Quantification of major phenolic and flavonoid markers in forage crop Lolium multiflorum using HPLC-DAD

ABSTRACT The objective of this study was to perform preliminary screening of phytochemical compounds and quantification of major phenolics and flavonoid markers in Italian ryegrass extract using HPLC-DAD. Previously, LC-MS analysis has identified different phenolic acids, including caffeic acid, ferulic acid, p-coumaric acid, chlorogenic acid, dihydroxy benzoic acid, propyl gallate, catechin, and six flavonoids including rutin hydroxide, luteolin, kaemferol, vitexin, narcissoside, and myricetin from Italian ryegrass extract. In the present study, Italian ryegrass silage powder was extracted with ethanol: water for 20 min at 90 °C. The extract targeted optimum yield of phenolic acids and flavonoids. Crude phenolic acid and flavonoids were then purified by solid phase extraction method. Purified fractions were then injected into HPLC with a diode-array detector. Quantified concentrations of isolated phenolic acids and flavonoids ranged from 125 to 220 µg/g dry weight. Limits of detection and limits of quantification for all standards (unknown compounds) ranged from 0.38 to 1.71 and 0.48 to 5.19 µg/g dry weight, respectively. Obtained values were compared with previous literatures, indicating that our HPLC-DAD quantification method showed more sensitivity. This method showed better speed, accuracy, and effectiveness compared to previous reports. Furthermore, this study could be very useful for developing phenolic acids and flavonoids from compositions in Italian ryegrass silage feed for pharmaceutical applications and ruminant animals in livestock industries.

Stability in chromosome number and DNA content in synthetic tetraploids of Lolium multiflorum after two generations of selection / Estabilidade no número cromossômico e no conteúdo de DNA em tetraploides sintéticos de Lolium multiflorum após duas gerações de seleção

ABSTRACT: Chromosome doubling of Italian ryegrass genotypes ( Lolium multiflorum Lam.) adapted to the brazilian edaphoclimatic conditions is an important strategy used by breeders and aims to obtain more vigorous genotypes with better forage quality and disease resistance. The effectiveness of chromosome doubling can be measured by genetic stability and fertility rates of plants over generations. However, a common problem in the polyploidization process is the regeneration of mixoploid plants that have impaired fertility and genetic stability. The objective of this study was to verify if progenies of recently tetraploidized plants remain stable regarding DNA content and chromosome number, over two generations. Progenies of L. multiflorum plants artificially tetraploidized with colchicine treatment were evaluated. Chromosome counting and estimates of the DNA content were used to evaluate the genetic stability. The percentage of tetraploid plants (4X) increased over generations (18%, 34% and 91% in cycle 0, 1 and 2, respectively). All progenies identified as tetraploid by flow citometry showed variation in chromosome number (mixoploidy), but produced viable seeds. Results showed that stabilization in chromosome number and DNA content in tetraploidized plant progenies requires time and that the success of this procedure depends on a continuous and accurate screening and selection.

RESUMO: A duplicação cromossômica de genótipos de azevém anual ( Lolium multiflorum Lam.) adaptados às condições edafoclimáticas brasileira é uma estratégia importante usada pelos melhoristas e visa a obtenção de genótipos mais vigorosos com melhor qualidade de forragem e resistência a doenças. A eficiência da duplicação cromossômica pode ser medida pela estabilidade genética e taxas de fertilidade das plantas ao longo das gerações. No entanto, um dos problemas comumente encontrados no processo de poliploidização é a regeneração de plantas mixoploides que apresentam comprometimento na fertilidade e instabilidade genética. O objetivo deste estudo foi verificar se progênies oriundas de plantas tetraploidizadas recentemente se mantêm estáveis quanto ao conteúdo de DNA e ao número cromossômico, ao longo de duas gerações. Foram avaliadas progênies de plantas de L. multiflorum tetraploidizadas artificialmente com tratamento de colchicina. A estabilidade genética foi avaliada por meio de contagens cromossômicas e estimativa da quantidade de DNA. A porcentagem de plantas tetraploides (4X) aumentou ao longo das gerações (18%, 34% e 91% no ciclo de 0, 1 e 2, respectivamente). Todas as progênies identificadas como tetraploides por meio de citometria de fluxo apresentaram variação no número cromossômico (mixoploidia), entretanto, produziram sementes viáveis. Os resultados demonstraram que a estabilização no número cromossômico e no conteúdo de DNA em progênies de plantas tetraploidizadas requer tempo e que o sucesso desse procedimento depende de um contínuo e rigoroso monitoramento e seleção.

Genotypic variation of Agrobacterium-mediated transformation of Italian ryegrass

In the present study, genotypic variation of Agrobacterium-mediated transformation of Korean Italian ryegrass has been evaluated. Mature seed-derived calli of seven cultivars were inoculated and co-cultured with Agrobacterium tumefaciens carrying the binary vector pCAMBIA1301, which contains a reporter gene (gus) and a plant selectable marker gene conferring resistance to hygromycin (hpt) in the T-DNA region. The effects of several factors such as callus type and callus age on transformation effectiveness and the expression of the GUS gene were investigated. The highest transformation effectiveness (6.7 percent) was obtained with the Hwasan 101 cultivar when 9-week-old calli (type-I) were inoculated with Agrobacterium. The overall transformation rates of the examined cultivars ranged from 0.4 percent to 6.7 percent. GUS histochemical assays, PCR, and southern analysis of transgenic plants demonstrated that transgenes were successfully integrated into the genome of Italian ryegrass. Thus, evaluation of transformation effectiveness and selection of a suitable cultivar of Italian ryegrass may improve molecular breeding of this species.

Produção animal em misturas forrageiras de estação fria semeadas em uma pastagem natural / Animal production in mixtures of temperate species seeded in a natural pasture

Com o objetivo de estimar alguns parâmetros de produção e qualidade de forragem e a análise econômica da pastagem, realizou-se um trabalho na área experimental do Departamento de Zootecnia da UFSM, entre maio e novembro de 1993. Os tratamentos foram aveia (Avena strigosa Schreb) + azevém (Lolium multiflorum Lam.) com: l) trevo vesiculoso (Trifolium vesiculosum Savi) e 2) ervilhaca (Vicia sativa L.), sobressemeadas em campo nativo e sendo pastejadas por terneiras cruza Nelore x Charolês. O delineamento experimental foi o inteiramente casualisado, com duas repetições, que foram potreiros de 1,5ha aproximadamente. O sistema de pastejo foi o contínuo com lotação variável, mantendo-se a disponibilidade de forragem entre 1500 e 2000kg/ha de Matéria Seca (MS), com uma pressão de pastejo real em tomo de 10 por cento. O período experimental foi de 84 dias, sendo os animais pesados a cada 28 dias, para o ajuste de lotação. Para os parâmetros ganho médio diário de peso, ganho de peso vivo por área, taxa de acúmulo e produção de MS da pastagem, não houve diferença entre os tratamentos. O tratamento com ervilhaca mostrou-se superior quanto ao teor de proteína bruta e digestibilidade in vitro da matéria orgânica (P < 0,05). No que se refere à análise econômica, o tratamento com trevo vesiculoso apresentou uma relação benefício/custo de 2,23, sendo superior àquela do tratamento com ervilhaca que foi de 1,11.

Aiming to evaluate parameters of forage production and quality and the economic analysis of pasture, a trial was conducted in the experimental área of the Animal Science Department of UFSM, Brazil, between May and November of 1993. The mixtures were constituited by oat (Avena strigosa Schreb) + Italian ryegrass (Lolium multiflorum Lam.) with: 1) arrowleaf clover (Trifolium vesiculosum Savi) and 2) Yetch (Vicia sativa L.), seeded in a natural pasture and grazed by Nelore x Charolês calves. The experiment was a completely randomized design, with two replications, characterized by paddocks of 1.5ha approximately. The grazing system was continuous with stocking rate variable, for mantaining the forage availability between 1500 and 2000kg Dry Matter (DM)/ha, with a actual grazing pressure of 10 percent approximately. The experimental period was of 84 days. Animais were weighted at a 28 days of interval, for adjusting the stocking rate. For the parameters daily liveweight gain, liveweight gain per area, growth rate and pasture DM production, there was no significant difference from the treatments. The treatment with vetch was higher in the crude protein content and the in vitro organic matter digestibility (P < 0.05). In economia analysis, the treatment with arrowleaf clover presented a benefit/cost relation of 2.23 and was higher than the treatment with vetch of 1.11.