The influence, was investigated, of abiotic parameters on the isolation of protoplasts from in vitro seedling cotyledons of white lupin. The protoplasts were found to be competent in withstanding a wide range of osmotic potentials of the enzyme medium, however, -2.25 MPa (0.5 M mannitol), resulted in the highest yield of protoplasts. The pH of the isolation medium also had a profound effect on protoplast production. Vacuum infiltration of the enzyme solution into the cotyledon tissue resulted in a progressive drop in the yield of protoplasts. The speed and duration of orbital agitation of the cotyledon tissue played a significant role in the release of protoplasts and a two step (stationary-gyratory) regime was found to be better than the gyratory-only system.

Optimisation of Protoplast Production in White Lupin

Using the pathosystem Phaseolus vulgaris-tobacco necrosis virus (TNV), we demonstrated that PD-L1 and PD-L4, type-1 ribosome inactivating proteins (RIPs) from leaves of Phytolacca dioica L., possess a strong antiviral activity. This activity was exerted both when the RIPs and the virus were inoculated together in the same leaf and when they were inoculated or applied separately in the adaxial and abaxial leaf surfaces. This suggests that virus inhibition would mainly occur inside plant cells at the onset of infection. Histochemical studies showed that both PD-L1 and PD-L4 were not able to induce oxidative burst and cell death in treated leaves, which were instead elicited by inoculation of the virus alone. Furthermore, when RIPs and TNV were inoculated together, no sign of H2O2 deposits and cell death were detectable, indicating that the virus could have been inactivated in a very early stage of infection, before the elicitation of a hypersensitivity reaction. In conclusion, the strong antiviral activity is likely exerted inside host cells as soon the virus disassembles to start translation of the viral genome. This activity is likely directed towards both viral and ribosomal RNA, explaining the almost complete abolition of infection when virus and RIP enter together into the cells.

Antiviral Activity of PD-L1 and PD-L4, Type 1 Ribosome Inactivating Proteins from Leaves of Phytolacca dioica L. in the Pathosystem Phaseolus vulgaris- Tobacco Necrosis Virus (TNV)

Phytolacca dodecandra L’Heit (Endod var. E-44) is a medicinal plant of high saponin content used as a snail control agent in schitosomiasis. The aim of this study was to develop a micrpropagation protocol for P. dodecandra . Nodal and shoot tip explants were sterilized and cultured on MS medium supplemented with different types of concentrations and combinations of auxin and cytokinin for shoot induction, multiplication and root formation. Sodium hypochlorite at a concentration of 1.56% exposure of 12 min gave high percentages (81.67%) of survived explants for nodal followed by 1.3% concentration of NaOCl exposure of 10 min (73%) for shoot tip. 6-Benzylaminopurine (2.22 μM) was found to be an optimum concentration for shoot induction, yielding 80% for nodal and 70% for shoot tip explants. The combination of BAP (2.22 μM) with kinetin (2.33 μM) was obtained as optimum concentration yielding 13.4 and 11.03 shoots per explants for nodal and shoot tip, respectively for shoot multiplication. Half - strength MS medium with IBA (2.45 μM) and IAA (2.85 μM) yielded more than 90% root formation with optimum root number and length. Sterilized soil mix of 2:1:1 (top forest soil: coffee husk: sand) was optimized yielding 80% on transparent polyethylene plastic box. This developed micropopagation protocol is highly significant for large scale propagation of this species as a source of saponin extraction for drug development against endemic schitosomiasis. Key words: Explants, E-44 cultivar, plant growth regulators, micropropagation, detergents.

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Micropropagation of Phytolacca dodecandra L'Herit (Endod var. E-44)

Ribosome-inactivating proteins (RIPs) are a class of plant defense proteins with N-glycosidase activity (EC 3.2.2.22). Pokeweed antiviral protein (PAP) is a Type I RIP isolated from the pokeweed plant, Phytolacca americana, thought to confer broad-spectrum virus resistance in this plant. Through a combination of standard molecular techniques and RNA sequencing analysis, we report here that a small RNA binds and cleaves the open reading frame of PAP mRNA. Additionally, sRNA targeting of PAP is dependent on jasmonic acid (JA), a plant hormone important for defense against pathogen infection and herbivory. Levels of small RNA increased with JA treatment, as did levels of PAP mRNA and protein, suggesting that the small RNA functions to moderate the expression of PAP in response to this hormone. The association between JA and PAP expression, mediated by sRNA299, situates PAP within a signaling pathway initiated by biotic stress. The consensus sequence of sRNA299 was obtained through bioinformatic analysis of pokeweed small RNA sequencing. To our knowledge, this is the first account of a sRNA targeting a RIP gene.

A small RNA targets pokeweed antiviral protein transcript

n of Ph (Endod

Pokeweed (Phytolacca americana L.) and endod (P. dodecandra L'Herit) produce ribosome-inactivating proteins which are sequestered in leaf cell walls. These proteins display strong antiviral activity. To aid in studying the antiviral mechanism, we developed protocols to isolate protoplasts from suspension culture cells and leaves. Ninety-five percent of pokeweed or endod culture cells were converted to protoplasts using 2% cellulase, 0.25% pectinase, 0.2 M mannitol, 2% sucrose, 15 mM CaCl2 Murashige and Skoog salts, pH 5.7. Viability was >85% after 24 h. Culture-derived protoplasts were purified by centrifugation through a 15% sucrose pad. Protoplasts collected from the supernatant were then pelleted in 0.3 M mannitol. Pokeweed leaves provided respectable yields (4×10(6) protoplasts/g f w) of partially-purified viable protoplasts when digested in solution containing 1% cellulase, 0.2% Pectolyase, 0.4 M mannitol, CPW salts, 0.5 mM MES, pH 5.6. We were unable to completely separate cell debris from mesophyll protoplasts, which were small and easily damaged by centrifugation. Endod leaves were found to be resilient to several digestion enzymes tested.

Hong Lu

Papers

Protoplasts from Phytolacca dodecandra L'Herit (endod) and P. americana L. (pokeweed).