Influence of exogenous putrescine on somatic embryogenesis and regeneration in litchi (Litchi chinensis Sonn.)

Litchi (Litchi chinensis Sonn.), like other ligneous plants, has long been considered a recalcitrant embryogenic species. Our previous research has shown that adding putrescine (Put) or D-Arginine (D-Arg) to the subculturing medium of embryonic callus (EC) affects EC proliferation and subsequent somatic embryogenesis (SE) in litchi. In this paper, we further confirmed that EC proliferation was significantly increased when either 0.17 mM putrescine (P3) or 2 mM D-Arginine (Ar3) was added to the control medium (M3, MS supplement with 4.52 µM 2,4-D). The subsequent induction of opalescent embryos (OEs) and opalescent dicotyledonous embryos (ODEs) was partially inhibited by Put, and the number of plantlets germinated from the OEs on the P3 medium were lower than those on the M3 medium; however, that was increased by D-Arg. Histomorphological analyses verified various developmental stages of EC proliferation in the M3, P3, and Ar3 media. On the M3 medium, an EC cell was divided into two cells first and then sequentially differentiated through multicell proembryo, globular, heart-shaped, and cotyledonary embryo stages. The EC cells on P3 and Ar3 medium were enlarged more significantly, undergoing repeated cell divisions and then forming a meristematic mass, from which OEs were initiated. The supplementation of Put into the M3 medium promoted the synthesis of endogenous Put and its conversion to Spd and Spm. The PA content in EC on the P3 medium was higher than that on the M3 medium, and the P3 medium enhanced the activity of arginine decarboxylase (ADC), ornithine decarboxylase (ODC), and diamine oxidase (DAO); however, it decreased the activity of polyamine oxidase (PAO). The PA content in the Ar3 medium was higher than that in the M3 medium. The supplementation of D-Arg to the M3 medium enhanced ADC and DAO activity but decreased ODC and PAO activity. In the other experiment, EC from the P3 medium was subsequently cultured on M3 (P3M3), P3 (P3P3), and Ar3 (P3Ar3) medium, using EC from M3 medium, and then cultured on M3 (M3M3) medium as a control. The EC proliferation rate of the P3Ar3 treatment was significantly higher than that of the other two treatments. The OEs, ODEs, and plantlets were all most elevated in the P3Ar3 treatment, followed by the P3M3 and P3P3 treatments. EC proliferation and plantlets were significantly higher than in the M3M3 treatment. When ECs were first cultured on Ar3 medium and transferred to M3 (Ar3M3), P3 (Ar3P3), and Ar3 (Ar3Ar3) media for 20 d, the EC proliferation of the Ar3Ar3 treatment was significantly higher than that of the other two treatments. Among the three treatments, EC from the Ar3Ar3 treatment showed the highest OE, ODE, and plantlet induction, followed by the Ar3M3 and P3P3 treatments. The Ar3Ar3 treatment also had the highest induction of OEs, ODEs, and plantlets compared with the other six treatments. EC from the Ar3P3 treatment had the highest Spd, Spm, and PA content, and the M3M3 treatment had the highest Put content. The Spm and PA content of EC from the M3M3 treatment was lower than in the other treatments. Among all treatments, the highest ADC, ODC, DAO, and PAO activity was found in ECs from the M3M3, P3P3, P3P3, and Ar3Ar3 treatments. These results indicate that exogenous Put or D-Arg could stimulate Put synthesis of endogenous Put by regulating the enzyme activities and then affecting the EC proliferation and SE of litchi.