Genetic study of Camelina sativa oilseed crop and selection of a new variety by the bulk method.

Camelina sativa, commonly referred to as camelina or false flax, has emerged as a promising cover crop with the potential to mitigate climate change-a pressing global challenge that demands urgent and sustainable solutions. Belonging to the Brassicaceae family and native to Europe and Central Asia, camelina is an oilseed crop known for its resilience in diverse climates, including arid and semi-arid regions, making it adaptable to various environments. A breeding program started from a study of six winter varieties and five spring varieties of camelina is described: these genetic materials were characterized by SSRs molecular markers and by GBS technique. Molecular data clearly showed all spring varieties were genetically similar and distinguishable from the winter varieties, which, in turn, clustered together. Using molecular data, parental varieties belonging to the two different clusters were selected to generate new genetic variability. The new variety obtained, selected through the bulk method based on three parameters: yield, earliness, and weight of 1000 seeds, has allowed the generation of the new genetic material provisionally named C1244. Chemical characterization was performed (bromatological and glucosinolates analysis) to better describe C1244 in comparison with benchmark varieties. The new variety exhibited early maturity, similar to spring varieties, making this genetic material promising for use in intercropping systems, a high weight of 1000 seeds (1.46 g) which improves and facilitates seeding/harvesting operations and a high oil content (33.62%) akin to winter varieties making it valuable for human and animal food purposes.

Brachytic2 mutation is able to counteract the main pleiotropic effects of brown midrib3 mutant in maize.

Maize is the basis of nutrition of domesticated herbivores and one of the most promising energy crops. The presence of lignin in the cell wall, tightly associated to carbohydrates, prevents the physical access of enzymes such as cellulase, limiting the carbohydrate degradability and consequently the energy value. To increase the utilization of the biomass cellulose content, the challenge of breeding programs is to lower or modify the lignin components. In maize several mutations are able to modify the lignin content and in particular the mutation in brown midrib3 (bm3) gene appeared as one of the most promising in breeding programs. Unfortunately this mutation has several negative pleiotropic effects on various important agronomic traits such as stay green, lodging and susceptibility to several infections.The maize Brachyitic 2 (br2) gene encodes for a putative protein involved in polar movement of auxins. br2 mutant plants are characterized by shortening of lower stalk internodes, unusual stalk strength and tolerance to wind lodging, darker leaves persisting longer in the active green state in comparison to wild type plants, suggesting a possible utilization of br2 plants to counteract the negative effects of the bm3 mutation. In this work, we report the generation and a preliminary characterization of the double mutant bm3 br2, suggesting the potential use of this new genetic material to increase biomass cellulose utilization.

Phlobaphenes modify pericarp thickness in maize and accumulation of the fumonisin mycotoxins.

Phlobaphenes are insoluble phenolic compounds which are accumulated in a limited number of tissues such as seed pericarp and cob glumes, conferring on them a typical red-brown pigmentation. These secondary metabolites, derived from 3-deoxy flavonoids, are thought to have an important role in plants' resistance against various pathogens, e.g. by reducing fungal infection, and also to have beneficial effects on human and animal health due to their high antioxidant power. The aim of this work was to determine the role of phlobaphenes in reducing mycotoxin contamination on maize kernels. We analysed the effect of the P1 (pericarp color 1) gene on phlobaphenes accumulation, pericarp thickness and fumonisins accumulation. Analysing fumonisins accumulation in different genetic backgrounds through three seasons, we found a clear decrease of these toxins through the three years (Wilcoxon test, Z = 2.2, p = 0.0277) in coloured lines compared with the isogenic non-coloured ones. The coloured lines, carrying P1 allele showed an increase of phlobaphenes (about 10-14 fold) with respect to colourless lines. Furthermore there was a correlation between phlobaphenes accumulation and pericarp thickness (R = 0.9318; p = 0.0067). Taken together, these results suggest that the P1 gene plays a central role in regulating phlobaphenes accumulation in maize kernels, and indirectly, also tackles mycotoxins accumulation. The development and cultivation of corn varieties rich in phlobaphenes could be a powerful tool to reduce the loss of both quality and yield due to mycotoxin contamination, increasing the safety and the quality of the maize product.

lpa1-5525: A New lpa1 Mutant Isolated in a Mutagenized Population by a Novel Non-Disrupting Screening Method.

Phytic acid, or myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the main storage form of phosphorus in plants. It is localized in seeds, deposited as mixed salts of mineral cations in protein storage vacuoles; during germination, it is hydrolyzed by phytases to make available P together with all the other cations needed for seed germination. When seeds are used as food or feed, phytic acid and the bound cations are poorly bioavailable for human and monogastric livestock due to their lack of phytase activity. Therefore, reducing the amount of phytic acid is one strategy in breeding programs aimed to improve the nutritional properties of major crops. In this work, we present data on the isolation of a new maize (Zea mays L.) low phytic acid 1 (lpa1) mutant allele obtained by transposon tagging mutagenesis with the Ac element. We describe the generation of the mutagenized population and the screening to isolate new lpa1 mutants. In particular, we developed a fast, cheap and non-disrupting screening method based on the different density of lpa1 seed compared to the wild type. This assay allowed the isolation of the lpa1-5525 mutant characterized by a new mutation in the lpa1 locus associated with a lower amount of phytic phosphorus in the seeds in comparison with the wild type.

Study and characterization of an ancient European flint white maize rich in anthocyanins: Millo Corvo from Galicia.

In the second half of the last century, the American dent hybrids began to be widely grown, leading to the disappearance or marginalization of the less productive traditional varieties. Nowadays the characterization of traditional landraces can help breeders to discover precious alleles that could be useful for modern genetic improvement and allow a correct conservation of these open pollinated varieties (opvs). In this work we characterized the ancient coloured cultivar "Millo Corvo" typical of the Spanish region of Galicia. We showed that this cultivar accumulates high amounts of anthocyanins (83.4 mg/100g flour), and by TLC (Thin Layer Chromatography) and HPLC (High Pressure Liquid Chromatography) analysis, we demonstrated that they mainly consisted of cyanidin. Mapping and sequencing data demonstrate that anthocyanin pigmentation is due to the presence of the red color1 gene(r1), a transcription factor driving the accumulation of this pigment in the aleurone layer. Further chemical analysis showed that the kernels are lacking in carotenoids, as confirmed by genetic study. Finally a DPPH (2,2-diphenyl-1-picrylhydrazyl) radical scavenging ability test showed that Millo Corvo, even though lacking carotenoids, has a high antioxidant ability, and could be considered as a functional food due to the presence of anthocyanins.

Low phytic acid 1 mutation in maize modifies density, starch properties, cations, and fiber contents in the seed.

Monogastric animals are unable to digest phytic acid, so it represents an antinutritional factor and also an environmental problem. One strategy to solve this problem is the utilization of low phytic acid (lpa) mutants that accumulate low levels of phytic P and high levels of free phosphate in the seeds; among the lpa maize mutants lpa1 exhibited the highest reduction of phytic acid in the seed. This study indicated that the low phytic acid mutations exerted pleiotropic effects not directly connected to the phytic acid pathway, such as on seed density, content of ions, and the antioxidant compounds present in the kernels. Furthermore some nutritional properties of the flour were altered by the lpa1 mutations, in particular lignin and protein content, while the starch does not seem to be modified as to the total amount and in the amylose/amylopectin ratio, but alterations were noticed in the structure and size of granules.

Study of low phytic acid1-7 (lpa1-7), a new ZmMRP4 mutation in maize.

Phytic acid (PA), myo-inositol 1,2,3,4,5,6-hexakisphosphate, is the main storage form of phosphorus in plants. It is localized in seeds, deposited as mixed salts of mineral cations in protein storage vacuoles; during germination, it is hydrolyzed by phytases. When seeds are used as food/feed, PA and the bound cations are poorly bioavailable for human and monogastric livestock due to their lack of phytase activity. Reducing the amount of PA is one strategy to solve these problems and is an objective of genetic improvement for improving the nutritional properties of major crops. In this work, we present data on the isolation of a new maize (Zea mays L.) low phytic acid 1 (lpa1) mutant allele obtained by chemical mutagenesis. This mutant, named lpa1-7, is able to accumulate less phytic phosphorus and a higher level of free inorganic phosphate in the seeds compared with wild type. It exhibits a monogenic recessive inheritance and lethality as homozygous. We demonstrate that in vitro cultivation can overcome lethality allowing the growth of adult plants, and we report data regarding embryo and leaf abnormalities and other defects caused by negative pleiotropic effects of this mutation.

A defective ABC transporter of the MRP family, responsible for the bean lpa1 mutation, affects the regulation of the phytic acid pathway, reduces seed myo-inositol and alters ABA sensitivity.

• We previously identified the lpa1 (low phytic acid) 280-10 line that carries a mutation conferring a 90% reduction in phytic acid (InsP(6) ) content. In contrast to other lpa mutants, lpa1(280-10) does not display negative pleiotropic effects. In the present paper, we have identified the mutated gene and analysed its impact on the phytic acid pathway. • Here, we mapped the lpa1(280-10) mutation by bulk analysis on a segregating F(2) population, an then, by comparison with the soybean genome, we identified and sequenced a candidate gene. The InsP(6) pathway was analysed by gene expression and quantification of metabolites. • The mutated Pvmrp1(280-10) cosegregates with the lpa1(280-10) mutation, and the expression level of several genes of the InsP(6) pathway are reduced in the lpa1(280-10) mutant as well as the inositol and raffinosaccharide content. PvMrp2, a very similar paralogue of PvMrp1 was also mapped and sequenced. • The lpa1 mutation in beans is likely the result of a defective Mrp1 gene (orthologous to the lpa genes AtMRP5 and ZmMRP4), while its Mrp2 paralog is not able to complement the mutant phenotype in the seed. This mutation appears to down-regulate the InsP(6) pathway at the transcriptional level, as well as altering inositol-related metabolism and affecting ABA sensitivity.

Effect of flavonoid pigments on the accumulation of fumonisin B1 in the maize kernel.

Mycotoxins are secondary metabolites with potential dangers for animal and human health. In particular, maize (Zea mays L.) infection caused by Fusarium and the consequent fumonisin contamination is widespread in several countries such as Italy. We developed six maize populations differing in their constitution of regulatory genes able to accumulate respectively anthocyanins in the aleurone layer (r1 gene), pericarp (b1 and pl1 genes) and phlobaphene in the pericarp (p1 gene). These coloured populations, with the related control colourless populations were analysed for mycotoxin content in the kernels during three field seasons with the aim of understanding if there were any correlations with their ability to accumulate flavonoids in kernel tissues. Our results indicate that accumulation of flavonoid pigments in the seeds, in particular phlobaphenes, is able to reduce the level of fumonisin B1. This finding could be used to minimize kernel mycotoxin contamination in this crop, in particular, the development of sweet, pop and polenta coloured corn varieties will help the farmer to keep the level of fumonisin under the threshold of contamination established for human corn consumption.

The low phytic acid1-241 (lpa1-241) maize mutation alters the accumulation of anthocyanin pigment in the kernel.

The lpa1 mutations in maize are caused by lesions in the ZmMRP4 (multidrug resistance-associated proteins 4) gene. In previous studies (Raboy et al. in Plant Physiol 124:355-368, 2000; Pilu et al. in Theor Appl Genet 107:980-987, 2003a; Shi et al. Nat Biotechnol 25:930-937, 2007), several mutations have been isolated in this locus causing a reduction of phytic acid (myo-inositol-1,2,3,4,5,6-hexakisphosphate, or InsP(6)) content and an equivalent increasing of free phosphate. In particular, the lpa1-241 mutation causes a reduction of up to 90% of phytic acid, associated with strong pleiotropic effects on the whole plant. In this work, we show, for the first time to our knowledge, an interaction between the accumulation of anthocyanin pigments in the kernel and the lpa mutations. In fact the lpa1-241 mutant accumulates a higher level of anthocyanins as compared to wild type either in the embryo (about 3.8-fold) or in the aleurone layer (about 0.3-fold) in a genotype able to accumulate anthocyanin. Furthermore, we demonstrate that these pigments are mislocalised in the cytoplasm, conferring a blue pigmentation of the scutellum, because of the neutral/basic pH of this cellular compartment. As a matter of fact, the propionate treatment, causing a specific acidification of the cytoplasm, restored the red pigmentation of the scutellum in the mutant and expression analysis showed a reduction of ZmMRP3 anthocyanins' transporter gene expression. On the whole, these data strongly suggest a possible interaction between the lpa mutation and anthocyanin accumulation and compartmentalisation in the kernel.

Phytic acid prevents oxidative stress in seeds: evidence from a maize (Zea mays L.) low phytic acid mutant.

A maize mutant defective in the synthesis of phytic acid during seed maturation was used as a tool to study the consequences of the lack of this important reserve substance on seed survival. Data on germinability, free iron level, free radical relative abundance, protein carbonylation level, damage to DNA, degree of lipid peroxidation, alpha- and gamma-tocopherol amount and antioxidant capacity were recorded on seeds of maize B73 and of an isogenic low phytic acid mutant (lpa1-241), either unaged or incubated for 7 d in accelerated ageing conditions (46 degrees C and 100% relative humidity). The lpa1-241 mutant, compared to wild type (wt), showed a lower germination capacity, which decreased further after accelerated ageing. Whole lpa1-241 mutant kernels contained about 50% more free or weakly bound iron than wt ones and showed a higher content of free radicals, mainly concentrated in embryos; in addition, upon accelerated ageing, lpa1-241 seed proteins were more carbonylated and DNA was more damaged, whereas lipids did not appear to be more peroxidated, but the gamma-tocopherol content was decreased by about 50%. These findings can be interpreted in terms of previously reported but never proven antioxidant activity of phytic acid through iron complexation. Therefore, a novel role in plant seed physiology can be assigned to phytic acid, that is, protection against oxidative stress during the seed's life span. As in maize kernels the greater part of phytic acid (and thus of metal ions) is concentrated in the embryo, its antioxidant action may be of particular relevance in this crop.