Development of an in vitro protocol for a difficult-to-propagate endemic Australian dryland sedge species Mesomelaena pseudostygia (Cyperaceae)

Abstract

In vitro propagation for Mesomelaena pseudostygia a difficult-to-propagate dryland sedge species (Cyperaceae) endemic to Western Australia is described. Multiple avenues to in vitro propagation were investigated: shoot culture, organogenesis and somatic embryogenesis, with zygotic embryos as initiation material. The highest multiplication rate for shoots was 3.4 ± 1.0 after six weeks on basal medium (½ strength Murashige and Skoog) with 2.5µM kinetin and 0.5µM 6benzylaminopurine. Shoots achieved peak rooting (83%) following a pulse treatment on basal medium containing 10 µM indolebutyric acid and 2 µM α-naphthaleneacetic acid for seven weeks, followed by transfer to medium (without growth regulators) for a further seven weeks. Alternatively, in vitro grown shoots were pulse treated on basal medium with both 100 µM indolebutyric acid and 20µM α-naphthaleneacetic acid for one week then placed in Rockwool plugs (under propagation house conditions) for another seven weeks resulting in 63% root induction. Rooted plantlets were also successfully transferred to potting mixture either in Rockwool plugs or bare rooted and maintained in propagation house conditions with ≥95% survival after seven weeks. These results indicate that micropropagation of M. pseudostygia is feasible for small to medium scale restoration purposes. The highest frequency of callus induction was from cultured zygotic embryos on basal medium with 5µM α-naphthaleneacetic acid, whereas 2,4-dichlorophenoxacetic acid (2 or 5µM) produced the largest callus sizes. A low frequency of shoot regeneration occurred in zygotic callus tissues in basal medium treatments containing cytokinin (kinetin or thidiazuron at 1µM). A small proportion (<20%) of zygotic embryo callus explants from 2,4-dichlorophenoxyacetic acid treatments were found to be embryogenic, firstly developing embryo-like structures after two weeks on basal medium (minus plant growth hormones), that continued to develop with approximately one in twenty germinating after a further four weeks on basal medium to form small plantlets. Further optimisation is needed to improve somatic embryogenesis efficiency for mass propagation.

In vitropropagation of temperate Australian terrestrial orchids: revisiting asymbiotic compared with symbiotic germination

“…Asymbiotic culture was based on the following basal media: Pa5 (Collins & Dixon, 1992), Murashige and Skoog basal salts (Murashige & Skoog, 1962) at halfstrength (½MS) with modifications (as described by Lai et al, 2014), Knudson C (KC) (Knudson, 1946), and Vacin and Went (VW) (Vacin & Went, 1949). Pa5 medium was used without further modification.…”