Initiation of somatic embryogenesis in Pinus caribaea var. hondurensis using mature female gametophytes as explants

Abstract

Due to the difficulties of harvesting immature cones in genetic breeding orchards, such as high costs and the considerable time required, this study aimed to evaluate the somatic embryogenesis (SE) initiation of Pinus caribaea var. hondurensis using the mature female gametophyte as the initial explant. The basic medium for embryogenesis initiation was composed of QL (Quoirin and Lepoivre, 1977) nutrients, sucrose, myo-inositol, glutamine, casein hydrolisate, thiamine, 2,4-D, BAP and agar. This medium was compared with DCR (Gupta and Durzan, 1985) and WV5 (Coke, 1996) media, supplemented or not with 0.1 g L−1 folic acid (FA). Afterwards, three concentrations of 2,4-D (10, 20 and 30 μM) combined or not with BAP (2, 4 and 8 μM) were tested. After 90 days the percentages of explants with embryogenic tissue (ET), embryo-like structures and oxidation were evaluated. We observed somatic tissue initiation` on mature female gametophytes. On QL medium supplemented with FA, the initiation was observed on up to 42% of female gametophytes. The combination of 10 μM 2,4-D and 4 μM BAP resulted in the highest initiation rate. The formation of ET was confirmed in all treatments by a histochemical test. Embryo-like structures developed on QL medium supplemented with FA, were first transferred to basic medium supplemented with 1.5 g L−1 AC for 30 days and finally to the same medium supplemented with 10% polyethylene glycol 3350 and 120 µM abscisic acid. The expression of SERK-1 and GER-1 genes was analyzed. SERK-1 was expressed only in the ET, while GER-1 was expressed in both the embryogenic and non-embryogenic tissues (non-ET). However, the somatic embryos studied through morphological and histochemical tests proved to be abnormal and did not develop further.