Study on environmental factors for specific functional component increase in mulberry (Morus alba L.) leaves with selection and breeding of suitable cultivars

The benefits of functional components of mulberry (Morus alba L.) leaves have been attracting the attention of the health food industry, with an increasing market demand. To more effectively obtain a mulberry leaf yield and high levels of functional components, the following factors were investigated: 1) selection and crossbreeding of mulberry cultivars to obtain high levels of target components, 2) effect of solar radiation and nitrogen fertilization, and 3) optimum harvesting timing using our experimental settings and common culture fields in Shimane Prefecture. 1. Varietal differences in the functional components of mulbeery leaves and breeding. The varietal differences in the flavonol glycosides contained in mulberry leaves were elucidated, including rutin, isoquercitrin, kaempferol 3-(6-rhamnosylglucoside) (K3RG), quercetin 3-(6-malonylglucoside) (Q3MG), astragalin, quercetin 3-(6-acetylglucoside) (Q3AG), and kaempferol 3-(6-malonyl glucoside) (K3MG). This information was used for breeding mulberry cultivars with high concentrations of functional components. The content, composition, and proportion of flavonols in leaves varied widely. 'Kobuchizawa 1' had the highest level of total flavonols, five times greater than that of 'Mikurasima 15'. Q3MG was the most abundantly contained flavonol in many of the cultivars, although it was not contained at all in some cultivars. Q3AG was found only in 'Keguwa'. Study of the mode of genetic inheritance and the content of Q3MG in crossbred offspring showed that malonyl transferase, an enzyme involved in the synthesis of Q3MG, was transcribed through a single gene according to Mendelian inheritance. An offspring with a higher Q3MG level than both parents could be obtained from the crossing, suggesting that crossbreeding was effective. Then, crossbreeding was conducted using 'Yonbaiseisou (tetraploid)' as a seedling parent and 'Kokusou 21 (diploid)' as a pollen parent, and obtained a new triploid cultivar 'Souraku'. Consequently, compared with 'Ichinose', 'Souraku' was found to have 1.4 times higher content of Q3MG and greater leaf yield. 'Souraku' was applied for a new variety registration in April 2013, and was accepted as a new variety in 2015. 2. Effect of environmental conditions on the functional components of mulberry leaves. Effects of solar radiation, which may influence production of flavonol and 1-deoxynojirimycin (DNJ) in mulberry leaves, were investigated by comparing greenhouse (poor solar radiation) and outdoor (rich solar radiation) settings. The level of flavonol in leaves cultivated in the greenhouse was markedly decreased when compared with those cultivated outdoors. In contrast, the DNJ content of plants cultivated in greenhouse was only slightly increased when compared with those cultivated outdoors. Interestingly, the flavonol content was markedly increased in the upper leaves of mulberry trees that were transferred from a greenhouse to the outdoors, compared with those cultivated only in the outdoors. Under high solar radiation, the flavonol level became very high but the DNJ level became slightly lower, suggesting that the impact of solar radiation is great on flavonols, but is small on the DNJ synthesis. Then, effects of applied nitrogen amounts were investigated regarding specific functional components in mulberry leaves. The relationships between mineral elements and the functional components in mulberry leaves were examined using mulberry trees cultivated in different soil conditions in four cultured fields. Then, the relationships between the nitrogen levels and functional components in leaves were studied by culturing mulberry in plastic pots and experimental fields. In the common cultured fields, total nitrogen was negatively correlated with the chlorogenic acid content (R2 = -0.48) and positively correlated with DNJ (R2 = 0.60). Additionally, differences in nitrogen application levels impacted each functional component in mulberry leaves. For instance, with increased nitrogen levels, the chlorogenic acid and flavonol contents significantly decreased, but the DNJ content significantly increased. The selection of the optimal nitrogen application level is important to obtain the desired functional components from mulberry leaves. 3. Seasonal changes in functional component contents in mulberry leaves. Optimum harvesting timing of mulberry leaves was investigated to obtain maximum yields of specific functional components. The components of mulberry leaves were analyzed to assess seasonal changes in the concentrations of functional components, including flavonols, chlorogenic acid, and DNJ. During the experimental period (from May 26 to October 16 in 2015), the ranges of flavonols, chlorogenic acid, and DNJ contents were 1134-2230 mg/100 g dry weight (DW), 616-1014 mg/100 g DW, and 53-199 mg/100 g DW, respectively. There were high flavonols contents in mulberry leaves from late May to early July, from early to mid-August, and after late September. There were high chlorogenic acid contents from late May to early July, and after late September. From late September, the flavonols contents increased over time, and showed the highest level on October 16, the last day of the experimental period. The increased flavonols contents after late September were due to the synergistic effects of solar radiation and lower temperature. The DNJ content showed a bell-shaped curve, peaking in mid-August, and the DNJ content was strongly correlated with total temperature. The flavonols and chlorogenic acid contents in mulberry leaves were higher after late September when the temperatures decreased and there was still high solar radiation, while the DNJ content peaked in August when temperatures were highest in the year. Based on our experiment findings, August is considered the best time to harvest mulberry leaves to obtain the optimal yields of beneficial functional components. If mulberry leaves are harvested twice per year, then the first harvest should be in July to August when temperatures and solar radiation hours are high, and the second harvest should be completed by the end of September. With a series of experiment results, the conditions to obtain high levels of specific functional components in mulberry leaves were identified pertaining to the selection mulberry cultivars, the effect of solar radiation, applied nitrogen amounts, and harvest timing. This knowledge is considered useful for producing high functional components of mulberry leaves in the future.