Pea-derived vaccines demonstrate high immunogenicity and protection in rabbits against rabbit haemorrhagic disease virus

Vaccines against rabbit haemorrhagic disease virus (RHDV) are commercially produced in experimentally infected rabbits. A genetically engineered and manufactured version of the major structural protein of RHDV (VP60) is considered to be an alternative approach for vaccine production. Plants have the potential to become an excellent recombinant production system, but the low expression level and insufficient immunogenic potency of plant-derived VP60 still hamper its practical use. In this study, we analysed the expression of a novel multimeric VP60-based antigen in four different plant species, including Nicotiana tabacum L., Solanum tuberosum L., Brassica napus L. and Pisum sativum L. Significant differences were detected in the expression patterns of the novel fusion antigen cholera toxin B subunit (CTB)::VP60 (ctbvp60(SEKDEL)) at the mRNA and protein levels. Pentameric CTB::VP60 molecules were only detected in N. tabacum and P. sativum, and displayed equal levels of CTB, at approximately 0.01% of total soluble protein (TSP), and traces of detectable VP60. However, strong enhancement of the CTB protein content via self-fertilization was only observed in P. sativum, where it reached up to 0.7% of TSP. In rabbits, a strong decrease in the protective vaccine dose required from 48-400 microg potato-derived VP60 [Castanon, S., Marin, M.S., Martin-Alonso, J.M., Boga, J.A., Casais, R., Humara, J.M., Ordas, R.J. and Parra, F. (1999) Immunization with potato plants expressing VP60 protein protects against rabbit hemorrhagic disease virus. J. Virol. 73, 4452-4455; Castanon, S., Martin-Alonso, J.M., Marin, M.S., Boga, J.A., Alonso, P., Parra, F. and Ordas, R.J. (2002) The effect of the promoter on expression of VP60 gene from rabbit hemorrhagic disease virus in potato plants. Plant Sci. 162, 87-95] to 0.56-0.28 microg antigenic VP60 (measured with VP60 enzyme-linked immunosorbent assay) of crude CTB::VP60 pea extracts was demonstrated. Rabbits immunized with pea-derived CTB::VP60 showed anti-VP60-specific antibodies, similar to RikaVacc((R))-immunized rabbits, and survived RHDV challenge

Editorial: Accelerating Genetic Gains in Pulses

Legumes, members of the Fabaceae/Leguminosae family, are the third largest family of higher plants with almost 20,000 species belonging to 650 genera, and are ubiquitous all over the world. Among all legumes, pulse crops or food legumes fall into the four clades of the subfamily Papilionoideae which include aeschynomenoids/dalbergiods, genistoids, hologalegina, and phaseoloids/millettoids. They are distinctive due to their positive impact on agricultural and environmental sustainability and have a prominent role in promoting human and animal health, soil amelioration, cropping system diversification, and sustenance of rural livelihoods (Pratap et al., 2021a). These also provide protein isolates that are increasingly being used in the food industry as functional ingredients suitable for vegan diets (Robinson et al., 2019). The inclusion of pulses in rotation with cereals helps to improve system yields, enhance net carbon sequestration, and lower the carbon footprint. Nonetheless, in addition to being an excellent source of protein, starch, and micronutrients, pulses also contain anti-nutritional compounds that can interfere with the absorption of minerals (Moore et al., 2018) and also the digestion of protein (Clemente et al., 2015). Realizing their importance, significant research has been dedicated to their genetic amelioration, thereby turning them into mainstream crops from so-called “orphan legumes”. Classical plant breeding methods led to the development of more than 3,800 improved varieties of different pulse crops globally, with improved attributes of grain yield, crop duration, stress resistance, nutrition quality, etc. However, despite this effort, the increase in average pulse yields (from 637 to 1,009 kg/ha) has been modest compared to dramatic increases in cereal productivity (from 1,353 to 4,074 kg/ha) between 1961 and 2017 (Kumar et al., 2020). Among legumes, Koester et al. (2014) studied 80 years of historical data of soybean breeding at the Crop Research and Education Center in Urbana, USA and reported a genetic gain of 26.5 kg ha−1 year−1 , attributing the gain in grain yield to increases in light interception, energy conversion, and partitioning efficiencies. Productivity gains in pulses have been recorded when especially considered along with the markedly reduced duration of the improved varieties, leading to increased cropping intensity, while genetic gains have been recorded for traits imparting resistance to major biotic and abiotic stresses, herbicide tolerance, larger seeds, and improved nutritional quality. This resulted in the growth, in terms of production and productivity, in major pulse-producing countries. For example, India witnessed the highest growth in production in mung bean (178%), followed by chickpea (125%), urdbean (90%), pigeonpea (51%), and lentil (34%) in the last 15 years (Gaur, 2021). Notably, breeding in most pulses has remained confined to the exploitation of genetic variation within the primary gene pool, which has resulted in a narrow genetic base in most of them

Agrobacterium-mediated transformation of tarwi (Lupinus mutabilis Sweet), a potential platform for the production of plant-made proteins

A robust, reproducible method of Agrobacterium-mediated transformation was developed for Lupinus mutabilis Sweet (tarwi), a large-seeded Andean legume. Initially, a regeneration and transformation protocol was developed using a plasmid which contained a bifunctional fusion gene conferring both \u3b2-glucuronidase (gus) and neomycin phosphotransferase activities, under the control of a constitutive 35S35SAMV promoter. The tissue explants consisted of longitudinal slices from embryonic axes of imbibed, mature seed. Using a series of tissue culture media for cocultivation, shoot initiation, shoot elongation, and rooting, kanamycin-resistant transgenic plants were recovered from approximately 1% of the explants. This transformation protocol was further used with a construct that contained the human adenosine deaminase (hADA) gene under the control of a legumin seed-specific promoter, also with a kanamycin resistance cassette for chemical selection. Changes made during the course of this study, which included adjustments to the antibiotic concentration during the shoot elongation and rooting phases plus the incorporation of techniques to improve ventilation in the tissue culture system, resulted in major improvements in shoot quality and, most significantly, rooting. The outcome was an increased frequency of transgenic plant recovery (7.4%), with a low (9.6%) rate of plants that escaped selection. The inheritance of the hADA gene was documented and showed the expected Mendelian segregation pattern. The produced hADA protein was a fully functional enzyme and localized only in the seed, as expected. Thus, this legume species is an excellent candidate for a nonfood plant host platform for the production of plant-made proteins.Peer reviewed: YesNRC publication: Ye

Editorial: Accelerating Genetic Gains in Pulses

Legumes, members of the Fabaceae/Leguminosae family, are the third largest family of higher plants with almost 20,000 species belonging to 650 genera, and are ubiquitous all over the world. Among all legumes, pulse crops or food legumes fall into the four clades of the subfamily Papilionoideae which include aeschynomenoids/dalbergiods, genistoids, hologalegina, and phaseoloids/millettoids. They are distinctive due to their positive impact on agricultural and environmental sustainability and have a prominent role in promoting human and animal health, soil amelioration, cropping system diversification, and sustenance of rural livelihoods (Pratap et al., 2021a). These also provide protein isolates that are increasingly being used in the food industry as functional ingredients suitable for vegan diets (Robinson et al., 2019). The inclusion of pulses in rotation with cereals helps to improve system yields, enhance net carbon sequestration, and lower the carbon footprint. Nonetheless, in addition to being an excellent source of protein, starch, and micronutrients, pulses also contain anti-nutritional compounds that can interfere with the absorption of minerals (Moore et al., 2018) and also the digestion of protein (Clemente et al., 2015). Realizing their importance, significant research has been dedicated to their genetic amelioration, thereby turning them into mainstream crops from so-called “orphan legumes”. Classical plant breeding methods led to the development of more than 3,800 improved varieties of different pulse crops globally, with improved attributes of grain yield, crop duration, stress resistance, nutrition quality, etc. However, despite this effort, the increase in average pulse yields (from 637 to 1,009 kg/ha) has been modest compared to dramatic increases in cereal productivity (from 1,353 to 4,074 kg/ha) between 1961 and 2017 (Kumar et al., 2020). Among legumes, Koester et al. (2014) studied 80 years of historical data of soybean breeding at the Crop Research and Education Center in Urbana, USA and reported a genetic gain of 26.5 kg ha−1 year−1 , attributing the gain in grain yield to increases in light interception, energy conversion, and partitioning efficiencies. Productivity gains in pulses have been recorded when especially considered along with the markedly reduced duration of the improved varieties, leading to increased cropping intensity, while genetic gains have been recorded for traits imparting resistance to major biotic and abiotic stresses, herbicide tolerance, larger seeds, and improved nutritional quality. This resulted in the growth, in terms of production and productivity, in major pulse-producing countries. For example, India witnessed the highest growth in production in mung bean (178%), followed by chickpea (125%), urdbean (90%), pigeonpea (51%), and lentil (34%) in the last 15 years (Gaur, 2021). Notably, breeding in most pulses has remained confined to the exploitation of genetic variation within the primary gene pool, which has resulted in a narrow genetic base in most of them

Seed specific expression and analysis of recombinant human adenosine deaminase (hADA) in three host plant species

The plant seed is a leading platform amongst plant-based storage systems for the production of recombinant proteins. In this study, we compared the activity of human adenosine deaminase (hADA) expressed in transgenic seeds of three different plant species: pea (Pisum sativum L.), Nicotiana benthamiana L. and tarwi (Lupinus mutabilis Sweet). All three species were transformed with the same expression vector containing the hADA gene driven by the seed-specific promoter LegA2 with an apoplast targeting pinII signal peptide. During the study, several independent transgenic lines were generated and screened from each plant species and only lines with a single copy of the gene of interest were used for hADA expression analysis. A stable transgenic canola line expressing the ADA protein, under the control of 35S constitutive promoter was used as both as a positive control and for comparative study with the seed specific promoter. Significant differences were detected in the expression of hADA. The highest activity of the hADA enzyme (Units/g seed) was reported in tarwi (4.26 U/g) followed by pea (3.23 U/g) and Nicotiana benthamiana (1.69 U/g). The expression of mouse ADA in canola was very low in both seed and leaf tissue compared to other host plants, confirming higher activity of seed specific promoter. Altogether, these results suggest that tarwi could be an excellent candidate for the production of valuable recombinant proteins.Peer reviewed: YesNRC publication: Ye

Proliferating floral organs (pfo), a Lotus japonicus gene required for specifying floral meristem determinacy and organ identity, encodes an F-box protein

To study flower development in the model legume Lotus japonicus, a population of transgenic plants containing a maize transposable element (Ac) in their genome was screened for floral mutants. One mutation named proliferating floral organs (pfo) causes plants to produce a large number of sepal-like organs instead of normal flowers. It segregates as a single recessive Mendelian locus, and causes sterility. Scanning electron microscopy revealed that pfo affects the identity, number and arrangement of floral organs. Sepal-like organs form in the first whorl, and secondary floral meristems are produced in the next whorl. These in turn produce sepal-like organs in the first whorl and floral meristems in the second whorl, and the process is reiterated. Petals and stamens are absent while carpels are either absent or reduced. The pfo phenotype was correlated with the presence of an Ac insertion yielding a 1.6-kb HindIII restriction fragment on Southern blots. Both the mutant phenotype and this Ac element are unstable. Using the transposon as a tag, the Pfo gene was isolated. Conceptual translation of Pfo predicts a protein containing an F-box, with high overall similarity to the Antirrhinum FIMBRIATA, Arabidopsis UNUSUAL FLORAL ORGANS and Pisum sativum Stamina pistilloida proteins. This suggests that Pfo may regulate floral organ identity and meristem determinacy by targeting proteins for ubiquitination

Transcript Profiling and Identification of Molecular Markers for Early Microspore Embryogenesis in Brassica napus1[W][OA]

Isolated microspores of Brassica napus are developmentally programmed to form gametes; however, microspores can be reprogrammed through stress treatments to undergo appropriate divisions and form embryos. We are interested in the identification and isolation of factors and genes associated with the induction and establishment of embryogenesis in isolated microspores. Standard and normalized cDNA libraries, as well as subtractive cDNA libraries, were constructed from freshly isolated microspores (0 h) and microspores cultured for 3, 5, or 7 d under embryogenesis-inducing conditions. Library comparison tools were used to identify shifts in metabolism across this time course. Detailed expressed sequence tag analyses of 3 and 5 d cultures indicate that most sequences are related to pollen-specific genes. However, semiquantitative and real-time reverse transcription-polymerase chain reaction analyses at the initial stages of embryo induction also reveal expression of embryogenesis-related genes such as BABYBOOM1, LEAFY COTYLEDON1 (LEC1), and LEC2 as early as 2 to 3 d of microspore culture. Sequencing results suggest that embryogenesis is clearly established in a subset of the microspores by 7 d of culture and that this time point is optimal for isolation of embryo-specific expressed sequence tags such as ABSCISIC ACID INSENSITIVE3, ATS1, LEC1, LEC2, and FUSCA3. Following extensive polymerase chain reaction-based expression profiling, 16 genes were identified as unequivocal molecular markers for microspore embryogenesis in B. napus. These molecular marker genes also show expression during zygotic embryogenesis, underscoring the common developmental pathways that function in zygotic and gametic embryogenesis. The quantitative expression values of several of these molecular marker genes are shown to be predictive of embryogenic potential in B. napus cultivars (e.g. ‘Topas’ DH4079, ‘Allons,’ ‘Westar,’ ‘Garrison’)