Improved Embryogenic Callus Induction and Histological Analysis of the Embryogenesis in Callus Culture from Loquat Anther (Eriobotrya japonica)

摘要

In this study, three loquat cultivars (Eriobotrya japonica L.) cultivars viz. ‘Longquan1’, ‘Dawuxing’ and ‘Zaozhong6’ were selected for in vitro anther culture. To improve the induction rate of microspore-derived callus, different pretreatments and carbohydrates were evaluated. Results showed that cold pretreatment significantly enhanced callus formation from anther microspores compared to other pretreatments. The average callus induction rate was 61.11% out of 450 anthers with cold pretreatment. The inoculation density of anthers had no significant effect on the survival of anthers and callus induction, but contacted faces of the anther brought different influence on the callus induction. Sucrose was more advantageous for embryonic callus induction than glucose. The capacity of dedifferentiation and calli derived from anther wall were seriously inhibited by the increased concentrations of sucrose. Media with more than 5% sucrose ensured most of calli to originate from microspores and inhibited anther wall to form calli. However, glucose failed to inhibit anther-wall-derived callus. Under the optimal concentration of sucrose the callus induction rate of ‘Zaozhong6’anthers was the highest (74%), followed by 'Longquan 1'(50.67%), and was the lowest in 'Dawuxing' (29.33%). Histological observations manifested great differences between the embryogenic and non-embryonic calli. The embryonic cells were arranged compactly with a larger karyoplasm and thick cytoplasm, and showed a vigorous division. The development of loquat anther embryos was similar with zygotic embryo, through the globular, the heart-shaped, the torpedo and the cotyledonary embryos stages. Between the heart shaped and the torpedo-shaped stage, vascular tissue initially formed. At the torpedo stage, the V-shaped vascular bundle appeared. Then at the cotyledonary stage, the V-shaped vascular bundle was clearly observed.