A Study On Germplasm Resources And Exogenous Induction Of Three Food Crop With High Melatonin Content

MT (short for Melatoninm,褪黑素in Chinese ) is a indole-trytamine(N-Acetyl-5-hydroxy tryptamine). It was initially found in the pineal of bovine. MT is a highly conserved molecule during bio-evolution, which exists extensively in animals including human, in plants, and in prokaryotes. It was approved that MT is an endogenous free radical scavenger, which is the most powerful antioxidant so far. More importantly, MT in food inevitably enters animals and human through eating the food with MT and plays efficacies in purging inner free radical, enhancing body immunity, delaying aging and improving sleep, etc.The maize(Zea mays L.), rice(Oryza sativa L.) and oat(Avena sativa L.) were utilized as the materials of the experiment, and the content and purpose of this study were as follows: Seeking for an effective method on melatonin isolating and extracting from crop seeds, and setting up a high performance liquid chromatography (thereafter abbreviated as HPLC) analysis method for determining the content of the active ingredient-melatonin in crop seeds; To determine MT content in the seeds of maize, rice and oat and to analyze its differences among various species and varieties; Study on MT content variation in crop seeds under different planting environments, from which to select crop germplasm resources with high and steady MT content; Study on the exogenous inductions method which could improve MT content in maize seeds.The main results of this study were as follows:1. To establish a HPLC- FD(whose full name was Fluorescence Detection) method for determining the content of MT in crop seeds. results were showed that the linearity relationship for MT was perfect within 0.01~1.20 mg/kg. The regression equation was Y=643436X-2446.1 and R2=0.9998, the average recovery rate was 94.11%. determination terms were required in this method: Alltima C18 , 4.6mm×250mm was as chromatographic column. The mobile phase was constituted by 50mM Na2HPO4 /H 3PO4 buffer(PH4.5):methanol=50:50, at a flow rate of 1.0ml/min,10μL extracted samples were injected for the analyses. The excitation wavelength was 280nm and the emission wavelength was 348 nm.Studied out the method which isolated and extracted effectively MT from maize seeds, that is, seeds was crushed by 800W or so supersonic power and 30-60min supersonic time. This method could effectively make MT release from seed cells and dissolve into methanol solvent.2. To determine MT level in seeds of 132 maize varieties, in unpolished rice of 145 rice varieties and in seeds of 35 oat varieties. The results were: mean content of MT in maize varieties was 96.5ng/g, range was 0～2034ng/g, variety with highest content of MT (2034ng/g) was YM056, which was 21.08 times of mean MT content in maize; mean content of MT in oat varieties was 98.7ng/g, range was 0～568ng/g, variety with highest content of MT (568ng/g) was OAT07, which was 5.75 times of mean MT content in oat; mean content of MT in rice varieties was 16.0ng/g, range was 0～264ng/g, variety with highest content of MT (264ng/g) was SD001, which was 16.5 times of mean MT content in rice. Results above showed that content of MT differed greatly among various crop species and varieties.3. To analyse MT content of seed in 10 kinds of minor cereals and legume crops such as millet(Setaria italica), buckwheat(Fagopyrum esculentum), soybean(Glycine mdx Merrill), black bean(Glycine max var), lotus seed(Nelumro nucifera), etc. Results: MT content was 302ng/g in black bean(Glycine max var), was 67 ng/g in lotus seed(Nelumro nucifera) and was 25ng/g, in millet(Setaria italica). MT content in the other 7 crops were all lower than 20 ng/g.4. There was a correlation between different seed coat colors of unpolished rice and MT content of them, that is, MT content in red (or black) unpolished rice was significantly higher than that in white unpolished rice. 22 red (or black) unpolished rice were analyzed, MT was present in 11 of them (50%), mean content of MT was 134.18ng/g; 123 white unpolished rice were analyzed, MT was present in 14 of them (11.34%), mean content of MT was 60.50ng/g.5. To select 3 maize varieties with high MT content and steadily in 3 years repeat: YM115(906.0 ng/g), YM046(792.0ng/g )and YM-N01(644.0ng/g), and 8 rice varieties with high MT content and steadily in 3 seasons planting repeat: SD001 (249.67ng/g),SD075(249.33ng/g),SD103(190.33ng/g),SD093(197.00ng/g),SD025 ( 189.33ng/g ),SD067 ( 170.00ng/g ),SD125 ( 157.00ng/g ),SD119(142.67ng/g).6. Selecting 15 rice varieties with high MT content, which were planted in early season and late season of year 2006 respectively. Results were that, mean MT content in unpolished rice planted in early and late season were 132.3ng/g and 144.2ng/g accordingly, and mean content of MT in unpolished rice planted in late season was significantly higher than that in early season , by significance comparison.7. Maize was sprayed by exogenous inductions, during seeding stage (leaves 6, core 1), and leaves of the maize was tooken to test the MT content after 5 days induction, results indicates that, as for 6 varieties of maize, with inducing formula No.3(inducer ID01, concentration 150mg/kg) and inducing formula No.4(inducer ID01, concentration 300mg/kg), effects of both were fine, MT content in maize leaves were improved averagely 50.59% and 69.05% respectively. And other induce treatments only had significant effects on given varieties. For example, inducing formula No.1(inducer ID01, concentration 5mg/kg) and inducing formula No.2(inducer ID01, concentration 50mg/kg) had significant effects on maize varieties YM148,YM115 and YM139; inducing formula No.5(inducer ID02, concentration 166.7mg/kg) could improve MT content in maize variety YM056 23.08%; inducing formula No.6(inducer ID01, concentration 1667mg/kg) and inducing formula No.7(inducer ID02, concentration 10000mg/kg) had significant effects on maize varieties YM056,YM049 and YM115, however, had non-significant effect on other varieties.Maize was sprayed by exogenous inductions, during seeding stage, MT content in maize seeds of maturity was all non-significantly differed from CK.8. maize during filling stage was Induced by exogenous inductions, and to determine MT contents in maize seeds, maize bracts and leaves in maturity stage (15d after inducing treatment), the results showed as follows:(1) Impacts on MT content in maize seeds by exogenous inductions: inducing formula No.7(inducer ID02 and concentration 10000mg/kg), inducing formula No.8(inducer ID02 and concentration 25000mg/kg), inducing formula No.6(inducer ID02 and concentration 1667mg/kg) could improve mean contents of MT in 4 maize varieties seeds to 15.43%,10.21% and 9.86% respectively;various maize varieties had different inducing effects, maize variety YM139 had significant inducing treatment effects with formula No.10, No.11, No.9, No.4, No.3, No.7, No.6, improved 25.20%, 24.28%, 22.06%, 16.48%, 13.01%, 12.29%, 10.74% accordingly;after inducing treatment with formula No.11, No.10, No.7, MT content in variety YM115 improved 12.44%, 11.77%, 11.08% accordingly;after inducing treatment with formula No.7, No.6, MT content in variety YM056 improved 13.57% and 12.05%, accordingly;after inducing treatment with formula No.7 and No.6, MT content in variety YM049 improved 20.27% and 10.75% respectively;(2) Impact on MT content in maize bracts by exogenous inductions: inducing formula No.3 (inducer ID01 and concentration 150 mg/kg), inducing formula No.4 (inducer ID01 and concentration 300 mg/kg) could increase mean content of MT in maize bracts 15.44% and 10.70% respectively. Of which, formula No.3 inducing treatment to maize varieties YM115, YM139, YM056 and YM049 could improve MT content in maize bracts 19.89%, 17.73%, 17.11% and 11.28%; inducing treatment to maize varieties YM115 and YM139 with inducing formula No.2 (inducer ID01 and concentration 50mg/kg) could improve MT content in maize bracts 15.94% and 13.01% accordingly; inducing treatment to maize variety YM056 with inducing formula No.4 could improve MT content in maize bracts 16.08%; inducing treatment to maize variety YM139 with inducing formula No.8 could improve MT content in maize bracts 10.33%;(3) Impact on MT content in maize leaves by exogenous inductions: all of 3 inducing treatments with inducing formula No.4, No.3, No.2 could improve MT content in maize leaves, mean contents of MT was improved 18.71%, 18.48% and 9.63% respectively.9. MT contents in different organs of maize were greatly different, MT content was, orderly, seeds>bracts>leaves>roots. Of which, MT content in seeds was higher greatly than that in bracts and leaves, there was no significant difference between bracts and leaves, MT content in roots was too low to be detected with HPLC.