To establish better embryo induction procedures in anther culture of apple, the relationships between the microspore stage and the flower bud stage, and between embryo induction and the developmental stage of microspores during cold pre-treatment of flower buds were investigated. We classified the flower bud stages into four: "Tight cluster", "First pink", "Pink", and "Full pink". Each stage was defined by a morphological characteristic. The microspore stages were classified into seven: "tetrads", "early uninucleate", "miduninucleate", "late uninucleate", "early binucleate", "late binucleate", and "mature grain". The morphological characteristic in each stage was clarified. The "Tight cluster" flower bud stage mainly corresponded with the microspore stage of tetrad to mid-uninucleate. The "First pink" of the flower cluster mainly corresponded with the mid-uninucleate and late uninucleate microspore. The "Pink" and "Full pink" of flowering corresponded respectively with the late binucleate and the mature grain. Cold pre-treatment for more than 25 days of flower buds was found to be an efficient means of increasing embryo formation in anther culture. The microspore of late uninucleate and early binucleate stages grew into embryos efficiently after 25 days of cold pre-treatment. To achieve that condition, flower buds at late "Tight cluster" to early "First pink" stage were collected because the microspore stage progresses during 25 days of cold pre-treatment. Most microspores were early uninucleate to early binucleate stages at the "Tight cluster" to "First pink" flower bud stages. The shoots regenerated from embryos were analyzed using SSR markers. The investigation revealed that all shoots had a haploid origin.

To select cultivars to produce doubled haploids (DHs) efficiently in an anther culture, a total of 28 apple cultivars (25 cultivars and 3 rootstock cultivars) were investigated to assess their callus formation rate, embryo formation rate, shoot formation rate from embryo, and efficiency of shoot multiplication and acclimatization.