Induction of embryonic callus from hypocotyl and needle of Pinus thunbergii Parl.

Abstract To lay the foundation for the establishment of somatic embryogenesis system in the vegetative organs of Pinus thunbergii Parl, the induction of somatic embryogenic callus(EC) was studied using hypocotyls as explants to find out the key factors affecting EC induction, and investigate the effects of medium type, concentration of auxin (2,4-D) and cytokinin (6-BA) on the EC induction. Orthogonal experiment design with three factors and four levels was adopted. MS, WPM, B5 and DCR were used as the basic medium, respectively, with different concentrations of 2,4-D (0.5 mg·L-1, 1.5 mg·L-1, 2.0 mg·L-1, 2.5 mg·L-1) and 6-BA (0.5 mg·L-1, 1.0 mg·L-1, 1.5 mg·L-1, 2.0 mg·L-1), with additional 0.2 mg·L-1 polyvinylpyrrolidone(PVP), 6 g·L-1 agar and 20 g·L-1 sucrose to induce callus. The interactions between the factors, the optimal combination and the best hormone concentration ratio were investigated by statistical methods. Callus were sampled for morphological observation under a stereoscopic microscope, followed by cytological observation under a biological microscope using a double staining method of acetate magenta and Evans blue to identify whether they were embryonic callus. The polyphenol oxidase (PPO) contents in the callus of different succession cultures were measured to test whether embryonic stage of the induced callus could be maintained. The results showed that the optimal combination was DCR + 2.5 mg·L-1 2,4-D + 1.0 mg·L-1 6-BA, and this protocol resulted in 98.7% (±1.9%) induction of callus. Using this protocol, callus of needle explants was also successfully induced, but the callus induction was slower. Microscopic observation reveals a distinct embryonic callus structure, an embryonic head with several suspensor cells. The analysis of variance showed that among the three factors, 2,4-D concentration significantly affected the callus induction rate (p<0.05), while the medium type and the 6-BA concentration had nonsignificant effect. Even so, the analysis of interaction showed that there was a significant interaction between the combination of 2,4-D and 6-BA (p<0.05). The PPO viability was significantly higher in the callus grown for 3 weeks, while it decreased after first and second succession. The above results inferred that 2,4-D plays an important role in embryotic callus induction process in the hypocotyl of P. thunbergii Parl. Although 6-BA concentration level by itself had no significant effect on callus induction rate, there may be complex links between the 6-BA and 2,4-D in the signal transduction pathways.