Cyclic secondary somatic embryogenesis and efficient plant regeneration in camphor tree (Cinnamomum camphora L.)

Abstract

An efficient protocol for secondary somatic embryogenesis in camphor tree is reported. Secondary somatic embryos (SSEs), initially obtained from the primary embryos of a nascent embryogenic culture in 2002, were proliferated and maintained for more than 4 yr via cyclic secondary somatic embryogenesis. Throughout this period, the embryo populations retained a high level of competence for plant regeneration. SSEs were produced on the surfaces of the cotyledons and radicular ends of maternal somatic embryos (MSEs). Histological observations of the various stages of secondary embryo development revealed four typical stages, namely, globular, heartshaped, torpedo, and cotyledonary. The process of secondary embryogenesis continued in a cyclic way, with each newly formed embryo producing a subsequent generation of secondary embryos. In order to progress developmentally beyond proliferation cycles, cotyledonary embryos from one of embryogenic lines (L14) were cultured on Murashige and Skoog (MS) medium with 0.1-3.0 mg l −1 abscisic acid (ABA) or 0.05-1.0 mg l −1 thidiazuron (TDZ) in darkness for 2 mo to achieve maturation. Matured embryos were then transferred to MS-based germination medium containing either 0.1 mg l −1 TDZ, 0.2 mg l −1 indole-3-butyric acid (IBA), and 0.5 mg l −1 6-benzylaminopurine (BA) or 0.1 mg l −1 TDZ and 0.2 mg l −1 IBA and were cultured in light for germination. Over 50% of embryos matured in the presence of 0.5 mg l −1 ABA were able to germinate with shoots and poor root system. Frequencies of embryos germinating normal shoots among different genotypes did not change significantly. A total of 93% of the shoots from the germinated embryos converted to plantlets on half strength MS medium with 0.5 mg l −1 IBA by 3 wk. Plantlets acclimatized successfully to ex vitro conditions and developed as field-grown plants with normal appearance.

Enhancement of somatic embryogenesis in camphor tree (Cinnamomum camphora L.): osmotic stress and other factors affecting somatic embryo formation on hormone-free medium

“…It was observed in this study that the primary somatic embryos derived from hormone-free medium could produce secondary somatic embryos on medium without PGR, which may be beneficial for plantlet conversion, as the secondary somatic embryos maintained on medium with auxin by repetitive embryogenesis failed to complete efficient plantlet conversion with shoots and roots simultaneously. Secondary somatic embryos of C. camphora that were cultured for [4 years required a maturation treatment before germinating on a medium containing PGRs for plantlet conversion (Shi et al 2009). It was also observed in this study that the primary somatic embryos derived from hormone-free medium needed a maturation treatment before plant regeneration and secondary somatic embryogenesis could occur on medium without PGR.…”

“…3c), could be inhibited by the addition of ABA (Shi et al 2009). Germination of maturation treated somatic embryos under light or in darkness (Fig.…”

“…Somatic embryos were cultured on MS medium with 0.5 mg l -1 ABA for maturation, and then transferred on to MS medium with 0.1 mg l -1 TDZ and 0.2 mg l -1 IBA for plantlet regeneration as described in Shi et al (2009).…”

“…The effects of PGRs on somatic embryo induction (Du et al 2007) and plant regeneration via secondary embryogenesis in C. camphora (Shi et al 2009) have been reported. In the present study, we show high-efficiency direct somatic embryogenesis in C. camphora induced by applying osmotic stress to immature zygotic embryos without hormonal treatment, as well as adventitious formation of embryogenic callus.…”

Thidiazuron Induces High Frequency Direct Somatic Embryogenesis Growth from Cotyledon Culture of Eurycoma longifolia

“…Shi et al (2010) also reported that secondary somatic embryos in camphor tree (Cinnamomum camphora) were produced on the surfaces of the cotyledons and radicular ends of maternal somatic embryos. According to Raemakers et al (1995), cells that were capable of direct somatic embryogenesis were physiologically similar to those in zygotic embryo.…”