Abstract The progress in protoplast technology and its application in fruit breeding were summarized， and the existing problems and application prospect were also discussed.
　　Key words Protoplast culture; Protoplast fusion; Cybrids; Fruit breeding
　　 Plant protoplasts are "naked cells" that are viable and surrounded by the plasma membrane and can induce regenerated plants under suitable culture conditions. Due to the absence of cell wall， protoplasts can directly and efficiently ingest foreign DNA or genetic materials， various organelles and even nuclei， and can also be fused with heterologous protoplasts under induction to form somatic hybrids， and the penetration and intersection of their culture techniques with related disciplines such as cytology， molecule biology and genetics has made the study of plant protoplasts more and more important[1]. Protoplast culture is the core content of cell engineering， as well as an important part of plant cell engineering technology， which is of great significance to the rapid propagation of plants， the realization of distant genetic recombination， transgenics， and new germplasm and breed improvement[2-3]. This paper reviewed the technique of protoplast culture and its application in fruit tree breeding， aiming at providing reference for relevant research.
　　Preparation and Culture of Protoplast Materials
　　Selection of protoplast separation materials
　　The selection of suitable materials for separation of protoplasts is the key to the success of isolation of protoplasts[4]. The genotype， type and physiological state of the materials are  different ， and the isolation and culture effects of protoplasts are also different. Genotype affects the yield and vigor of protoplasts and directly affects the regeneration of protoplasts[5-7]; and the type of materials and their physiological state directly affect the culture effect of protoplasts[8]. All kinds of organs， tissues and cells of plants can be used as explants， but for different species， different explants can be chosen. For instance， the leaves of dicotyledons can give a large number of relatively homogeneous protoplasts without causing damage to the mother cells. In addition， cotyledons， hypocotyls， shoot tips and calli， suspension cultures and  somatic  embryos all can be used as materials for isolating  protoplasts.
　　Many studies have suggested that pre treatment of materials before isolation has a positive effect on maintaining the integrity of protoplasts and increasing the yield of protoplasts. Pretreatment of materials can change the physiological state of the nucleus and cell wall， improve the strength of the cell membrane and the efficiency of cell wall enzymatic hydrolysis， and reduce protoplast damage. The material pretreatment methods mainly include branch dark treatment， leaf wilting pretreatment， leaf pre culture， pre  plasmolysis  and embryogenic callus pre cuture and suspension culture[9]. 　　Isolation and purification of protoplasts
　　The isolation process of protoplasts is an enzymatic hydrolysis process in a system with an appropriate enzyme composition， pH of the enzyme solution and enzymatic hydrolysis temperature for appropriate enzymatic hydrolysis time under a stable isotonic condition or at a pressure slightly lower than the intracellular pressure. Suitable enzymes and osmotic stabilizers are two prerequisites for obtaining a large number of protoplasts and maximally maintaining the vitality of the protoplasts.
　　The enzyme is an important substance in the isolation of protoplasts， including cellulase， hemicellulase， pectinase， driselase and macerozyme. The enzymes most commonly used are cellulase and pectinase. The former acts on the pectin in the intercellular space to separate cells; and the latter acts on the cell wall directly， causing the cell wall to decompose and release protoplasts. Different materials should be tested to determine the appropriate type and composition of the enzyme solution. Osmotic stabilizers not only play an important role in maintaining the integrity and stability of plant protoplasts， but also play an important role in subsequent culture and cell division. There are two kinds of osmotic stabilizers： saccharides and salts. Among them， the saccharides mainly include sorbitol， mannitol， glucose， sucrose， xylose and dextran. The salts mainly include magnesium sulfate， calcium chloride and potassium chloride. The two types of osmotic stabilizers may be used singly or in combination.
　　After enzymatic hydrolysis， the system is filtered through a nickel mesh of about 400 mesh， to remove incomplete tissue fragments （ducts， sieve tube， etc.） and cell clusters. The filtrate was then centrifuged， obtaining the supernatant which is discarded and the protoplasts which are resuspended in the wash solution （CPW） and centrifuged again. The above centrifugation was performed for three times repeatedly. The purification effect was checked under an inverted microscope. If the effect is good， the cells can be centrifuged with a protoplast medium; and if the effect is poor， centrifugation can be performed with sucrose with a mass fraction of 20% to float the intact protoplasts on the liquid surface[10].
　　Protoplast culture
　　The medium has a great effect on the division frequency of protoplasts， and plays a key role in protoplast culture. The medium components mainly include organic matter， inorganic substances and hormones. The commonly used protoplast media are mostly Km8P and NT medium. Km8P medium is based on B5 medium， and NT medium is improved in the basis of MS medium[11]. The pH of the medium is generally 5.6-5.8. Additives in the medium vary with the culture and culture organ. Different osmotic pressure regulators and carbon sources have certain effects on the development of protoplasts， and too high or too low osmotic pressure is unfavorable for tissue division[12]. Mannitol is commonly used as an osmotic pressure regulator and a carbon source， and sorbitol is also used[13]. The protoplast culture of different plants has different requirements for osmotic pressure regulators. After culturing for a period of time， the medium with reduced osmotic pressure must be added in time to gradually reduce the osmotic pressure of the medium， otherwise the formed cell clusters and small calli cannot continue to grow and develop. However， there are exceptions[14]. 　　In the early stage of protoplast culture， higher levels of auxin and cytokinin are required to initiate cell wall regeneration and cell division， and different types and concentrations of auxin and cytokinin are required for different plants and cells. For instance， sucrose is required for cell division of sour cherry protoplasts， while the culture of peach protoplasts requires no growth hormones， but components such as ME and CM. The auxins commonly used are NAA， 2， 4 D and IAA， and the cytokinins commonly used include 6 BA， ZT， KT， etc.[15].
　　The initial planting density of protoplasts has an effect on the planting effect， and generally， 10 000-100 000 protoplasts are planted in 1 ml. If the density is too large， the regenerated cell clusters will be small， which affects their growth; and if the density is too small， the protoplast body will extravasate.
　　Protoplasts of different tree species， different genotypes of the same species and different material sources of the same genotype differ greatly in planting density[16]. There are many methods for protoplast culture， such as culture in shallow liquid medium，  solid liquid  combined culture， nurse culture and agar island  culture [1，17-19]. Among them， culture in shallow liquid medium and solid liquid combined culture are commonly used. The culture in shallow liquid medium has the advantages of simple operation， easy addition of medium and good ventilation.  The solid culture method mainly includes agar or agarose embedding， and has the advantages of easy observation of protoplast growth process and prevention of protoplast aggregation. Agarose has a low melting point and can reduce damage to protoplasts and provide necessary nutrients[15]. What kind of culture method is used in the  protoplast  culture process should be determined depending on the material.
　　Regeneration of protoplast plants
　　There are usually three protoplast regeneration pathways： （1） calli are formed from the development of cell cluster and then differentiate into buds， and finally grow into plants; （2） embryoid bodies are formed directly and then develop into plants; and （3） calli are formed at first， and then differentiate to embryoid bodies， which finally develop into plants[20-21]. So far， the protoplast regenerated plants that have been obtained on fruit trees are all regenerated plants which are obtained through the pathway of inducing differentiation of callus organs derived from protoplasts. After the protoplasts are transferred to the proliferation medium to form calli， the induction of adventitious buds is a key issue in the regeneration of protoplast plants. Generally， stepwise induction is used to gradually increase the ratio of cytokinin to auxin to facilitate bud differentiation[12]， because it maintains the growth of calli and gradually makes their structure tighter， which leads to the gradually transformation of calli from the growing state to the differentiate state， that is to say it provides a stepwise adaptation condition at the hormone level for organ differentiation in the calli[22]. After the adventitious buds are induced and sub cultured for several times， long shoots can be cut to induce rooting on the rooting medium. 　　Application of Protoplast Culture in Fruit Tree Breeding
　　Acquiring and transferring exogenous genes， promoting variation of regenerated plants， enriching germplasm resources
　　Protoplasts are small in size and large in population， and are more sensitive to physical and chemical factors in the culture system. Therefore， the asexual mutation rate of protoplasts is higher than that of normal somatic clones. Artificial induction during protoplast culture will result in more variations， thus providing higher possibility for increasing germplasm resources. Protoplasts are also good receptors for non vector genetic manipulation. Protoplasts can directly ingest foreign substances including nuclei， organelle genome， DNA fragments and viral particles and directly transform genes， and are thus an ideal tool for genetic transformation research. There are many ways to introduce exogenous genes into protoplasts， the most common being the PEG method， and others using electroporation. There are many studies on the genetic transformation of kiwifruit. For instance， Rugini[23] integrated the Rirol gene of Agrobacterium tumefaciens into a late flowering Actinidia deliciosa male plant， and the transgenic plant showed typical hairy root characteristics with increased rooting ability. Guo[24] used LfycDNA to transform kiwifruit， which improved its early maturing property. The cytoplasm fusion technique is obviously superior to the sexual hybridization in that it can genetically manipulate the cytoplasm， thereby causing the reorganization of various organelles such as mitochondria and chloroplasts， providing a broad world for cultivating new varieties. It can be seen that somatic hybridization has great potential for creating mutations， and can enrich germplasm resources and maintain and promote biodiversity.
　　Realizing distant genetic recombination， creating distant  hybrids
　　Distal hybridization incompatibility is one of the important reasons for the distant hybridization carriers of fruit trees. Hybridization incompatibility is one of the main reasons for the low  success  rate of wide cross breeding， while protoplast fusion technology can eliminate hybrid incompatibility to form heterozygous bivalents to some extent. In theory， if the hybrid plant is fertile and stably inherited， it may form a new variety that is useful in agriculture. Protoplast fusion overcomes barriers such as incompatibility and sexual organ abortion in hybrid breeding to a certain extent， and obtains hybrids， but the farther the relationship is， the more difficult it is to obtain genetically stable hybrids. Liu et al.[25] induced the fusion of the suspension protoplasts of Microcitrus papuana Swingle and the mesophyll protoplasts of Citrus reticulata Blanco by electrofusion and obtained regenerated plants. Chromosomal examination revealed that the regenerated plants were tetraploid （2n=4x=36） somatic hybrids. In fruit trees， somatic hybrids have great potential as rootstocks， and obtaining resistant rootstocks by somatic cell hybridization is a promising breeding direction. 　　Protoplast fusion to obtain cytoplasmic hybrids
　　Nonsymmetrical crossing can produce cytoplasmic hybrids， i.e.， hybrids containing only the nuclei of the female parent， which are almost impossible to occur in sexual progeny. Cytoplasmic hybrids can be produced by three pathways： （1） fusion of one normal protoplast and one enucleated protoplast， （2） fusion of one normal protoplast and one nuclear inactivated protoplast， and （3） exclusion of the nucleus or chromosome of one parent at a certain stage after fusion[9]. Cytoplasmic hybrids have special significance in breeding， and especially， fertile heterotetraploids or diploid hybrids can be obtained within species and in some combinations of related species. Protoplast fusion can transfer cytoplasmic genes into a new nuclear background， and can recombine the chloroplast genome and the mitochondrial genome also， creating a new source of cytoplasmic variation. There are many ways to obtain cytoplasmic hybrids. For instance， Vardi and Poorman irradiated such three citrus type protoplasts as the hybrid of C. sinensis （L.） Osbeck and C. aurantium L.， C. aurantium L. and Virafranca lemon with X rays， respectively， and fused them with C. aurantium L. and Virafranca lemon treated with 0.25 mol/L iodovinylamine for 25 min， obtaining three combined cytoplasmic hybrids[26-27].
　　Xiangmin SUO et al. Protoplast Culture and Its Application in Fruit Breeding
　　Problems and Prospect
　　The protoplast regenerated plants and protoplast fusion techniques of fruit trees have made great progress in fruit tree breeding， but there are also many problems. Most scholars research is limited to the acquisition of regenerated plants， and the proportion of regenerated plants obtained is small; protoplast culture of fruit trees is poor in stability and reproducibility and is strongly empirical; the use of inducing substances in the induced fusion and the selection of the fusion method are very important， and need to be strengthened; and the loss of donor gene is a random process in somatic cell fusion， and the loss degree is unpredictable and difficult to control.
　　In view of the problems of fruit tree protoplasts in fruit tree breeding， the protoplast culture and fusion techniques should be strengthened in research. The genotypes of protoplast cultured plants should be expanded. Excellent individuals should be screened on the basis of regenerated plants from protoplast culture. The research on induced fusion and various physiological， biochemical， and genetic mechanisms of hybrid cells and the research on electrofusion fusion program control should be strengthened. Based on protoplast technology， specific trait genes should be transferred specifically. Distant hybridization barriers should be overcome， to breed fertile offspring. 　　In conclusion， the application potential of fruit tree protoplasts is great. In the future work， protoplast fusion should be used for gene mapping， cell hybridization and fruit tree improvement， as well as exogenous gene introduction， gene regulation and expression by protoplast transformation， to breed breeding materials with higher practical value and boost the process of breeding.
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