不同类型作物种子对系统性荧光化合物吸收的特性及防伪研究
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摘要
种子质量对农业生产起着至关重要的作用,但市场上假冒种子不断出现,给农业生产的优质高产带来了严重的危害,同时也损害了种子产业和农民的利益。目前,种子防伪技术多集中在外包装防伪等易被复制和淘汰的技术上,单纯针对种子本身且比较稳定的防伪手段目前国内外鲜有报道。采用荧光化合物种子标记防伪至今国内外研究甚少。因此,本文利用种子常用处理方法,对不同类型种子进行荧光化合物标记,通过幼苗生理变化和荧光表现,筛选出适宜的荧光标记化合物；针对生产上种子普遍进行包衣的特点,探讨了荧光防伪在种子包衣技术上的应用生理效应和动力学特性,并对标记后包衣种子进行贮藏,探讨贮藏时间与荧光标记物有效性之间的关系;同时研究了荧光物质在土壤中的吸附和降解行为,为种子荧光防伪的田间实际应用提供理论依据。本研究对不同类型作物种子提出了有效的防伪理论和技术体系,研究结果填补了国内外主要农作物种子荧光防伪研究的空白。也为其他林木、牧草种子的防伪提供理论借鉴。得到的主要结果如下：
1、筛选了荧光标记方法和适用的荧光标记物。选取不同种类的荧光化合物采用浸种、引发方法对不同类型作物种子进行荧光标记,结果表明,荧光化合物浸种处理后的荧光表现显著高于引发处理后的效果,且筛选出的荧光化合物在0.1mg/ml浓度浸种处理时对种子活力和幼苗生长均无不良影响,荧光表现明显。不同类型作物种子适宜采用的荧光化合物分别为：油菜为罗丹明B(RB)、丽丝胺罗丹明B(LRB)臧红T(ST)；花椰菜为罗丹明B(RB)、丽丝胺罗丹明B (LRB)葱为罗丹明B(RB)、丽丝胺罗丹明B (LRB)臧红T(ST)；洋葱为罗丹明B(RB)、丽丝胺罗丹明B (LRB)臧红T(ST)：水稻为罗丹明B(RB)、硫酸二氨基吖啶黄(DH)、臧红T(ST)；玉米为罗丹明B(RB)、硫酸二氨基吖啶黄(DH)、臧红T(ST);豌豆为罗丹明B(RB)、丽丝胺罗丹明B(LRB)臧红T(ST)；蚕豆为罗丹明B(RB)、丽丝胺罗丹明B(LRB)臧红T(ST)。
2、确定了荧光标记物进入种子的途径。荧光标记物进入种子的途径因种子类型的不同而存在差异,其中受种子结构的影响较大。荧光标记物主要通过发芽口进入油菜和花椰菜种子,豌豆和玉米主要是通过种皮渗透的方式进入,蚕豆种子主要是通过种脐和发芽口进入,水稻种子可通过稃壳、稃尖和护颖渗透进入。洋葱和葱种子呈棱形,种皮坚硬,荧光标记物难以进入种子内部：荧光标记物在种子及幼苗中的荧光表现因种皮颜色和种子类型不同而存在差异。豌豆、蚕豆、玉米、水稻果种皮颜色较浅,荧光表现显著。油菜、花椰菜、葱和洋葱种皮颜色较深,荧光表现相对较弱;一些双子叶作物种子如油菜、花椰菜、豌豆和蚕豆幼苗的根、茎、叶均可观察到明显的荧光,一些单子叶百合科植物如葱和洋葱在幼苗根,茎可观察到荧光,某些单子叶禾本科植物如水稻、玉米等不易在茎和叶片观察到荧光。
3、研究了荧光标记防伪在包衣种子上的可行性。采用种子包膜技术,将RB分别标记在不同类型作物种子上,通过种子萌发和幼苗生长期间的生理变化及荧光表现,筛选与不同类型作物种子包膜处理相适宜的荧光标记剂量。结果表明,水稻、玉米、葱,洋葱种子经RB药种比1：10包膜处理后,幼苗干重和苗高均受到一定程度的伤害,药种比1：20和1：30包膜处理后,种子萌发和幼苗生长均未受到不良影响。油菜、花椰菜、蚕豆种子经RB药种比1：10、1：20和1：30药种比包膜处理后,种子萌发和幼苗生长均未受到伤害。豌豆种子经RB药种比1：10和1：20包膜处理后,幼苗干重、苗高、POD、SOD活性显著降低,1：30药种比包膜处理对种子萌发和幼苗生长等均无不良影响；RB标记后的包衣种子及幼苗的荧光表现与浸种处理结果相一致,表明RB荧光表现未受包衣材料显著影响,为包衣种子荧光标记防伪提供了理论依据。
4、探讨了不同贮藏时间与荧光效果的关系。每隔一个月对不同条件下贮藏的荧光标记包衣种子进行RB含量测定和荧光观察,结果表明：1：10和1：20药种比处理后的玉米和豌豆种子,贮藏5个月后的RB含量显著低于贮藏1个月的RB含量,1：30药种比处理后种子上的RB含量在贮藏期间无显著差异。水稻和葱种子在1：10、1：20、1：30药种比处理后的RB含量在贮藏期内均无显著差异。蚕豆种子1：10包膜贮藏6个月后,与贮藏1个月种子RB含量存在显著差异,1：20和1：30包膜处理的种子RB含量在贮藏期间无显著差异。花椰菜种子1：30包膜贮藏6个月后,与贮藏1个月种子RB含量存在显著差异,1：10和1：20包膜处理的种子RB含量在贮藏期间无显著差异。洋葱和油菜种子在1：10、1：20、1：30药种比处理后贮藏6个月后的RB含量均与其贮藏1个月含量存在显著差异；相同种子同等药种比处理时,每个月在常温和15℃条件下贮藏后的荧光量均无显著差异,说明经荧光标记物包膜处理的种子未受温度显著影响,为荧光标记物种子防伪研究的实际应用提供了理论指导；贮藏期间,不同药种比处理的种子荧光表现均比对照明显,相同药种比处理的种子荧光表现随着贮藏时间的延长,并没有显著的区别。豌豆、玉米、水稻、蚕豆等种皮颜色较浅的种子荧光表现最为明显；油菜和花椰菜种皮颜色呈深褐色,荧光表现也较为明显；葱和洋葱种子为棱形,种皮呈黑色,坚硬,荧光表现最弱。
5、研究了荧光标记包衣种子贮藏后在田间土壤中的生理特性和荧光表现。经荧光标记物包膜处理后的不同类型作物种子在土壤中种植时,不同药种比处理条件下,种子萌发和幼苗生长情况有所不同。结果表明,油菜种子在1：20和1：30药种比处理后,幼苗根长和苗高显著增加,花椰菜种子在1：20药种比处理后幼苗根长、苗高和叶绿素含量显著提高,蚕豆种子在1：30药种比处理后,幼苗干重、根长和苗高均显著高于裸种。豌豆种子在1：30处理后种子发芽率和发芽势显著高于裸种。葱和洋葱种子经荧光标记物1：10药种比处理后,幼苗苗高显著低于裸种。水稻种子在荧光标记物1：10药种比处理后,幼苗根长和苗高显著低于裸种,1：20药种比处理后,幼苗苗高显著提高。玉米种子经荧光标记物1：10药种比处理后,叶绿素含量和POD活性显著低于裸种,1：30药种比处理后,幼苗苗高显著提高。油菜种子萌发第7天可在幼苗根部、叶片、茎部纵切面维管束组织观察到明显的荧光。花椰菜种子萌发第10天在根部、叶片、叶脉、茎部纵切面维管束组织可观察到明亮的荧光。葱和洋葱分别从荧光标记至种子萌发第8天和第10天时根部和茎部维管束组织均可观察到明显荧光。水稻仅在种子上荧光表现强烈,茎部和叶片均无荧光表现,种子上的荧光表现持续到第16天时与对照无差异。玉米仅在种子和根部荧光表现强烈,持续到第14天时,荧光表现减弱至对照水平,且叶片和茎部均无荧光表现。蚕豆和豌豆种子、茎部维管束组织、根部、叶片、叶脉处均可观察到明显的荧光,但幼苗分别发育至第16天和15天时,各部位荧光表现与对照相比无明显差异。
6、探讨了罗丹明B在田间土壤中的吸附和降解。田间土壤能有效的吸附荧光化合物RB,吸附遵循Langmuir等温吸附方程,饱和吸附量是1.716mg/g,吸附平衡常数为0.042L/mg。吸附的罗丹明B可有效的进行光-Fenton降解反应(通过光氧化与催化技术)。土壤中存在与光-Fenton降解反应类似的条件,因此,吸附在土壤中的的罗丹明B能够通过光氧化与催化技术进行有效降解。同时,种子包衣上使用的罗丹明B量极少,所以罗丹明B的使用不会对土壤带来污染。可作为各种种子的防伪标记使用。
Seed quality plays a crucial role in seed production. Fake and shoddy seeds in market were often found, which brought great harm for high quality and high yield of agricultural production, and damage the benefits of farmers and seed enterprises. Nowadays, the anti-counterfeiting technologies of seed are mainly focus on seed packaging, which are easy to be reproducibility and immitigability. A new seed anti-counterfeiting technology, aiming at seed itself rather than the packing are barely reported at home and broad. Meanwhile, there were few studies on seed anti-counterfeiting by using fluorescent compounds. Therefore, fundamental research on fluorescence anti-counterfeiting methods was carried out in present study. Different types of crop seeds were marked by fluorescent compounds with seed treatment methods, and the suitable fluorescent dyes and methods were selected according the physiological changes and fluorescence of seedling. Film coating seeds with fluorescent compounds were stored for various time, their physiological effects of application and dynamics feature of fluorescence anti-counterfeiting were studied aiming at relationship of storage time and fluorescence effects. Moreover, the absorption and degradation of fluorescent compound in soil were also studied in order to provide theoretical basis for field reality. Thus, this study provides an effective anti-counterfeiting theory and a integrate technical system for different types of crop seeds, and the results fill up the vacant of fluorescent anti-counterfeiting for field crop seeds at home and abroad. Meanwhile, the results also supplied theoretical references for anti-counterfeiting of forest and forage seeds. Achieved main results were as follows:
1. Suitable fluorescent compounds and their labling methods were selected. After treated with soaking and priming methods, the suitable soaking concentration0.1mg/ml for different types crop seeds was selected out. Seed vigor and seedling performance were not affected when seeds soaking in solution of0.1mg/ml Rhodamine B (RB), and the fluorescence of seed and seedling were both stronger than that of others and controls. The optimum fluorescence dyes for different crop seeds were:rape (RB. Lissamine RB. Safranine T); cauliflower (RB. Lissamine RB); chive (RB, Lissamine RB, Safranine T); onion (RB, Lissamine RB, Safranine T); rice (RB.3,6-Diamincacridine Hulmisulfate. Safranine T):maize (RB.3,6-Diamincacridine Hulmisulfate, Safranine T):pea (RB. Lissamine RB, Safranine T); broad bean (RB. Lissamine RB, Safranine T).
2. The paths of fluorescent compound entering seeds were determined. The path of fluorescent compound enters seeds varied in crop seed, which were mainly affected by seed coat. Fluorescent compound entered into seeds of rape and cauliflower through micropyle; fluorescent compound penetrated into pea and maize seeds through seed coat; fluorescent dye entered broad bean seed through hilum and micropyle; fluorescent compound entered rice seed through lemma, apiculus and glume. Fluorescent compound was difficult to enter chive and onion seeds as their seed coats were really hard. In addition, fluorescence was observed obviously in root, stem, leaf and vain in some dicotyledonous such as rape, cauliflower, pea and broad bean.'Fluorescence was also observed obviously in root and stem of some monocotvledon such as chive and onion. Fluorescence was not observed in leaf and stem of some grass such as maize and rice.
3. The feasibility of fluorescence anti-counterfeiting in coated seed was studied. RB was marked in different crop seeds by seed film coating, and the suitable doses of RB were selected according to the physiological changes and fluorescence during seed germination and seedling growth. The results showed that the seedling dry weight and height of rice, maize, chive, and onion seedlings were injured at the ratio of1:10(RB to seeds), and seed germination and seedling growth were not affected with the ratio of1:20and1:30. After seeds of rape, cauliflower and broad bean were treated with ratio of1:10,1:20and1:30. germination and seedling growth were not affected significantly. After treated with ratio of1:10and1:20. seedling dry weight, height. POD and SOD activity of pea decreased significantly, and1:30were no harm for seed germination and seedling growth. The fluorescence of seedling when seeds coated with RB were consistent to the result of seeds soaked with RB. indicating that the fluorescence of RB were not affected significantly by the coating materials, so,the results provide theoretical basis for fluorescence anti-counterfeiting of coated seeds.
4. The relationship of storage time and the fluorescence of RB were researched. The content and fluorescence of RB marked on coated seeds were measured each mouth during six-month storage for studying the effect of RB fluorescence. The results showed that, for seeds of maize and pea coated with1:10and1:20. the RB content of seeds stored for5months was lower significantly than that of stored for1month, and the RB content from pea seeds coated with1:30had no differences among different storage times. The RB content of rice and chive seeds coated with1:10,1:20and1:30had no significant differences among different storage times. For broad bean seeds coated with1:10. the RB content of6month storage was lower significantly than that of stored for1month, and the RB content of seeds coated with1:20and1:30had no differences among different storage times. For cauliflower seeds coated with1:30, the RB content of seeds stored for6month was lower significantly than that of stored for1month, and the RB content of seeds coated with1:10and1:20had no differences among different storage times. The RB content of rice and chive seeds coated with1:10,1:20and1:30stored for6months were lower significantly than that of stored for1month. Meanwhile, the RB content of the same crop seeds coated with the same ratio had no signifigent difference between normal temperature and15℃, indicating that the content of RB were not affected significantly by the temperature, which supplied theoretical basis for field application of fluorescence anti-counterfeiting. Fluorescence of seeds coated with different ratio was significantly higher than that of the control seeds, and for seeds with the same ratio, the fluorescence had no obvious differences among different storage time. The seed fluorescence of pea. maize, rice and broad bean were stronger because of having light seed coat. For rape, cauliflower, chive and onion, the fluorescence was weak as the seed coat color was depth.
5. Their physiological characteristics and the fluorescence in soil were researched after seeds coated with RB were stored for6months. The results showed that root length and seedling height of rape were higher significantly than those of the control after the seeds coated with1:20and1:30, and the root length, seedling height and chlorophyll content of cauliflower were improved significantly after seeds coated with1:20. After broad bean seeds coated with1:30, the root length, dry weight and height of seedling, germination percentage and germination energy of pea seeds were higher significantly than those of the control. The shoot length of chive and onion was significantly decreased. The root and shoot length of rice were decreased significantly after seed coated with1:10, however, the shoot length was increased significantly after seed coated with1:20. For maize seed coated with1:10. chlorophyll content and POD activity were lower significantly than those of the control, and shoot height increased significantly after seeds coated with1:30. When rape seeds coated with RB germinated for7days, the fluorescence was observed in root, leaf, vain and vascular of stern. When cauliflower seeds coated with RB germinated for10days, the fluorescence was observed in root, leaf, vain and vascular of stem. When chive and onion seeds with RB germinated for8,10days respectively, the fluorescence was observed in root and vascular of stem. When rice seeds coated with RB germinated for16days, the fluorescence was only showed in seeds and not found in stem and leaf. The fluorescence was only showed in seed and root, and not found in stem and leaf when maize seed coated with RB germinated for14days. For pea and broad bean seeds coated with RB, when germinated for16,15days respectively, the fluorescence was observed in root, leaf, vain and vascular of stem.
6. The absorption and degradation of RB in soil were studied. The fluorescent compound of RB could be absorbed by field soil effectively and the absorption process in accordance with the Langmuir isotherm, in which the amount of saturated absorption was1.716mg/g and the adsorption equilibrium constant, was0.042L/mg. Then the adsorptive RB was degraded effectively under light-Fenton condition. There are similar conditions to light-Fenton in soil, therefore, RB absorbed by field soil can be degraded. Meanwhile, dose of RB in coated treatment was less, so the RB could not pollute soil and be used as an anti-counterfeiting marker for different crop seeds.
1、筛选了荧光标记方法和适用的荧光标记物。选取不同种类的荧光化合物采用浸种、引发方法对不同类型作物种子进行荧光标记,结果表明,荧光化合物浸种处理后的荧光表现显著高于引发处理后的效果,且筛选出的荧光化合物在0.1mg/ml浓度浸种处理时对种子活力和幼苗生长均无不良影响,荧光表现明显。不同类型作物种子适宜采用的荧光化合物分别为：油菜为罗丹明B(RB)、丽丝胺罗丹明B(LRB)臧红T(ST)；花椰菜为罗丹明B(RB)、丽丝胺罗丹明B (LRB)葱为罗丹明B(RB)、丽丝胺罗丹明B (LRB)臧红T(ST)；洋葱为罗丹明B(RB)、丽丝胺罗丹明B (LRB)臧红T(ST)：水稻为罗丹明B(RB)、硫酸二氨基吖啶黄(DH)、臧红T(ST)；玉米为罗丹明B(RB)、硫酸二氨基吖啶黄(DH)、臧红T(ST);豌豆为罗丹明B(RB)、丽丝胺罗丹明B(LRB)臧红T(ST)；蚕豆为罗丹明B(RB)、丽丝胺罗丹明B(LRB)臧红T(ST)。
2、确定了荧光标记物进入种子的途径。荧光标记物进入种子的途径因种子类型的不同而存在差异,其中受种子结构的影响较大。荧光标记物主要通过发芽口进入油菜和花椰菜种子,豌豆和玉米主要是通过种皮渗透的方式进入,蚕豆种子主要是通过种脐和发芽口进入,水稻种子可通过稃壳、稃尖和护颖渗透进入。洋葱和葱种子呈棱形,种皮坚硬,荧光标记物难以进入种子内部：荧光标记物在种子及幼苗中的荧光表现因种皮颜色和种子类型不同而存在差异。豌豆、蚕豆、玉米、水稻果种皮颜色较浅,荧光表现显著。油菜、花椰菜、葱和洋葱种皮颜色较深,荧光表现相对较弱;一些双子叶作物种子如油菜、花椰菜、豌豆和蚕豆幼苗的根、茎、叶均可观察到明显的荧光,一些单子叶百合科植物如葱和洋葱在幼苗根,茎可观察到荧光,某些单子叶禾本科植物如水稻、玉米等不易在茎和叶片观察到荧光。
3、研究了荧光标记防伪在包衣种子上的可行性。采用种子包膜技术,将RB分别标记在不同类型作物种子上,通过种子萌发和幼苗生长期间的生理变化及荧光表现,筛选与不同类型作物种子包膜处理相适宜的荧光标记剂量。结果表明,水稻、玉米、葱,洋葱种子经RB药种比1：10包膜处理后,幼苗干重和苗高均受到一定程度的伤害,药种比1：20和1：30包膜处理后,种子萌发和幼苗生长均未受到不良影响。油菜、花椰菜、蚕豆种子经RB药种比1：10、1：20和1：30药种比包膜处理后,种子萌发和幼苗生长均未受到伤害。豌豆种子经RB药种比1：10和1：20包膜处理后,幼苗干重、苗高、POD、SOD活性显著降低,1：30药种比包膜处理对种子萌发和幼苗生长等均无不良影响；RB标记后的包衣种子及幼苗的荧光表现与浸种处理结果相一致,表明RB荧光表现未受包衣材料显著影响,为包衣种子荧光标记防伪提供了理论依据。
4、探讨了不同贮藏时间与荧光效果的关系。每隔一个月对不同条件下贮藏的荧光标记包衣种子进行RB含量测定和荧光观察,结果表明：1：10和1：20药种比处理后的玉米和豌豆种子,贮藏5个月后的RB含量显著低于贮藏1个月的RB含量,1：30药种比处理后种子上的RB含量在贮藏期间无显著差异。水稻和葱种子在1：10、1：20、1：30药种比处理后的RB含量在贮藏期内均无显著差异。蚕豆种子1：10包膜贮藏6个月后,与贮藏1个月种子RB含量存在显著差异,1：20和1：30包膜处理的种子RB含量在贮藏期间无显著差异。花椰菜种子1：30包膜贮藏6个月后,与贮藏1个月种子RB含量存在显著差异,1：10和1：20包膜处理的种子RB含量在贮藏期间无显著差异。洋葱和油菜种子在1：10、1：20、1：30药种比处理后贮藏6个月后的RB含量均与其贮藏1个月含量存在显著差异；相同种子同等药种比处理时,每个月在常温和15℃条件下贮藏后的荧光量均无显著差异,说明经荧光标记物包膜处理的种子未受温度显著影响,为荧光标记物种子防伪研究的实际应用提供了理论指导；贮藏期间,不同药种比处理的种子荧光表现均比对照明显,相同药种比处理的种子荧光表现随着贮藏时间的延长,并没有显著的区别。豌豆、玉米、水稻、蚕豆等种皮颜色较浅的种子荧光表现最为明显；油菜和花椰菜种皮颜色呈深褐色,荧光表现也较为明显；葱和洋葱种子为棱形,种皮呈黑色,坚硬,荧光表现最弱。
5、研究了荧光标记包衣种子贮藏后在田间土壤中的生理特性和荧光表现。经荧光标记物包膜处理后的不同类型作物种子在土壤中种植时,不同药种比处理条件下,种子萌发和幼苗生长情况有所不同。结果表明,油菜种子在1：20和1：30药种比处理后,幼苗根长和苗高显著增加,花椰菜种子在1：20药种比处理后幼苗根长、苗高和叶绿素含量显著提高,蚕豆种子在1：30药种比处理后,幼苗干重、根长和苗高均显著高于裸种。豌豆种子在1：30处理后种子发芽率和发芽势显著高于裸种。葱和洋葱种子经荧光标记物1：10药种比处理后,幼苗苗高显著低于裸种。水稻种子在荧光标记物1：10药种比处理后,幼苗根长和苗高显著低于裸种,1：20药种比处理后,幼苗苗高显著提高。玉米种子经荧光标记物1：10药种比处理后,叶绿素含量和POD活性显著低于裸种,1：30药种比处理后,幼苗苗高显著提高。油菜种子萌发第7天可在幼苗根部、叶片、茎部纵切面维管束组织观察到明显的荧光。花椰菜种子萌发第10天在根部、叶片、叶脉、茎部纵切面维管束组织可观察到明亮的荧光。葱和洋葱分别从荧光标记至种子萌发第8天和第10天时根部和茎部维管束组织均可观察到明显荧光。水稻仅在种子上荧光表现强烈,茎部和叶片均无荧光表现,种子上的荧光表现持续到第16天时与对照无差异。玉米仅在种子和根部荧光表现强烈,持续到第14天时,荧光表现减弱至对照水平,且叶片和茎部均无荧光表现。蚕豆和豌豆种子、茎部维管束组织、根部、叶片、叶脉处均可观察到明显的荧光,但幼苗分别发育至第16天和15天时,各部位荧光表现与对照相比无明显差异。
6、探讨了罗丹明B在田间土壤中的吸附和降解。田间土壤能有效的吸附荧光化合物RB,吸附遵循Langmuir等温吸附方程,饱和吸附量是1.716mg/g,吸附平衡常数为0.042L/mg。吸附的罗丹明B可有效的进行光-Fenton降解反应(通过光氧化与催化技术)。土壤中存在与光-Fenton降解反应类似的条件,因此,吸附在土壤中的的罗丹明B能够通过光氧化与催化技术进行有效降解。同时,种子包衣上使用的罗丹明B量极少,所以罗丹明B的使用不会对土壤带来污染。可作为各种种子的防伪标记使用。
Seed quality plays a crucial role in seed production. Fake and shoddy seeds in market were often found, which brought great harm for high quality and high yield of agricultural production, and damage the benefits of farmers and seed enterprises. Nowadays, the anti-counterfeiting technologies of seed are mainly focus on seed packaging, which are easy to be reproducibility and immitigability. A new seed anti-counterfeiting technology, aiming at seed itself rather than the packing are barely reported at home and broad. Meanwhile, there were few studies on seed anti-counterfeiting by using fluorescent compounds. Therefore, fundamental research on fluorescence anti-counterfeiting methods was carried out in present study. Different types of crop seeds were marked by fluorescent compounds with seed treatment methods, and the suitable fluorescent dyes and methods were selected according the physiological changes and fluorescence of seedling. Film coating seeds with fluorescent compounds were stored for various time, their physiological effects of application and dynamics feature of fluorescence anti-counterfeiting were studied aiming at relationship of storage time and fluorescence effects. Moreover, the absorption and degradation of fluorescent compound in soil were also studied in order to provide theoretical basis for field reality. Thus, this study provides an effective anti-counterfeiting theory and a integrate technical system for different types of crop seeds, and the results fill up the vacant of fluorescent anti-counterfeiting for field crop seeds at home and abroad. Meanwhile, the results also supplied theoretical references for anti-counterfeiting of forest and forage seeds. Achieved main results were as follows:
1. Suitable fluorescent compounds and their labling methods were selected. After treated with soaking and priming methods, the suitable soaking concentration0.1mg/ml for different types crop seeds was selected out. Seed vigor and seedling performance were not affected when seeds soaking in solution of0.1mg/ml Rhodamine B (RB), and the fluorescence of seed and seedling were both stronger than that of others and controls. The optimum fluorescence dyes for different crop seeds were:rape (RB. Lissamine RB. Safranine T); cauliflower (RB. Lissamine RB); chive (RB, Lissamine RB, Safranine T); onion (RB, Lissamine RB, Safranine T); rice (RB.3,6-Diamincacridine Hulmisulfate. Safranine T):maize (RB.3,6-Diamincacridine Hulmisulfate, Safranine T):pea (RB. Lissamine RB, Safranine T); broad bean (RB. Lissamine RB, Safranine T).
2. The paths of fluorescent compound entering seeds were determined. The path of fluorescent compound enters seeds varied in crop seed, which were mainly affected by seed coat. Fluorescent compound entered into seeds of rape and cauliflower through micropyle; fluorescent compound penetrated into pea and maize seeds through seed coat; fluorescent dye entered broad bean seed through hilum and micropyle; fluorescent compound entered rice seed through lemma, apiculus and glume. Fluorescent compound was difficult to enter chive and onion seeds as their seed coats were really hard. In addition, fluorescence was observed obviously in root, stem, leaf and vain in some dicotyledonous such as rape, cauliflower, pea and broad bean.'Fluorescence was also observed obviously in root and stem of some monocotvledon such as chive and onion. Fluorescence was not observed in leaf and stem of some grass such as maize and rice.
3. The feasibility of fluorescence anti-counterfeiting in coated seed was studied. RB was marked in different crop seeds by seed film coating, and the suitable doses of RB were selected according to the physiological changes and fluorescence during seed germination and seedling growth. The results showed that the seedling dry weight and height of rice, maize, chive, and onion seedlings were injured at the ratio of1:10(RB to seeds), and seed germination and seedling growth were not affected with the ratio of1:20and1:30. After seeds of rape, cauliflower and broad bean were treated with ratio of1:10,1:20and1:30. germination and seedling growth were not affected significantly. After treated with ratio of1:10and1:20. seedling dry weight, height. POD and SOD activity of pea decreased significantly, and1:30were no harm for seed germination and seedling growth. The fluorescence of seedling when seeds coated with RB were consistent to the result of seeds soaked with RB. indicating that the fluorescence of RB were not affected significantly by the coating materials, so,the results provide theoretical basis for fluorescence anti-counterfeiting of coated seeds.
4. The relationship of storage time and the fluorescence of RB were researched. The content and fluorescence of RB marked on coated seeds were measured each mouth during six-month storage for studying the effect of RB fluorescence. The results showed that, for seeds of maize and pea coated with1:10and1:20. the RB content of seeds stored for5months was lower significantly than that of stored for1month, and the RB content from pea seeds coated with1:30had no differences among different storage times. The RB content of rice and chive seeds coated with1:10,1:20and1:30had no significant differences among different storage times. For broad bean seeds coated with1:10. the RB content of6month storage was lower significantly than that of stored for1month, and the RB content of seeds coated with1:20and1:30had no differences among different storage times. For cauliflower seeds coated with1:30, the RB content of seeds stored for6month was lower significantly than that of stored for1month, and the RB content of seeds coated with1:10and1:20had no differences among different storage times. The RB content of rice and chive seeds coated with1:10,1:20and1:30stored for6months were lower significantly than that of stored for1month. Meanwhile, the RB content of the same crop seeds coated with the same ratio had no signifigent difference between normal temperature and15℃, indicating that the content of RB were not affected significantly by the temperature, which supplied theoretical basis for field application of fluorescence anti-counterfeiting. Fluorescence of seeds coated with different ratio was significantly higher than that of the control seeds, and for seeds with the same ratio, the fluorescence had no obvious differences among different storage time. The seed fluorescence of pea. maize, rice and broad bean were stronger because of having light seed coat. For rape, cauliflower, chive and onion, the fluorescence was weak as the seed coat color was depth.
5. Their physiological characteristics and the fluorescence in soil were researched after seeds coated with RB were stored for6months. The results showed that root length and seedling height of rape were higher significantly than those of the control after the seeds coated with1:20and1:30, and the root length, seedling height and chlorophyll content of cauliflower were improved significantly after seeds coated with1:20. After broad bean seeds coated with1:30, the root length, dry weight and height of seedling, germination percentage and germination energy of pea seeds were higher significantly than those of the control. The shoot length of chive and onion was significantly decreased. The root and shoot length of rice were decreased significantly after seed coated with1:10, however, the shoot length was increased significantly after seed coated with1:20. For maize seed coated with1:10. chlorophyll content and POD activity were lower significantly than those of the control, and shoot height increased significantly after seeds coated with1:30. When rape seeds coated with RB germinated for7days, the fluorescence was observed in root, leaf, vain and vascular of stern. When cauliflower seeds coated with RB germinated for10days, the fluorescence was observed in root, leaf, vain and vascular of stem. When chive and onion seeds with RB germinated for8,10days respectively, the fluorescence was observed in root and vascular of stem. When rice seeds coated with RB germinated for16days, the fluorescence was only showed in seeds and not found in stem and leaf. The fluorescence was only showed in seed and root, and not found in stem and leaf when maize seed coated with RB germinated for14days. For pea and broad bean seeds coated with RB, when germinated for16,15days respectively, the fluorescence was observed in root, leaf, vain and vascular of stem.
6. The absorption and degradation of RB in soil were studied. The fluorescent compound of RB could be absorbed by field soil effectively and the absorption process in accordance with the Langmuir isotherm, in which the amount of saturated absorption was1.716mg/g and the adsorption equilibrium constant, was0.042L/mg. Then the adsorptive RB was degraded effectively under light-Fenton condition. There are similar conditions to light-Fenton in soil, therefore, RB absorbed by field soil can be degraded. Meanwhile, dose of RB in coated treatment was less, so the RB could not pollute soil and be used as an anti-counterfeiting marker for different crop seeds.
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