Wheat egg in vitro fusion with wheat and green bristlegrass sperm

Summary Isolated gametes can be used to investigate fertilization mechanisms, and probe distant hybridization between different species. Pollen grains of wheat and Setaria viridis are tricellular, containing sperm cells at anthesis. Sperm from these plants were isolated by breaking open pollen grains in a osmotic solution. Wheat ovules were digested in an enzyme solution for 20 min, and then transferred to an isolation solution without enzymes to separate egg cells from ovules. The fusion of wheat egg cells with wheat and S. viridis sperm was conducted using an electro-fusion apparatus. Under suitable osmotic pressure (10% mannitol), calcium concentration of 0.001% (CaCl2·2H2O), and a 30–35 V alternating electric field for 15 s, egg cells and sperm adhered to each other and became arranged in a line. Electroporation of the plasma membrane of egg cells and sperm using a 300–500 V direct-current electric field (45 µs amplitude pulse) caused them to fuse.

[1]  W. R. Sharp,et al.  Plant Regeneration from Cell Cultures , 2011 .

[2]  T. Okamoto,et al.  Establishment of an in vitro fertilization system in rice (Oryza sativa L.) , 2007, Planta.

[3]  S. Russell,et al.  In vitro fertilization as a tool for investigating sexual reproduction of angiosperms , 2006, Sexual Plant Reproduction.

[4]  U. Grossniklaus,et al.  Delayed activation of the paternal genome during seed development , 2022 .

[5]  K. Glimelius,et al.  Characterization of sexual progenies of male-sterile somatic cybrids between Brassica napus and Brassica tournefortii , 1999, Theoretical and Applied Genetics.

[6]  T. Dresselhaus,et al.  A transcript encoding translation initiation factor eIF-5A is stored in unfertilized egg cells of maize , 1999, Plant Molecular Biology.

[7]  B. Barnabás,et al.  Electro-fused isolated wheat (Triticum aestivum L.) gametes develop into multicellular structures , 1995, Plant Cell Reports.

[8]  E. Kranz,et al.  Early cytological events after induction of cell division in egg cells and zygote development following in vitro fertilization with angiosperm gametes , 1995 .

[9]  R. Skirvin,et al.  Sources and Frequency of Somaclonal Variation , 1994 .

[10]  E. Kranz,et al.  In vitro fertilisation of maize by single egg and sperm cell protoplast fusion mediated by high calcium and high pH , 1994, Zygote.

[11]  C. Dumas,et al.  An in Vitro System for Adhesion and Fusion of Maize Gametes , 1994, Science.

[12]  E. Kranz,et al.  In Vitro Fertilization with Isolated, Single Gametes Results in Zygotic Embryogenesis and Fertile Maize Plants. , 1993, The Plant cell.

[13]  Yukun Liu,et al.  The Summary of Plant Cell Fusion and Its Application * , 2013 .

[14]  张亚楠,et al.  The Primary Attempt of in Vitro Fertilization of Torenia fournieri , 2012 .

[15]  N. Haq,et al.  Isolation and in vitro fusion of egg and sperm cells in Oryza sativa. , 2005, Plant physiology and biochemistry : PPB.

[16]  E. Kranz,et al.  In vitro fertilization of single, isolated gametes of maize mediated by electrofusion , 2004, Sexual Plant Reproduction.

[17]  C. Dumas,et al.  Use of in vitro fertilization and zygote culture in crop improvement , 1995 .