhesion and fusion by different chemical reagents. Inducers can be divided into various salts [including NaNO3, Ca(NO3)2, KNO3, MgCl2,etc.], polycompounds (including polylysine, poly-l-ornithine hydrochloride, polyethylene glycol, polyvinyl alcohol,etc.) and other compounds (including ATP, ADP, animal glue, lysozyme, inactivated Sendai virus, dextran sulfate,etc.). And polyethylene glycol PEG fusion method is the most widely used chemical fusion method. This method can obtain high induced-fusion rate (about 10%), good repeatability and little side effects on protoplasts, but the fusion treatment time, PEG molecular weight and the concentration of induction solution are difficult to grasp, so it is easy to form multiple fusion bodies[2].

2.2 Electrofusion methodElectrofusion was produced in the 1980s and has become one of the effective means of cell fusion. The reversible electrical penetration of the cell membranes in the contact areas of two adjacent cells under the action of a short-term strong electric field can induce their membranes to fuse with each other, resulting in cell fusion. The electrofusion technique is simple, rapid and non-toxic to cells, and the whole process of fusion can be observed under microscope.

2.3 Laser fusion methodIn the mid-1980s, a laser fusion device appeared, that is, a laser beam was used to destroy or disturb the cell membrane in the contact area of adjacent cells, and two different cells were fused under the microscope[3]. Compared with other fusion methods, the laser-induced fusion method has high selectivity and can fuse any two different cells. In addition, it can also achieve non-contact, safe and long-distance operation, but this operation requires the processing of cells one by one rather than a large number of cells at the same time, and the equipment is expensive.

3 Screening and identification of hybrid cells

3.1 ScreeningAfter induction treatment, protoplasts will form fusant, non-fusant, multi-fusant and chimera populations. The screening of hybrid cells is the key to protoplast fusion. At present, the main methods for hybrid cell screening are complementary selection, physical selection, growth differential selection, asymmetric fusion selection and so on.

Fig.1 Picture of protoplast fusion of Haematococcus pluvialis

3.2 IdentificationThe selected fusants usually contain heterokaryons, partial heterozygotes and partial diploids, so further identification is needed. At present, the commonly used methods for hybrid cell identification are phenotype identification, cytological identification, isozyme identification and molecular biological identification. In order to enhance the reliability of hybrid cell identification, several methods are usually used together.

4 Research progress of protoplast-induced fusion of microalgae

Since the 1970s, researchers first used some wall-deficient varieties of microalgae to study cell fusion, which opened the prelude to the study of microalgae cell fusion. Subsequently, researchers reported the factors affecting the efficiency of protoplast fusion. In 1992, Mizukamietal.[4]usedP.yezoensisas material to study the effect of cell wall hydrolase on cell fusion efficiency. The results showed that the crude enzyme mixture fromA.crassispinaandPseudomonassp. had the best effect on protoplast electrofusion. In addition, the results of the study on the survival rate of protoplasts showed that the activity of protoplasts greatly affected the efficiency of electrofusion in the process of cell wall hydrolase treatment. Mizukamietal.[5]studied the factors affecting the cell fusion efficiency ofP.yezoensis. The results showed that the concentration of osmotic regulator, calcium ion and cell membrane specific digestive enzymes all affected the cell fusion efficiency ofP.yezoensis.

With the maturity of protoplast fusion conditions, some successful cases of algal protoplast fusion have been reported. Dai Jixunetal.[6]fused the newly isolated protoplasts ofP.haitanensisandP.yezoensisby PEG fusion method in 1990. The single fusant began to divide for the first time after a week of culture, and developed into a cell mass after four weeks, but finally did not differentiate into a complete plant. In 1992, Liu Guangfaetal.[7]isolatedDunaliellasp. by using Streptomyces protease. The protoplast was fused with the globule ofE.coli(containing chloramphenicol acetyltransferase plasmid) by PEG fusion method, and the fusant was very similar toDunaliellaSalina. After protein component analysis, it was found that there were significant differences between fusants,Dunaliellasp. andEscherichiacoli. In addition, the fusant has chloramphenicol resistance, and it can be inferred that the plasmid ofEscherichiacolihas been transferred to the fusant cells. However, in the subsequent subculture, the resistance of the fusant to chloramphenicol decreased gradually, and the chloramphenicol acetyltransferase plasmid was not isolated from the fusant. In 1994, Tjahjonoetal.[8]carried out protoplast fusion between inhibitor-resistant mutants of green algae (U.plucialis), and obtained hybrids with higher carotenoid forming ability than both parents and wild types. In 1995, Sivanetal.[9]fused the protoplasts of the low-phycoprotein mutant ofPorphyridiumsp. with the green mutant, and obtained 8 red fusants, and the phycobiliprotein and chlorophyll content of the fusant was higher than that of the mutant parent. In 2001, Shen Jihongetal.[10]fused autotrophic microalgae rich in EPA and DHA and fast-growing heterotrophic microalgaeTetraflagellumusing cell fusion technology, and selected co-cultured fusion algae strains. In 2004, Jiang Shengtaoetal.[11]fused the protoplasts ofH.pluvialisandS.cerevisiae, and analyzed their fusants and wild parents by RAPD. The results showed that the fusants may be hybrids ofH.pluvialisandS.cerevisiae.

5 Problems in algal protoplast fusion

Although the technology of protoplast fusion and hybridization of microalgae has made great progress, there are still many difficulties and challenges. For example, although there is no interspecies incompatibility at the stage of cell fusion, there is still serious incompatibility and exclusion among distant species in the subsequent development of nuclear fusion, chromosome hybridization and fusion hybrid cells. Moreover, in the process of fusion, a considerable number of fusants did not have real nuclear fusion, and the chromosomes of the two parents were conserved or even rejected, so that the genes they carried could not be expressed in the same cell at the same time. Finally, segregation occurred in the subsequent subculture.

Another difficulty in protoplast fusion and hybridization is the identification of fusants. At present, the basis of physiology and biochemistry is still widely used in hybrid identification. Therefore, the two parents should also have some biochemical or genetic markers, but there are few microalgae with such markers in nature, coupled with the complex genetic background of microalgae, which undoubtedly increases the difficulty of hybrid identification. At present, the study of microalgae cell hybridization still falls into the category of basic theoretical research. Because of the problems such as parental hybridization incompatibility and fusant identification difficulty, it is not easy to obtain true nuclear fusion hybrids through cell fusion.