Investigation of browning factors in embryogenic callus culture of Pinus tabulaeformis and optimization of proliferation medium

Objective  To study the effects of different factors on browning of embryogenic callus of Pinus tabulaeformis during proliferation, and optimize the culture medium based on proliferation rate for lower browning rate and higher efficiency.

Method  Embryogenic callus of P. tabulaeformis from nine cell lines and three different year were used as material.We performed a serious of single factor experiments and calculated the browning rates and proliferation rates of embryogenic callus under the treatments of different callus number, pH, phytagel content, type and content of sugars, and culturing time. Orthogonal experiment was then conducted to screen out the best culture conditions.

Result  Results of the single factor experiments showed that browning rate was significantly different among different genotypes, and it was significantly positively correlated with the age of subculture. The browning rate was relatively low and the proliferation rate was relatively high when the callus number in each petri dish was 6–7, medium pH was 5.8–6.0, phytagel was 2.0–3.0 g·L^–1, and sucrose was 10 g·L^–1. Sucrose was better than glucose and maltose.

Conclusion  Based on the orthogonal design, placing 7 callus in each 90 mm petri dish, adding 2.5 g·L^–1 phytagel and 10 g·L^–1 sucrose to the medium, and adjusting the pH to 5.9 could achieve the preferred browning rate, proliferation rate and efficiency.