Gramineae is one of the largest families with the widest distribution in the world, including the world's most important crops ,such as rice, corn, wheat, barley, sorghum, sugar cane, and so on. There is a important practical significance to learn of the mechanism of in vitro fertilization of gramineae because we can further improve the yield and quality of the gramineae. To date, the first successful case of in vitro fertilization is Kranz et al (1991) making the fusion of corn sperm cells and egg cells by power fusion, and resulting in some plants. Since then, there has been ongoing studies on the gramineae family in vitro fertilization from other researchers, who have made some prog- resses. In this article, we will briefly introduce recent advances in in vitro fertilization of gramineae.

1 Isolation of sperm cells
Isolation and purification of sperm cells arethe bases of physiological and biochemical in vitro fertilization study. They are also the premise of physiology, biochemistry and molecular biology on the sperm and egg identification process in higher plants. There are two types of Angiosperm pollens: bicellular pollen type, a generative cell and a vegetative cell in a mature pollen grain. tricellular pollen type, two sperm cells and a vegetative cell in a mature pollen grain.The pollen tubes of bicellular pollen type grains would be firstly induced in vitro cultivation to isolate sperm cells in the pollen tube, after the generative cell of pollen tube divides to form two sperm cells, The mature pollens of gramineae of a tricellular pollen cell could be used directly to isolate sperm cells. There are 2 main ways to separate sperm cells from tricellular pollen cells: the impact of infiltration and grinding. In 1973, Cass soaked pollens of barley in hypotonic solution of 10% BK sucrose solution, and obtained some sperm cells successfully (Cass, 1973). The impact of infiltration method is relatively simple, and it has high ratio of efficiency separation. To date the method was adopted in the sperm cells isolation of the graminea, such as corn (Dupuis et al., 1987; Yang and Zhou, 1989), rice (Guo et al., 1999), and so on, but different sperm cells masters are required and different optimum concentrations of separation are observed depending on the specific plants. There is little application of grinding on the isolation of the sperm cells of gramineae.

2 Isolation of egg cell
Egg cells are located in the embryo sac, and the embryo sac is born in the ovule, which made it more difficult to isolate the egg cells. We can isolate embryo sac, and then isolate the egg sac, or we can also isolate egg cell directly from the ovule. At present, we obtained the egg cell from gramineae mainly by enzyme or anatomical dissection. Enzymatic dissection methods which began in the 1980s have been relied on for the isolation of egg cell from maize (Kranz and Lorz, 1993), wheat (Kovacs et al., 1995), Rice (Zhao et al., 2000). Corn is by far the only case that achieved in vitro fertilization using isolated egg cell obtained from enzyme dissection method. Another method, micro-dissection was developed in the 1990s, which is performed universally for the gramineae in particular. At present, micro-dissection has been applied successfully to isolate the egg cell of barley (Holm et al., 1994), wheat (Holm et al., 1994; Kovacs et al., 1995), maize (Kovacs et al., 1995), Rice (Zhao et al., 2000). Zhao et al (2000) further attempted to compare the effects of the ovule from the vertical profile, crossprofile extrusion and separation of rice on egg and found cross-sectional ovule very easy to maintain egg cell in situ, to identify and to operate. 

It is generally believed that dissection enzyme should be used more in the plants (such as tobacco) with thin beads heart, and easily separated ovules, while Microdissection applies to plants (such as corn) with bulky ovules and small numbers of ovules. The two methods can complement each other, for example, in the separation of rice and corn, the ovule of the short-term digestion of the egg will help improve the efficiency of separation, but if the enzyme is not thoroughly cleaned or the over-all follow-up on the homozygous which would be a negative impact on culture. 

3 Egg, zygote and sperm in vitro culture, sperm cells and egg cell fusion
The embryo sac cells which have been in vitro culture include egg cell ,help cells, central cell, zygote, and artificial insemination central cells. The most eyecatching ones are egg cell and zygote in vitro culture. Kranz et al (1995) first reported that the egg cell of corn starts splitting and developing small cell mass in high concentration of 2,4-D, but it can not further grow. Subsequently, Zhao et al (2000) inducted the first split in the cultivation of egg cells. Zygote can be divided into homozygous and artificial zygote. Homozygous zygote was easier to get than artificial zygote, so homozygous zygote has been separated in many species for studies. Homozygous of the four natural crops, barley (Holm et al., 1994), wheat (Holm et al., 1994; Kumlehn et al., 1998; Pónya et al., 1999), maize (Leduc et al., 1996) and Rice (Han et al., 1998; Zhang et al., 1999) in vitro culture have been able to regenerate plants. Zhang et al (1999) found the homozygous development of rice is not in accordance with normal embryonic development mode in the cultivation of rice’s homozygous, but generated plants after by the bipolar embryoid or the formation of adventitious root. Zhao et al (2002) used embryonic cell lines of rice as suspension cell husbandries, obtained the desired result in inducing the growth of zygote by micro-feeding room culture, the rice zygote successfully splits into multi-cell mass. Artificial zygote is induced in vitro by a combination of methods. The methods by which the egg and sperm cells were induced to form artificial zygote are as following: electric fusion, calcium-induced fusion, and PEG-induced fusion. Zygote obtained from corn after sperm-egg fusion, formed the plant in vitro culture through electric fusion (Kranz et al., 1991). Faure et al (1994) put the egg and sperm cells of the corn into 5 mmol/L cacl₂, and found that the sperm cells and the egg cell fused in a very short time. By increasing the concentration to 0.05 mol/L cacl₂, the frequency of corn sperm-egg fusion had also increased, but the zygote formed multi-cell mass and no plant was obtained after the fusion. Kovacs et al (1995) found slightly inequal split in the cultivation artificial homozygous of wheat. Such successful studies in vitro culture will form a quality test system for genetic transformation and improvement of crops.

4 Molecular biology research
The use of in vitro fertilization system in the process of gene expression is a new field. The two major technological bases of study have matured, they are, access to low-volume manual egg cells and zygote, and the method of building a micro-cell cDNA library. RT_PCR application technology, 128 egg cells by the separation of corn and 104 artificial zygote in vitro fertilization after 18 hours of construction of cDNA library, and a number of specific expression in the egg or the fertilization induced expression gene were isolated (Leduc et al., 1996). Gou et al (2001) first built a CDNA library of rice sperm cells, and isolated the expressional gene from the sperm cells. Bai et al (2002) first isolated the full-length gene RSG6f from rice sperm cells. RT-PCR results show that the gene is expressed in the roots, leaves, the pollen cells and mature pollen, pollination ovary and sperm cell with particularly high volume in the sperm cells, thus it is a differentially expressed genes. Miao et al (2003) used cloning which has been expressed in the sperm cells as a probe to screen rice sperm cell CDNA library, and got a two full-length CDNA cloning. They then successfully constructed expression vector PRGFP1 for Agrobacterium-mediated genetic transformation of rice. In addition to building a library of cDNA to study gene expression, the researchers also tried to introduce foreign gene into the zygote experimentby microinjection of two genes (GUS gene, the gene regulation of anthocyanin) were separated from the corn import Zygote, the train after a short period of time, prove to be imported in the homozygous gene expression in the moment (Leduc et al., 1996). 

5 Conclusion
After the model Corn fertilization succeeded, the researchers mainly aimed to create more systems of in vitro operation. At present, as an important food crop, rice has a better foundation for embryo culture. The culture of renewable natural homozygous plants has also been successful. The large number of sperm cells of rice can be isolated, but separation of egg cells in rice quantitatively restricts the result of in vitro fertilized. In addition to the kinds of in vitro fertilization, the distant vitro fertilization, the use of an egg, a zygote with genetically engineered receptosr also enjoyed great vitality. Whether natural or artificial zygote, there are very high frequency splits in vitro, which have incomparable advantages with a somatic general and other cell systems. 

The in vitro fertilization has two major advantages which are the possibility of performing it live and with control. we can study the process of signal transduction in vivo of fertilization and embryo which will not be as effective if studied by physiology and biophysics methods, Additionally, we can apply biological methods to study the process of gene expression, find out the various aspects of the development of functional genes, and then apply transgenic technology to change the process of fertilization and embryo development. If we find the mechanism that activates the egg-related genes, there is the hope that we can use the gene to create operating new plant type without fertilization and embryogenesis. Its significance is self-evident. 

Authors' Contributions
ZAW performed the experiment, wrote and modified the manuscript. The author has read and approved the final manuscript.

Acknowledgements
I thanked two anonymous reviewers for their strict criticism on this paper.

References
Bai Y., Gou X.P., Xv Y., Yan F., Tang L., and Chen F., 2002, Cloning of a differentially expressed gene in rice sperm cells and preparation of antiserum against expressed, Chinese Journal of Applied & Environmental Biology, 8(4): 378-382 

Cass D.D., 1973, An ultrastrucurral and Nomarski interfernce study of the sperm bareley, Can. J. Bot., 51(3): 601-605 
http://dx.doi.org/10.1139/b73-072

Dupuis I., Roeckel P., Matthys-Rochon E., and Dumas C., 1987, Procedure to isolate viable sperm cells from corn (Zea mays L.) pollen grains, Plant Physiol., 85(4): 876-878
http://dx.doi.org/10.1104/pp.85.4.876 PMid:16665823 PMCid: 1054361

Faure J.E., Digonnet C., and Dumas C., 1994, An in vitro system for adhesion and fusion of maize gametes, Science, 263(5153): 1598-1600 
http://dx.doi.org/10.1126/science.263.5153.1598 PMid:17744790

Gou X.P., Wang S.H., and Chen F., 1999, Isolation and Cytological observation of viable sperm cells of rice, Journal of Integrative Plant Biology, 41(6): 669-671 

Gou X.P., Xv Y., Yan F., and Chen F., 2001, Representative cDNA library from isolated rice sperm cells, Journal of Integrative Plant Biology, 43(10): 1093-1096

Han H.M., Zhao J., Shi H.Z., Yang H.Y., and Zhou C., 1998, Isolation of egg cells and zygotes in oryza sativa, Journal of Integrative Plant Biology, 40 (2): 186-188

Holm P.B., Knudsen S., Mouritzen P., Negri D., Olsen F.L., and Roue C., 1994, Regeneration of fertile barley plants from mechanically isolated protoplasts of the fertilized egg cell, The Plant Cell, 6(4): 531-543 
PMid:12244247 PMCid: 160456

Kovacs M., Barnabàs B., and Kranz E., 1995, Electro-fused isolated wheat (Triticum aestivum L.) gametes develop into multicellular structures, Plant Cell Rep., 15(3-4): 178-180 
http://dx.doi.org/10.1007/BF00193715

Kranz E., and Lörz H., 1993, In vitro fertilization with isolated, single gametes resulted in zygotic embryogenesis and fertile maize plants, Plant Cell, 5(7): 739-746 
PMid:12271084 PMCid:160312

Kranz E., Bautor J., and Lörz H., 1991, Eletrofusion-mediated transmission of cytoplasmic organelles through the in vitro fertilization process, fusion of sperm cells with synergids and central cells, and cell reconstitution in maize, Sex Plant Report, 4(1): 17-21 
http://dx.doi.org/10.1007/BF00194566

Kranz E., Von Wiegen, P., and Lörz H., 1995, Early cytological events after induction of cell division in egg cells and zygote development following in vitro fertilization with angiosperm gametes, Plant J., 8(1): 9-23 
http://dx.doi.org/10.1046/j.1365-313X.1995.08010009.x

Kumlehn J., Lörz H., and Kranz E., 1998, Differentiation of isolated wheat zygotes into embryos and normal plants, Planta, 205(3): 327-333 
http://dx.doi.org/10.1007/s004250050327

Leduc N., Mattys-Rochon E., Rougier M., Mogensen L., Holm P.B., Magnard J.L., and Dumas C., 1996, Isolated maize zygotes mimic in vivo embryonic development and express microinjected genes when cultured in vitro, Dev. Biol., 177: 190-203 http://dx.doi.org/10.1006/dbio.1996.0155 PMid:8660887

Miao C., Gou X.P., Lan L.Q., Bao J.L., Tang L., Xu Y., Wang S.H., Chen F., 2003, Molecular cloning and expression of RSSG58 gene in rice sperm cells, Acta Botanica Sinica, 45(2): 234-241

Pónya Z., Finy P., Fehér A., Mitykó J., Dudits D., and Barnabás B., 1999, Optimization of introducing foreign genes into egg cells and zygotes of wheat via microinjection, Protoplasma, 208(1-4): 163-172 
http://dx.doi.org/10.1007/BF01279087

Yang H.Y., and Zhou C., 1989, Isolation of viable sperms from pollen of Brassica napus, Zea mays and Secale cereale, Chinese Journal of Botany, 1(1): 80-84 

Zhang J., Dong W.H., Galli A., and Potrykus I., 1999, Regeneration of fertile plants from isolated zygotes of rice (Oryza sativa L.). Plant Cell Rep., 9(2): 321-326 

Zhao J., Yao C.Y., Zhou C., and Yang H.Y., 2002, Growth of zygotes and early proembryos in rice and wheat using microculture system with feeder cells, Acta Agronomica Sinica, 28(3): 289-293 

Zhao J., Zhou C., and Yang H.Y., 2000, Isolation and in vitro culture of zygotes and central cells in Oryza sativa L., Plant cell Rep., 19(3): 321-326
http://dx.doi.org/10.1007/s002990050020