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.

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.

Maize streak virus-resistant transgenic maize: A first for Africa

In this article, we report transgene-derived resistance in maize to the severe pathogen maize streak virus (MSV). The mutated MSV replication-associated protein gene that was used to transform maize showed stable expression to the fourth generation. Transgenic T 2 and T 3 plants displayed a significant delay in symptom development, a decrease in symptom severity and higher survival rates than non-transgenic plants after MSV challenge, as did a transgenic hybrid made by crossing T 2 Hi-II with the widely grown, commercial, highly MSV-susceptible, white maize genotype WM3. To the best of our knowledge, this is the first maize to be developed with transgenic MSV resistance and the first all-African-produced genetically modified crop plant. © 2007 The Authors.

Riding the tide of biopharming in Africa: Considerations for risk assessment

In the past few years, plant biotechnology has gone beyond traditional agricultural production of food, feed and fibre, and moved to address more complex contemporary health, social and industrial challenges. The new era involves production of novel pharmaceutical products, speciality and fine chemicals, phytoremediation and production of renewable energy resources to replace non-renewable fossil fuels. Plants have been shown to provide a genuine and low-cost alternative production system for high-value products. Currently, the principal plant-made products include antibodies, feed additives, vaccine antigens and hormones for human and animal health, and industrial proteins. Despite the unique advantages of scalability, cost and product safety, issues of politics, environmental impact, regulation and socioeconomics still limit the adoption of biopharmaceuticals, especially in the developing world. Plant-based production systems have further complicated biosafety, gene flow and environmental impact assessments with generally genetically modified plants, topics that are already partially understood. This article provides a background to biopharming, highlighting basic considerations for risk assessment and regulation in developing countries, with an emphasis on plant-based vaccine production in South Africa.

Marker gene elimination from transgenic barley, using co-transformation with adjacent 'twin T-DNAs' on a standard Agrobacterium transformation vector

We have tested a methodology for the elimination of the selectable marker gene after Agrobacterium-mediated transformation of barley. This involves segregation of the selectable marker gene away from the gene of interest following co-transformation using a plasmid carrying two T-DNAs, which were located adjacent to each other with no intervening region. A standard binary transformation vector was modified by insertion of a small section composed of an additional left and right T-DNA border, so that the selectable marker gene and the site for insertion of the gene of interest (GOI) were each flanked by a left and right border. Using this vector three different GOIs were transformed into barley. Analysis of transgene inheritance was facilitated by a novel and rapid assay utilizing PCR amplification from macerated leaf tissue. Co-insertion was observed in two thirds of transformants, and among these approximately one quarter had transgene inserts which segregated in the next generation to yield selectable marker-free transgenic plants. Insertion of non-T-DNA plasmid sequences was observed in only one of fourteen SMF lines tested. This technique thus provides a workable system for generating transgenic barley free from selectable marker genes, thereby obviating public concerns regarding proliferation of these genes.

Analysis of genetically modified red-fleshed apples reveals effects on growth and consumer attributes

Consumers of whole foods, such as fruits, demand consistent high quality and seek varieties with enhanced health properties, convenience or novel taste. We have raised the polyphenolic content of apple by genetic engineering of the anthocyanin pathway using the apple transcription factor MYB10. These apples have very high concentrations of foliar, flower and fruit anthocyanins, especially in the fruit peel. Independent lines were examined for impacts on tree growth, photosynthesis and fruit characteristics. Fruit were analysed for changes in metabolite and transcript levels. Fruit were also used in taste trials to study the consumer perception of such a novel apple. No negative taste attributes were associated with the elevated anthocyanins. Modification with this one gene provides near isogenic material and allows us to examine the effects on an established cultivar, with a view to enhancing consumer appeal independently of other fruit qualities. © 2012 Society for Experimental Biology, Association of Applied Biologists and Blackwell Publishing Ltd.

Product stability and sequestration mechanisms in Solanum tuberosum engineered to biosynthesize high value ketocarotenoids

To produce commercially valuable ketocarotenoids in Solanum tuberosum, the 4, 4′ β-oxygenase (crtW) and 3, 3′ β-hydroxylase (crtZ) genes from Brevundimonas spp. have been expressed in the plant host under constitutive transcriptional control. The CRTW and CRTZ enzymes are capable of modifying endogenous plant carotenoids to form a range of hydroxylated and ketolated derivatives. The host (cv. Désirée) produced significant levels of nonendogenous carotenoid products in all tissues, but at the apparent expense of the economically critical metabolite, starch. Carotenoid levels increased in both wild-type and transgenic tubers following cold storage; however, stability during heat processing varied between compounds. Subcellular fractionation of leaf tissues revealed the presence of ketocarotenoids in thylakoid membranes, but not predominantly in the photosynthetic complexes. A dramatic increase in the carotenoid content of plastoglobuli was determined. These findings were corroborated by microscopic analysis of chloroplasts. In tuber tissues, esterified carotenoids, representing 13% of the total pigment found in wild-type extracts, were sequestered in plastoglobuli. In the transgenic tubers, this proportion increased to 45%, with esterified nonendogenous carotenoids in place of endogenous compounds. Conversely, nonesterified carotenoids in both wild-type and transgenic tuber tissues were associated with amyloplast membranes and starch granules.

Transcriptional regulation of a pineapple polyphenol oxidase gene and its relationship to blackheart.

Two genes encoding polyphenol oxidase (PPO) were isolated from pineapple (Ananas comosus[L.] Merr. cv. Smooth Cayenne). Sequence analyses showed that both contained a single intron and encoded typical chloroplast-localized PPO proteins, the sequences of which corresponded to two pineapple PPO cDNAs, PINPPO1 and PINPPO2, recently described by Stewart et al. (2001). Southern blot analyses suggested that pineapple contained only two PPO genes. Analysis of expression of PINPPO1 promoter GUS fusion constructs showed this promoter had a low basal activity and was cold- and wound-inducible, consistent with known mRNA expression profiles. Striking homologies to gibberellin response complexes (GARC) were observed in sequences of both the PINPPO1 and PINPPO2 promoters. Transient assays in mature pineapple fruit and stable expression in transgenic tobacco showed that PINPPO1 promoter-GUS fusions were indeed gibberellin (GA) responsive. A role for the element within the putative GARCs in mediating GA-responsiveness of the PINPPO1 promoter was confirmed by mutational analysis. PINPPO2 was also shown to be GA-responsive by RT-PCR analysis. Mutant PINPPO1 promoter-GUS fusion constructs, which were no longer GA-inducible, showed a delayed response to cold induction in pineapple fruit in transient assays, suggesting a role for GA in blackheart development. This was supported by observations that exogenous GA3 treatment induced blackheart in the absence of chilling. Sequences showing homology to GARCs are also present in some PPO promoters in tomato, suggesting that GA regulates PPO expression in diverse species.

The use of an acetoacetyl-CoA synthase in place of a -ketothiolase enhances poly-3-hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C-4 plants for the production of poly[(R)-3-hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield-potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing -ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl-CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB-producing sugarcane containing the -ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2x10(6)Da. These results are a major step forward in engineering a high biomass C-4 grass for the commercial production of PHB.

Stable expression of silencing-suppressor protein enhances the performance and longevity of an engineered metabolic pathway

Transgenic engineering of plants is important in both basic and applied research. However, the expression of a transgene can dwindle over time as the plant's small (s)RNA-guided silencing pathways shut it down. The silencing pathways have evolved as antiviral defence mechanisms, and viruses have co-evolved viral silencing-suppressor proteins (VSPs) to block them. Therefore, VSPs have been routinely used alongside desired transgene constructs to enhance their expression in transient assays. However, constitutive, stable expression of a VSP in a plant usually causes pronounced developmental abnormalities, as their actions interfere with endogenous microRNA-regulated processes, and has largely precluded the use of VSPs as an aid to stable transgene expression. In an attempt to avoid the deleterious effects but obtain the enhancing effect, a number of different VSPs were expressed exclusively in the seeds of Arabidopsis thaliana alongside a three-step transgenic pathway for the synthesis of arachidonic acid (AA), an ω-6 long chain polyunsaturated fatty acid. Results from independent transgenic events, maintained for four generations, showed that the VSP-AA-transformed plants were developmentally normal, apart from minor phenotypes at the cotyledon stage, and could produce 40% more AA than plants transformed with the AA transgene cassette alone. Intriguingly, a geminivirus VSP, V2, was constitutively expressed without causing developmental defects, as it acts on the siRNA amplification step that is not part of the miRNA pathway, and gave strong transgene enhancement. These results demonstrate that VSP expression can be used to protect and enhance stable transgene performance and has significant biotechnological application.

Genome-wide association analysis and differential expression analysis of resistance to Sclerotinia stem rot in Brassica napus

Brassica napus is one of the most important oil crops in the world, and stem rot caused by the fungus Sclerotinia sclerotiorum results in major losses in yield and quality. To elucidate resistance genes and pathogenesis-related genes, genome-wide association analysis of 347 accessions was performed using the Illumina 60K Brassica SNP (single nucleotide polymorphism) array. In addition, the detached stem inoculation assay was used to select five highly resistant (R) and susceptible (S) B. napus lines, 48 h postinoculation with S. sclerotiorum for transcriptome sequencing. We identified 17 significant associations for stem resistance on chromosomes A8 and C6, five of which were on A8 and 12 on C6. The SNPs identified on A8 were located in a 409-kb haplotype block, and those on C6 were consistent with previous QTL mapping efforts. Transcriptome analysis suggested that S. sclerotiorum infection activates the immune system, sulphur metabolism, especially glutathione (GSH) and glucosinolates in both R and S genotypes. Genes found to be specific to the R genotype related to the jasmonic acid pathway, lignin biosynthesis, defence response, signal transduction and encoding transcription factors. Twenty-four genes were identified in both the SNP-trait association and transcriptome sequencing analyses, including a tau class glutathione S-transferase (GSTU) gene cluster. This study provides useful insight into the molecular mechanisms underlying the plant's response to S. sclerotiorum.

Sugarcane-Based Biofuels and Bioproducts

Sugarcane has garnered much interest for its potential as a viable renewable energy crop. While the use of sugar juice for ethanol production has been in practice for years, a new focus on using the fibrous co-product known as bagasse for producing renewable fuels and bio-based chemicals is growing in interest. The success of these efforts, and the development of new varieties of energy canes, could greatly increase the use of sugarcane and sugarcane biomass for fuels while enhancing industry sustainability and competitiveness. Sugarcane-Based Biofuels and Bioproducts examines the development of a suite of established and developing biofuels and other renewable products derived from sugarcane and sugarcane-based co-products, such as bagasse. Chapters provide broad-ranging coverage of sugarcane biology, biotechnological advances, and breakthroughs in production and processing techniques. This text brings together essential information regarding the development and utilization of new fuels and bioproducts derived from sugarcane. Authored by experts in the field, Sugarcane-Based Biofuels and Bioproducts is an invaluable resource for researchers studying biofuels, sugarcane, and plant biotechnology as well as sugar and biofuels industry personnel.

Produção in vitro, análise fitoquímica e avaliação da atividade antineoplásica de metabólitos de Hovenia dulcis Thunb. (Rhamnaceae) obtidos por diferentes estratégias biotecnológicas

Hovenia dulcis Thunberg, natural da Ásia Oriental, é cultivada no Brasil onde é conhecida como uva-do-japão. A espécie possui várias indicações na medicina popular e alguns estudos apontam o seu potencial antineoplásico, tripanocida e hepatoprotetor. Metabólitos secundários são substâncias não essenciais para a sobrevivência celular, mas que fornecem vantagens adaptativas aos vegetais, sendo atribuído, para algumas delas, atividades biológicas importantes. Substâncias de interesse medicinal têm sido obtidas por técnicas da cultura de tecidos vegetais, como a calogênese e a cultura de células em suspensão, que permitem a síntese de matéria-prima de forma contínua e homogênea, independentemente de fatores ambientais e sazonais. O presente estudo objetivou o estabelecimento de culturas in vitro de H. dulcis, visando à produção de metabólitos de interesse, com vistas à avaliação do seu potencial antineoplásico sobre células K562. Foram testados protocolos para o estabelecimento de diferentes sistemas, como culturas de calos, de células em suspensão (CCS) e compact callus clusters (CCC) e ainda a avaliação do uso de elicitores na otimização de metabólitos produzidos in vitro. Foi verificado que a adição dos fitorreguladores KIN e TDZ, substituindo o BAP, não foi capaz de induzir a formação de calos friáveis, bem como a manutenção das culturas em ausência de luz. O uso do nitrato de prata promoveu a friabilidade de calos em todas as concentrações testadas, considerando-se 2,0 mg.L-1 a melhor concentração. Foram alcançadas taxas de 100% de formação de CCS tanto na presença, quanto em ausência de AgNO3. O maior acúmulo de biomassa foi verificado na concentração mais baixa de PIC (0,625 mg.L-1). A análise dos espectros de RMN indicou a presença de (+)-dihidromiricetina, (+)-galocatequina, hovenitina II, hovenosideo G, hodulosideo III, hodulosideo IV, hodulosideo I e hovenidulciosideo B1 nas culturas de calos friáveis. No estabelecimento de culturas CCC, observou-se a formação de calos compactos verdes em todas as concentrações de ANA testadas. O aumento da velocidade de rotação para 135 rpm aumentou a dispersão das células com consequente formação dos agregados celulares desejados. A seleção de linhagens celulares demonstrou ser um método eficiente na uniformização do tamanho desses agregados e tal uniformidade se manteve estável por mais de cinco subcultivos em 100% das culturas. Uma fração rica em saponinas foi obtida a partir dos agregados celulares, correspondendo a 1,46% da massa seca. A análise por RMN sugeriu a presença das saponinas Hovenosideo G e dos hovenidulciosideos A2 e B2. O uso de elicitores em cultura de calos mostrou-se adequado à produção de metabólitos secundários, sem alterações morfológicas nos mesmos. A elicitação alterou o perfil cromatográfico analisado por HPLC. Na elicitação com 5,0 mg.L-1 de extrato de levedura foi verificado um aumento de quase três vezes (12,280 3,396 equivalentes de quercetina/mg de extrato) na síntese de flavonoides. Finalmente, os estudos de ação antitumoral in vitro demonstraram citotoxicidade dos extratos de calos não elicitados de H. dulcis sobre linhagem de leucemia mieloide crônica (IC50 de 74,05 μg.mL-1.) e inibição do crescimento de tais células (K562), sugerindo o potencial antineoplásico para um produto biotecnológio (calo) desta espécie.