Microspore Culture in Eleven Cultivars of Loquat

In plant breeding, microspore embryogenesis via isolated microspore culture is increasing as a method to obtain in a single-step doubled haploids and homozygosity. In this study, isolated microspore culture of eleven cultivars of loquat (Eriobotrya japonica (Thunb.) Lindl.) has been carried out. An analysis of anthers from different flower bud sizes, has been carried out, to identify and to select the flower bud size with anthers containing the highest quantity of vacuolated microspores. After a pre-treatment of the panicles at 4 degrees C in dark for 4 days, flower buds were sterilized and microspores were isolated and cultured in two different media (P and MB). Moreover, only the isolated microspores cultured in P Medium were subjected to two additional thermal shocks: cold shock (1 h at -20 degrees C) and heat shock (7 days at 33 degrees C). All the cultures were placed in a growth cabinet at 26 +/- 1 degrees C in the dark for 30 days and later placed in the light. Samples from cultures were processed for microscopy analysis at specific times. Observations on microspore development were carried out by fluorescence and light microscopy. The sporophytic development of isolated microspores in culture has been confirmed by the presence in the cultures of binucleated with two equal-size vegetative-type nuclei that had just started their sporophytic pathway, multinucleated, pollen-derived multicellular structures, and calli. The results are an advancement towards the knowledge of pollen embryogenesis in loquat.