A cytological observation on the development of embryoid in cultured anther of apple

Abstract：【Objective】Anther culture is an important method to produce haploid from microspores, and is widely used in plant breeding research. Apple is a temperate fruit species which is characterized by a long reproductive cycle with a juvenile phase of several years, a large tree size and a high degree of heterozygosity derived from self-incompatibility. It is almost impossible to obtain a homozygous plant by the conventional hybrid breeding. Production of haploids by anther culture offers new possibilities for genetic studies and breeding. Because of presence of anther wall cells, the regenerated plants via anther culture often have different levels of polidy, such as haploids, diploids, triploids, tetraploids, and chimeras. Therefore, homozygous analysis must be conducted to confirm whether regenerated buds are derived from pollen grains. Cytological oberservations on the development of embryoids inside the cultured anthers is a direct evidence to prove that the plants are developed from the microspores. At the same time, embryoid induction procedures in anther culture of apple will be clarified at the cellular level.【Methods】The cultivar of‘Gala’was used in this paper. In early April, the full flower buds were selected from different directions in the upper and outer parts of the canopy and the buds of different developmental stages were used to observe the occurrence of microspores and the development of andro-genesis. The cultured anthers were used to study the process of embryoids development. Samples were taken at 0, 5, 10, 12, 15, 17, 20, 25, 30, 40, 50 and 60 days after inoculation. Then the flower buds and anthers at each period were fixed by FAA for 12-24 hours, and the conventional paraffin sections were obtained after series alcohol dehydration. The 6-8 m thick sections were double stained with hematoxylin and saffron. Morphological characteristics of anthers and their internal microsporocytes in different periods were observed under the light microscope.【Results】Based on the cytological oberservations, development of microspore in apple could be divided into seven stages: the tetrad, the early uninucleate, the mid- uninucleate, the late uniucleate, the early binucleate, the late binucleate and the mature grain stages, each demonstrating distinctive characteristics. During the tetrad and early uninucleate stages, the single nucleus was very bright and located in the center of the pollen grain. At the tetrad stage, there were four pollen grains in a cell but they appear as a tri-molecular complex. At the mid-uninucleate stage, the cell volume increased rapidly; the nucleus moved to one side because of the existence of vacuoles; and the microspores became rugby ball shape. During stages of the late uninucleate and the binucleate, the microspores became ball- shaped. At the early binucleate stage, just after pollen mitosis, the larger vegetative nucleus stained very diffusely, whereas the generative nucleus was very bright and compact. At the late binucleate stage, the nuclear stained deeper, and at the mature grain stage, the microspores became triangular. The microspores at the stage of mid-late uniucleate were better for the anther culture, and the embryoids were derived from the equal division of microspores. The first division of microspores was symmetric and began to split from about 5 days of anther culture. Then both nuclei divided repeatedly, 2-3 rounds of division had been performed at 10 days of culture and multi-cell clusters were observed from 20 days of culture. Then, as the number of culture days increased, they further developed into spherical embryoids, heart-shaped embryoids, torpedo-shaped embryoids and cotyledonary embryoids. The spherical embryos could be seen at 20 days of culture, and the cotyledonary embryos could be seen by naked eyes at 30 days of culture. During the culture, there were also several different stages of embryoids developed from an anther. The cells of callus were large and irregularly shaped with invisible nuclei.【Conclusion】The developmental stage of the microspore strongly affects anther culture success, and cold pre-treatment can greatly improve embryoid formation in apple anther culture. In apple anther culture, microspores at the stages of mid-late uninucleate after cold pre-treatment were most suitable. Therefore, the microspores of early-mid uninucleate are suitable for anther culture of apple. Our study provides cytological evidence for embryoid induction from the haploid microspores through anther culture.