In vitro breeding strategies in the development of Australian native plants

Plant tissue culture is a technique that exploits the ability of many plant cells to revert to a meristematic state. Although originally developed for botanical research, plant tissue culture has now evolved into important commercial practices and has become a significant research tool in agriculture, horticulture and in many other areas of plant sciences. Plant tissue culture is the sterile culture of plant cells, tissues, or organs under aseptic conditions leading to cell multiplication or regeneration or organs and whole plants. The steps required to develop reliable systems for plant regeneration and their application in plant biotechnology are reviewed in countless books. Some of the major landmarks in the evolution of in vitro techniques are summarised in Table 5.1. In this chapter the current applications of this technology to agriculture, horticulture, forestry and plant breeding are briefly described with specific examples from Australian plants when applicable.

Plastic traits of an exotic grass contribute to its abundance but are not always favourable

In herbaceous ecosystems worldwide, biodiversity has been negatively impacted by changed grazing regimes and nutrient enrichment. Altered disturbance regimes are thought to favour invasive species that have a high phenotypic plasticity, although most studies measure plasticity under controlled conditions in the greenhouse and then assume plasticity is an advantage in the field. Here, we compare trait plasticity between three co-occurring, C 4 perennial grass species, an invader Eragrostis curvula, and natives Eragrostis sororia and Aristida personata to grazing and fertilizer in a three-year field trial. We measured abundances and several leaf traits known to correlate with strategies used by plants to fix carbon and acquire resources, i.e. specific leaf area (SLA), leaf dry matter content (LDMC), leaf nutrient concentrations (N, C:N, P), assimilation rates (Amax) and photosynthetic nitrogen use efficiency (PNUE). In the control treatment (grazed only), trait values for SLA, leaf C:N ratios, Amax and PNUE differed significantly between the three grass species. When trait values were compared across treatments, E. curvula showed higher trait plasticity than the native grasses, and this correlated with an increase in abundance across all but the grazed/fertilized treatment. The native grasses showed little trait plasticity in response to the treatments. Aristida personata decreased significantly in the treatments where E. curvula increased, and E. sororia abundance increased possibly due to increased rainfall and not in response to treatments or invader abundance. Overall, we found that plasticity did not favour an increase in abundance of E. curvula under the grazed/fertilized treatment likely because leaf nutrient contents increased and subsequently its' palatability to consumers. E. curvula also displayed a higher resource use efficiency than the native grasses. These findings suggest resource conditions and disturbance regimes can be manipulated to disadvantage the success of even plastic exotic species.

EPG monitoring of the probing behaviour of the common brown leafhopper Orosius orientalis on artificial diet and selected host plants

The common brown leafhopper Orosius orientalis (Hemiptera: Cicadellidae) is a polyphagous vector of a range of economically important pathogens, including phytoplasmas and viruses, which infect a diverse range of crops. Studies on the plant penetration behaviour by O. orientalis were conducted using the electrical penetration graph (EPG) technique to assist in the characterisation of pathogen acquisition and transmission. EPG waveforms representing different probing activities were acquired from adult O. orientalis probing in planta, using two host species, tobacco Nicotiana tabacum and bean Phaseolus vulgaris, and in vitro using a simple sucrose-based artificial diet. Five waveforms (O1–O5) were evident when O. orientalis fed on bean, whereas only four waveforms (O1–O4) and three waveforms (O1–O3) were observed when the leafhopper fed on tobacco and on the artificial diet, respectively. Both the mean duration of each waveform and waveform type differed markedly depending on the food substrate. Waveform O4 was not observed on the artificial diet and occurred relatively rarely on tobacco plants when compared with bean plants. Waveform O5 was only observed with leafhoppers probing on beans. The attributes of the waveforms and comparative analyses with previously published Hemipteran data are presented and discussed, but further characterisation studies will be needed to confirm our suggestions.

Optimization of human papillomavirus type 16 (HPV-16) L1 expression in plants: Comparison of the suitability of different HPV-16 L1 gene variants and different cell-compartment localization

Virus-like particle-based vaccines for high-risk human papillomaviruses (HPVs) appear to have great promise; however, cell culture-derived vaccines will probably be very expensive. The optimization of expression of different codon-optimized versions of the HPV-16 L1 capsid protein gene in plants has been explored by means of transient expression from a novel suite of Agrobacterium tumefaciens binary expression vectors, which allow targeting of recombinant protein to the cytoplasm, endoplasmic reticulum (ER) or chloroplasts. A gene resynthesized to reflect human codon usage expresses better than the native gene, which expresses better than a plant-optimized gene. Moreover, chloroplast localization allows significantly higher levels of accumulation of L1 protein than does cytoplasmic localization, whilst ER retention was least successful. High levels of L1 (>17% total soluble protein) could be produced via transient expression: the protein assembled into higher-order structures visible by electron microscopy, and a concentrated extract was highly immunogenic in mice after subcutaneous injection and elicited high-titre neutralizing antibodies. Transgenic tobacco plants expressing a human codon-optimized gene linked to a chloroplast-targeting signal expressed L1 at levels up to 11% of the total soluble protein. These are the highest levels of HPV L1 expression reported for plants: these results, and the excellent immunogenicity of the product, significantly improve the prospects of making a conventional HPV vaccine by this means. © 2007 SGM.

Expression of HIV-1 antigens in plants as potential subunit vaccines

Background Human immunodeficiency virus type 1 (HIV-1) has infected more than 40 million people worldwide, mainly in sub-Saharan Africa. The high prevalence of HIV-1 subtype C in southern Africa necessitates the development of cheap, effective vaccines. One means of production is the use of plants, for which a number of different techniques have been successfully developed. HIV-1 Pr55Gag is a promising HIV-1 vaccine candidate: we compared the expression of this and a truncated Gag (p17/p24) and the p24 capsid subunit in Nicotiana spp. using transgenic plants and transient expression via Agrobacterium tumefaciens and recombinant tobamovirus vectors. We also investigated the influence of subcellular localisation of recombinant protein to the chloroplast and the endoplasmic reticulum (ER) on protein yield. We partially purified a selected vaccine candidate and tested its stimulation of a humoral and cellular immune response in mice. Results Both transient and transgenic expression of the HIV antigens were successful, although expression of Pr55Gag was low in all systems; however, the Agrobacterium-mediated transient expression of p24 and p17/p24 yielded best, to more than 1 mg p24/kg fresh weight. Chloroplast targeted protein levels were highest in transient and transgenic expression of p24 and p17/p24. The transiently-expressed p17/p24 was not immunogenic in mice as a homologous vaccine, but it significantly boosted a humoral and T cell immune response primed by a gag DNA vaccine, pTHGagC. Conclusion Transient agroinfiltration was best for expression of all of the recombinant proteins tested, and p24 and p17/p24 were expressed at much higher levels than Pr55Gag. Our results highlight the usefulness of plastid signal peptides in enhancing the production of recombinant proteins meant for use as vaccines. The p17/p24 protein effectively boosted T cell and humoral responses in mice primed by the DNA vaccine pTHGagC, showing that this plant-produced protein has potential for use as a vaccine.

High level protein expression in plants through the use of a novel autonomously replicating geminivirus shuttle vector

We constructed a novel autonomously replicating gene expression shuttle vector, with the aim of developing a system for transiently expressing proteins at levels useful for commercial production of vaccines and other proteins in plants. The vector, pRIC, is based on the mild strain of the geminivirus Bean yellow dwarf virus (BeYDV-m) and is replicationally released into plant cells from a recombinant Agrobacterium tumefaciens Ti plasmid. pRIC differs from most other geminivirus-based vectors in that the BeYDV replication-associated elements were included in cis rather than from a co-transfected plasmid, while the BeYDV capsid protein (CP) and movement protein (MP) genes were replaced by an antigen encoding transgene expression cassette derived from the non-replicating A. tumefaciens vector, pTRAc. We tested vector efficacy in Nicotiana benthamiana by comparing transient cytoplasmic expression between pRIC and pTRAc constructs encoding either enhanced green fluorescent protein (EGFP) or the subunit vaccine antigens, human papillomavirus subtype 16 (HPV-16) major CP L1 and human immunodeficiency virus subtype C p24 antigen. The pRIC constructs were amplified in planta by up to two orders of magnitude by replication, while 50% more HPV-16 L1 and three- to seven-fold more EGFP and HIV-1 p24 were expressed from pRIC than from pTRAc. Vector replication was shown to be correlated with increased protein expression. We anticipate that this new high-yielding plant expression vector will contribute towards the development of a viable plant production platform for vaccine candidates and other pharmaceuticals. © 2009 Blackwell Publishing Ltd.

Human papillomavirus vaccines in plants

Human papillomaviruses are the etiological agents of cervical cancer, one of the two most prevalent cancers in women in developing countries. Currently available prophylactic vaccines are based on the L1 major capsid protein, which forms virus-like particles when expressed in yeast and insect cell lines. Despite their recognized efficacy, there are significant shortcomings: the vaccines are expensive, include only two oncogenic virus types, are delivered via intramuscular injection and require a cold chain. Plant expression systems may provide ways of overcoming some of these problems, in particular the expense. In this article, we report recent promising advances in the production of prophylactic and therapeutic vaccines against human papillomavirus by expression of the relevant antigens in plants, and discuss future prospects for the use of such vaccines. © 2010 Expert Reviews Ltd.

Non-invasive identification of proteoglycans and chondrocyte differentiation state by Raman microspectroscopy

Proteoglycans (PGs) are crucial extracellular matrix (ECM) components that are present in all tissues and organs. Pathological remodeling of these macromolecules can lead to severe diseases such as osteoarthritis or rheumatoid arthritis. To date, PG-associated ECM alterations are routinely diagnosed by invasive analytical methods. Here, we employed Raman microspectroscopy, a laser-based, marker-free and non-destructive technique that allows the generation of spectra with peaks originating from molecular vibrations within a sample, to identify specific Raman bands that can be assigned to PGs within human and porcine cartilage samples and chondrocytes. Based on the non-invasively acquired Raman spectra, we further revealed that a prolonged in vitro culture leads to phenotypic alterations of chondrocytes, resulting in a decreased PG synthesis rate and loss of lipid contents. Our results are the first to demonstrate the applicability of Raman microspectroscopy as an analytical and potential diagnostic tool for non-invasive cell and tissue state monitoring of cartilage in biomedical research. ((c) 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim).