AU2007308582A1 - A plant growth regulator containing hemin - Google Patents

Description

A plant growth regulator containing hem in Technical Field The present invention relates to a plant growth regulator containing hemin, whi h belongs to the field of development and utilization of plant growth regulator, and is especially applicable for development of agricultural chemicals, chemical regulation and control of farmland, provenance agriculture, and tissue culture, storage of fruits a d vegetables, and preservation of cut flowers. Technical Background The plant growth and development are usually controlled and regulated by endogenc s plant hormone, and simultaneously affected by various adverse environmen al conditions (including numerous biotic/abiotic stress), which often cause reduction of growth rate and yield, wherein, the most closely related to human life is tie deterioration of agronomic traits , and one of the most important causes is tie metabolism deterioration of plant tissues and parts and oxidation resistance reducti n induced thereby. It is proved that yield increase can be achieved by improvi g agricultural environment, selecting crop strains with good adversity tolerance a d resistance, and developing transgenic crops, but those methods often have high co t, long development lifespan, and even bring other side effects, such as risk evaluation a d potential safety issue of transgenic crops. On the other hand, although quite a f w patents can be applied on improvement of plant adversity tolerance and resistance, a d of various agronomic traits, those patents usually obtain the effect by compoundii g several kinds of macro/micro elements and/or plant hormones or synthetic derivatives in proportion. Due to complicated components, the working mechanism is not clear, tie application method is not easy to master by producer, besides, some plant hormones are expensive and have certain side effects during application; therefore the applicati n effect is usually not stable. It is already known that when plants suffer stress from various adverse environmen s, their endogenetic signal molecules' contents and distribution in vivo, such as ABA aid 1-1202, will generate alteration trend, which shows that ABA and 1202, as important signal factors, can participate cross tolerance and adaption of various environmen ial factors; at the same time, if one of the signal factors (ABA and H202, etc) is adopted o induce stress to the plant, the plant can resist subsequent the same kinds or differ t kinds of stress. Recent research has already found that NO (nitric oxide) as plant gro th regulation/signal molecule may widely participate various physiological processes, and particularly plays an important regulation function on plant growth/development a d relates to the response of the adverse environment, moreover, relates to plat growth/development process and NO treatment concentration. For example, k1w concentration exogenous NO (within i4 M concentration range) can significant y improve saline tolerance of the plants, and is related to improvement of oxidation resistance of tissues and regulation of plant ion balance, and messengers like Ca 2 + and 11202 molecules participate the regulation as well. But high concentration NO treatment it has significant undermined effect (Ruan, etc., Chin Sci Bull, 2002; Uchida, etc., P t Sci, 2002; Zhao, etc., Plant Physioi, 2004). Additionally, plants' NO function is also related to interaction with ABA and other hormones/plant growth regulation substance s (Beligni and Lamattina, Planta, 2000; Delledonne, Curr Opin Plant Biol, 2005). n November 1999, Argentinean scientist Lamattina, etc. applied American patent related to plants' NO function development and utilization (Method of enhancing the metabol c function and the growing conditions of plants and seeds, Patent Application Number: 450192) and was granted the patent right in June 2001 (US Patent No.: 6242384). On the other hand, carbon monoxide (CO), which is also two-atom gaseous molecu e same as NO, has always been considered as toxic gas. According to its chemic 1 properties, pure CO is colorless, odorless, and non-irritating gas, with molecular weigh it 28.01, density 0,967g/L, freezing point -207'C, boiling point -190'C, low solubility n water, good solubility in ammonia water, and explosion limit in air mixture is 13-740. Usually CO is generated when carbon-containing substances are not complete y combusted, for example, there are more than 70 types of industrial production n operations in which CO is involved, such as coking, iron smelting, forging, casting ar d heat treatment in metallurgical industry, ammonia synthesis, and the production of acetone, phosgene, and methanol in chemical industry, blasting and gas explosion accident in mining industry, carbon graphite electrode production and production of metal carbonylate, such as nickel carbonylate or iron carbonylate, or production or application involving CO-containing combustible gas (such as water gas containing C D up to 40%, gas in blast furnace and generator containing CO 30% , and coal g s containing CO 5-15% ), all can contact or generate CO gas, and waste gas of intern I combustion engine using diesel or gasoline also contains CO about 1-8%. Additionally, hemin (C 34

H

32 C1FeN 4 0 4 , molecular weight 651.94), Hematin (C3 4 H33N 4 OsFc, 2 molecular weight 633.49), and CORM-3 are commonly used as artificial donors capat le of releasing CO gas, and manufactured CO gas can also be generated by coheati g reaction of concentrated sulfuric acid and formic acid. H2Cs CH3 H H3C/ -N N \ ClFe N N H3C \ // CH3 HO O HO O Chemical structure of Hemin It is already known that CO is the most frequent toxic substance causing domestic accidental death in many countries. Occurrence of acute CO intoxication is related 0 CO contact concentration and duration. It is learned that the reason why CO will cau e gas intoxication is that the inhalation of CO into human lung combines with hemoglob n in blood. Due to the existence of carboxy hemoglobin, oxygen carriage function f blood in humans will malfunction, which causes acute oxygen deficiency of human body. When CO concentration is high, CO can combine with iron in cytochro e oxidase, and thus inhibit inhalation process of the histiocytes and hamper its oxyg n utilization. As central nervous system is the most sensitive part to oxygen deficiency, different intoxication symptoms can be generated according to CO inhalation amour t. For example, it may be lethal when CO concentration in air is reached to 0.4%. A large amount of recent research work has proved that CO is also an importa it intracellular messenger in animals, which belongs to the scope of reactive carbon species (RCS), participates in regulation of various physiological and pathologic I processes in animals, and has a function similar to that of NO in animals (Verma, etq, Science, 1993). In animals, heme oxygenase (HO) is the initiation enzyme and rate-limiting enzyme which catalyzes and degrades heme into CO, Fe, and bilirubi 3 which has important biological function. And intracellular messenger function of t e CO has already attracted general attention. For example, CORM-3 (soluble liquid O donor) can promote growth of the cardiac cells and thus effectively treat heart attack of patients; CO gas released by CORM-3 during heart attack period can effectively prey nt the cardiac cells form compression and oxygen deficiency; at the same time, COR1 3 also can enhance physiological function of the transplanted organs, and reduce t e rejection of the body mechanism to the transplanted organs. Therefore, Prof Bri n Mann from University of Sheffield, UK said that it can be observed from experiment results that CO molecules have tremendous potential in medical application, a d physiological activities of CO will be applied to wider field. More interestingly, CO has the similar functions of maintaining blood vessel tension and protection cardiac mus le cell to those of NO in cardiovascular system. Usually, CO and NO both can aler conformation of soluble guanylate cyclase (sGC) by combining the Fe 2 " of active cen r of sGC, which lead to enhancement of enzyme activity, and thus increase cG P concentration in cell and exert function of relaxing endotheliocyte. Moreov r, generation and regulation systems of CO and NO are also very similar. For examp e, nitric oxide synthase (NOS), an important synthetase of HO and NO, has two forms, i e. constitutive type and inducible type; HO is also subdivided into HO-1 (iHO) and HO-2/3 (constitutive HO, cHO), and HO also has response effect to some stimulation factors of iNOS, such as endotoxin, cytokine, and active oxygen. Additionally, produ ts of NOS and HO systems all have very high affinity to heme molecules, and ha e complementary relationship on regulation function of cGMP concentration in cells. n the whole, study in animals shows that CO and NO have many similarities in aspects >f molecular structure, physiological function, signal transmission route, and syntheta e system. Correspondingly, study in plants also has preliminarily found that HO has capability >f synthesizing CO gas in vivo (Muramoto, etc., Plant Physiol, 2002). Additionally, CD generation phenomena (usually < 1,000ppm) often accompanies plant se d germination and seedling growth processes, in which the highest CO generation concentration .is 6 ,000ppm, and is preliminarily speculated to be related :o photorespiration, photosynthesis, and respiratory metabolism (Wilks, Science, 195); Siegel, etc., Science, 1962). This discovery shows that CO also might be nature I byproducts of the plant metabolism, and also provides practical basis and idea f r identification and utilization of control and regulation functions of CO in plant grow h 4 and development. Disclosure of the Invention Problem to be solved The object of the present invention is to overcome the disadvantages in availat le techniques which usually have unstable or certain harmful effects and high cost due o the adoption of various macro/micro-elements and plant hormones/growth regulati n substances (including NO) to improve plant stress tolerance and resistance and vario s agronomic traits; and increase the concentration of plant endogenous CO by and by ia hemin (also HO-1 activity inducer), and thus to provide a method for promoting gro h and evocation of the plants, enhance various metabolic capabilities, improve vario s agronomic traits like yield and quality, strengthen plant stress tolerance/resistance, a id improve oxidation resistance as well. Technical Scheme A plant growth regulator containing hemin, wherein the plant growth regulator contai s hemin, and effective final concentration of hemin is 0.001-1,000 11 M after the regulat r is prepared into solution. The plant growth regulator further contains Ca 2 " with fi 1 concentration of 0-1,000 P M, salicylic acid with final concentration of 0-1,000 P M, r other agricultural chemicals with final concentration of 0-1,000 1 M after prepared in to solution. The plant growth regulator can induce plant to release CO gas with a total concentration no more than 1,000ppm in vivo. The application of the said plant grow h regulator containing hemin is characterized by irrigating, spraying, immersing >r soaking plants, and plant seeds or tissues with the plant growth regulator containing hemin, or directly adding the regulator into culture medium containing the said pla t materials, to improve various agronomic traits or metabolic functions and biotic >r abiotic stress tolerance or resistance, then continuing the treatment for 1-60 days, 1 3 times per day. The said plants, and plant tissues and seeds include monocotyledons, dicotyledons, >r gymnosperms, and plants, and plant tissues and seeds of genetically modified organism is (GMO) cultivated in farmland and green house. The various abiotic stress such as, salt damage, osmotic stress, drought at d waterlogging stress, cold damage, heat shock, UV radiation, ozone, trauma, metal (iro 1 copper, lead, manganese, aluminum, and mercury, etc.) toxication, and excessive r 5 error dose of using weed killer (including weed killers like Paraquat and Diquat, e c. which induce excessive generation of active oxygen), and the biotic stress inclu e various pests and pathogenic bacteria infection. The said various agronomic traits, metabolic functions, and biotic/abiotic stress tolerance/resistance refer to that during various development and production periods :>f seeding, germination, root induction, growth, transplanting, stem cutting, flowering, fruit formation or ripening, and tissue culture and under various natural or artific ial biotic and abiotic stress, alpha/beta amylase activity is induced to alleviate vario s stress on inhibition of seed germination, improve seed germination rate under normal conditions, break hibernation, delay aging process, prolong shelf life, induce lateral ro t and adventitious root, improve various antioxidase gene expression and synthes s, increase enzyme activities, decrease running rate of various active oxygen generation systems, delay generation of lipid peroxide products, induce stoma closure of pla t blades, keep greenness, increase dry/fresh weight and elongation/growth rate f overground and underground organs, increase crop yield and improve quality, enhan e plant resistances to disease, pest, and heavy metals, and induce synthesis of vario s protective substances like proline, flavones, and phytoalexin. The method for regulating plant growth with hemin is characterized by: (1) solution preparation of the plant growth regulator containing hemin: the solution contains hemin with final effective concentration of 0.00 1-1,000 9 M, and also contains Ca2+ with final concentration of 0-1,000 9 M, salicylic acid with final concentration of 0-1,000 P M, or other agricultural chemical with final concentration of 0-1,000 M; (2) irrigating, spraying, immersing or soaking plants, and plant seeds or organs with the solution of the plant growth regulator containing hemin, or directly adding tl e solution into culture medium containing the said plant materials, to improve various agronomic traits or metabolic functions and biotic or abiotic stress tolerance >r resistance, and continuing the treatment for 1-60 days, 1-3 times per day. Benefits of the invention Although it has been reported that low concentration CO gas and Hematin can impro e plant seed germination (Dekker and Hargrove, Am J Bot, 2002; Xu, etc., J Integr Pla t Biol, 2006), adverse side effects to human can be easily induced due to direct use of C 6 gas; at the same time, compared with Hemin with Cl, Hematin with high iron count nt has unstable chemical properties and high cost. The difference is in that the present invention adopts the plant growth regulator containing henin which can directly indu e low concentrated CO gas release in vivo, as hemin with stable chemical properties c n directly induce activity of HO-1I (belonging to iHO), CO gas can be generated by and by and directly absorbed by plants, and plant organs and seeds, so as to effectively preve t direct harm of CO to humans; while CO generated in plants, and plant organs and see Is in vivo can further induce the synthesis of NO, so as to coordinately improve vario is agronomic traits or metabolic functions and biotic or abiotic stress tolerance and resistance. Study in plants has already proved that high concentration CO gas (> 6 ,000ppn, equivalent to CO gas >0.6% (V/V)) can significantly inhibit plant growth ar d development. The present invention is a method using a plant growth regulator containing hemin capable of releasing low concentrated CO gas for improvir g agronomic traits of plants, and plant organs and seeds and metabolic functions, including promoting growth and evocation of plants, accelerating seed germination, improving oxidation resistance, and strengthening plant stress tolerance/resistance. The present invention further proves that hemin, as CO donor/HO-I activity inducer, s similar to NO, and can also improve plant stress tolerance/resistance and agronomic traits, and enhance various metabolic functions, its action mechanism relates to using CO as signal/reduction molecule to improve plant stress tolerance/resistance and metabolic functions, or utilizing interaction with NO, H20 2 signal molecules and ABA, JAA and GA plant honnones to achieve regulation functions including generating lateral roots and adventitious root under normal or stress conditions, and inducing ro t under tissue culturing conditions, alleviating inhibition action of various stress to seed germination, improving germination rate under normal conditions, breaking seed hibernation, inducing stoma closure of plant leaf, keeping greenness, increasin . dry/fresh weight and elongation/growth rate of overground and underground organ, enhancing plant resistances to diseases and pest, delaying aging, and enhancing oxidation resistance. As CO is also endogenetic byproduct of plant physiological metabolism, the above research result shows that CO or its related donor also might b novel plant growth regulation substance, and can exert effective regulation function fo crop under stress or normal growth conditions. 7 As NO is a free radical itself, it is likely to cause oxidation damage during applicati n process; while CO is not a free radical, it is unlikely to induce oxidation damage. Additionally, the concentration of the released CO induced via hemin in the pres nt invention is far less than human sensitive concentration (for example, lethal dosage f CO to human is at least 4,000ppm, while the concentration in the present invention is o more than 1,000ppm), and hemin solution capable of inducing plant to release CO gas in vivo by and by is adopted, therefore it is more unlikely to cause direct signific nt influence on human. On the other hand, the resource of the present hemin as CO don or is abundant (usually hemin is the deep processed product of animal blood) and has I w cost. Therefore, compared with conventional generic engineering and other chemi al regulation method, adoption of CO donor hemin for improving crop stre ss tolerance/resistance, agronomic traits, and metabolic functions has bett er performance/cost ratio and environmental friendly advantage. On the whole, the pres t invention not only provides practical evidence for crop development physiological a id adverse environment physiological research, and also provides new practical idea inr development of reasonable deep processed products of animal blood. The present invention provides a method using a plant growth regulator containi g hemin for improving plant and seed agronomic traits and improving metabolic functio s, which has following advantages: 1) pollution free and environmental friendliness CO itself is not a free radial but a plant endogenic metabolic product (wi h concentration usually no more than 1,000ppm), all plants contain CO degradati n metabolic mechanism in vivo, which includes removing CO by generating CO 2 -ia oxidation or by using hemoglobin; at the same time, hemin itself is derived frot various animal bloods, usually used as hematic agent in food industry; therefore adoption of low concentration CO donor hemin solution under crop farmland and greenhouse conditions will not cause adverse side effect on ecological environmei t. Moreover, application concentration (- 1,000 P M) of hemin solution will not cau e human health safety issue since the release of CO gas induced in plant is generated by and by, thus the present invention is a nontoxic pollution free technique, ai d complies with requirement of green agriculture. 2) low cost CO donor hemin adopted in the present method is a deep processed product f various animal bloods, and has low cost; therefore application cost is low. 8 3) high stability Compared with high-iron-content hematin, hemin with Cl atom has stable chemical properties, and is also convenient for use. 4) wide application range The present method is especially applicable for agricultural chemical development, chemical regulation and control of farmland, provenance agriculture, tissue cultuie, storage of fruits and vegetables, and preservation of cut flowers. CO generated 'ia induction by hemin solution can be used as messenger/reduction molecule fr enhancing plant stress resistance/tolerance, improving various agronomic traits, a d enhancing various metabolic functions, and has multi-effect characteristi s compared with other chemical regulation substances. Description of the Drawings Fig. 1 shows that the plant growth regulator containing hemin can alleviate inhibition effect of osmotic stress on wheat seed germination. Fig. 2 shows the induction effect that the solution of the plant growth regulat r containing hemin has on generation of adventitious root of cucumbers. In the Fig. 1, C is germination picture (control group) of wheat seed after distilled water treatment for 2 days, P is 25% PEG-6000 for simulating draught stress, P+HmO.l, P+Hml.0 and P+HmlO.O are combination treatment with 25% PEG-6000 with 0.1, 1.0, and 10.0 9ii hemin respectively, which shows that CO donor hemin can alleviate inhibition effect f 2 day osmetic stress to wheat seed germination in concentration dependent form, n which 1.0 i' M hemin reaches the best effect; Fig. 2 shows that compared with the control group (distilled water, CK), generation of adventitious root of cucumbers (5m as indicated by ruler) can be induced after 5-day treatment with different concentratio s of CO donor hemin (0.1, 1.0, and 10.0 p1 M). Detailed description of the invention The present invention provides a method using a plant growth regulator contain g hemin for improving agronomic traits of plants, and plant organs and seeds (includii g generically modified organisms), and enhancing various metabolic functions and biotic/abiotic stress tolerance/resistance, which is characterized by preparing hem n solution with final concentration of 0.001-1,000 p M, which is capable of releasing low concentration CO gas (< 1,000ppm, CO gas release is detennined by GC-MS, Anderson, etc., J Agric Food Chem, 2005; or determined by hemoglobin colorimetr , 9 Chalmers, Clin Chem, 1991), and adding Ca 2 , salicylic acid, or other agriculture l chemicals 0-1,000 1 M to prepare a mixed solution; irrigating, spraying, immersing r soaking plants, and plant seeds and organs with the solution, or directly adding th e solution into culture medium containing the said plant materials; then continuing ti e treatment for 1-60 days, 1-3 times per day. Embodiment 1 Epidermis strip of Qinghai No. 9 broad bean is adopted as experimental material, AB (abscisic acid) and polyethylene glycol (PEG-6000) are adopted to simulate drought stress as positive control, Hemin (Fluka) is used to treat the epidermis strip for 3 hour compared with the control group (buffer solution), it is found that hemin has a function similar to that of ABA and PEG-6000, which can significantly induce stoma closure, and has effective concentration within 0.01-1.0 P M, and optimal hemin concentration s of 0.1 and 1.0 11 M respectively (low concentration CO gas release, 1,O00pp determined by GC-MS, Anderson, etc., J Agric Food Chem, 2005), which hints that lo-v concentration hemin can participate regulation of plant response to draught stress, the a induce stoma closure, and thus enhance plant draught resistance; CO gas saturate aqueous solutions with different dilution factors are further adopted for treating epidermis strips of the broad bean, it is found that CO solutions with saturation degre of 0.1% and 0.01% also can induce stoma closure of the broad bean leaf (CO I,000ppm, determined by hemoglobin colorimetiy, Chalmers, Clin Chem, 1991). Embodiment 2 25% (W/V) PEG-6000 is used to carry out osmotic stress treatment on "YANGMA 158" wheat seed, different concentrations (0.1, 1.0, and 10.0 V M) of hemin are added t> study influence of exogenous hemin on germination of wheat seed under osmotic stres (Fig. 1). The result shows that germination of wheat seed under osmotic stress i inhibited to various degrees (compared with normal water content condition germination is delayed by 1-2 days). 1.0 and 10.0 1 M hemin promotes germination o wheat seed under osmotic stress at early stage (treatment within two days), in which 1. 9L M hemin treatment (P+Hml.0) has the most significant effect which is shown by th significantly increased germination rate and root length after treatment with 2 day (P<0.05 or 0.01, Table 1), and bud length (data not listed) is significantly higher thai osmotic stress treatment (P) alone. Compared with osmotic stress treatment (P) alone 1.0 M hemin (P+Hml.0) also significantly improves amylase activity (includin 10 alpha/beta-amylase) (P<0.05, Table2), promotes starch degradation, significant y increases reducing sugar content as well as soluble sugar content (P<0.05, Table 2). Treatments using either CO synthesis inhibitor ZnPPIX (50 P M) or scaveng er hemoglobin (1-1b, lmg/ml) have no significant influence on germination of wheat seed (picture not listed), but reverse promotion action of 1.0 1' M hemin (P+Hrml.0) on whe t seed germination under osmotic stress, including reduction of amylase activity a d sugar and soluble sugar contents, and increase of starch content to some extents (Tab e 2), which shows that the effective substance in hemin for germination promotion s certainly CO. To prove aforementioned result, CO gas saturated solutions with differe it dilution factors are further adopted to treat wheat seed under osmotic stress. It is four d that all different concentrations of CO gas saturated solutions can alleviate inhibiting effect of osmotic stress on seed germination; wherein 0.1% CO gas saturated solution (P+0. t %CO, CO gas actual concentration j 1,000ppm) has the most significant effect L Compared with osmotic stress (P) alone, P+0.1% CO treatment can significantly increase wheat seed germination rate and root depth (detailed data is not listed). Embodiment 3 After soaked by water, "LUFENG" cucumber seeds (provided by Jiangsu Academy of Agricultural Sciences) are seeded in enamel dish covered by 2-3 layers of white gauzz, and double distilled water is added to preserve moisture. The seeds are cultured at 25:: I 'C tinder light radiation (light radiation intensity 200 w mol - 2 s', radiation cyce lightl4hr/dark 10hr) for 5 days, then taproot is cut off, and the seed is placed int> different concentrations of CO donor hemin solutions then cultivation for 5 day;, number of adventitious roots >1mm is measured, compared with control group (distille I water, CK), treatments using 0.1, 1.0, and 10.0 gM hemin solutions can all significantl induce generation of cucumber adventitious roots (Fig. 2, 5mm as indicated by ruler). Embodiment 4 Hemin (represented by Hm) is brought from Shanghai Hongru Technology Development Co., Ltd. Seeds of (WUYUGENG No. 3) rice with uniform size ar selected, surface sterilized with 0.1% KMnO4 for 3min, cleaned by washing, soaked fo 24hr with clean water, and accelerated for germination at 30'C in dark, and 50 seed; are placed in each culturing dish; following treatments are carried out: control group (CK), 0.IM NaCl; Hm 10.0, 0.1MNaC +10.0 P MHm+1.0 P MCaC 2 ; Hml.C, 0.1MNaCI +1.0 P MHm+1.0 i- MCaC 2 ; Hm 0.1, 0.1MNaCI +0.1 u MHm+1.0 P MCaC2 11 every treatment solution is changed once everyday. The result shows that Hrn within certain concentration can promote rice seed germination under salt stress, and Hm 1 0 and Hm 0.1 have most significant effects. For example, rice seed under salt stress staid s germination after Timl.0 treatment for 36hr, which is 6 hr earlier than the control grou ; after treatment for 48hr, root length and number and bud length of seeds are 11 significantly better than the control group. Compared with the control group, after H -A 1.0 and Hm 0.1 treatments for 48hr, rice seed germination vigors are improved by 59.2% and 39.4% respectively, germination rates are improved by 33.3% and 23.8 o respectively, and germination indexes are improved by 50.4% and 34.8% respective y (Table 3 and 4). Additionally, HmI.0 treatment also significantly improves activity f amylase during rice seed germination process under salt stress, and increases reducir g sugar and soluble total sugar contents (Table 5). Embodiment 5 "WUYUGENG No. 3" rice seedling under 0.1 MNaCl salt stress is treated with 1.0 M hemin for two days, it is found that hemin at that concentration can enhan< e greenness keeping level of the leaves, delay degradation rate of chlorophyll, reduce peroxidation level of root system and leaf membrane lipid, and lower accumulation of malonaldehyde (MDA), and also can perform protection action on plant cell oxidation damage under salt stress by enhancing rice seedling oxidation resistance. For example compared with those of the control group, activities of antioxidases like CAT (catalase), APX ascorbatee peroxidase), POD (peroxidase) and SOD (superoxide dismutase) <f leaves are respectively improved by 263.5%, 45.9%, 170.0%, and 78.5% (Table 6A), and activities of APX, POD, and SOD of root system are improved by 150.4%, 108.9*0, and 145.7% (Table 6B). Embodiment 6 Cut Chinese rose "YINGXING" bought from gardening market of Nanjing City s adopted as experimental material. The flower material is transported to the lab within hr and rewatered for 2hr, flowers with blossom grade 2 are selected and trimmed to oil keep two compound leaves and stem 30cm before treatment. 5 flowers are inserted ii each vase, 30 flowers are treated in each group, the treatment is repeated for three time solution in the vase is 250ml, room temperature is 25-30'C, relative humidity is 35-60* and experiment is carried out under scattered light. Various solutions in the vases ar provided as below: CK (control group): distilled water; HmO.]: 0.1 19 M Hemir 12 HmO.01: 0.01 P M -lemin; HmO.001: 0.001 u M Hemin; SA: 1.0 w M salicylic acid (common antioxidant in cut flower preservative); -ImO.01+SA: 0.01 y1 M Hemin+1.0 M salicylic acid; Hm0.001+SA: 0.001 vi M Hemin+1.0 vi M salicylic acid. The chemical reagents are directly added into the solution, and the vase is subjected to seal treatment The result shows that I-ImO.01 +SA has the best treatment effect, prolongs cut flower Ii by 4.4 days on average compared with the control group (6.1 days), and is consistent with slow growth of blossom grade after treatment for 6 days, but has no significant influence on flower diameter increase rate; HmO.01 also has good treatment effect , which prolongs cut flower life by 4.1 days on average compared with the control group (Table 7). Therefore, it shows that combined treatment using low concentration hemi and SA (HmO.01+SA) can be used as cut flower preservative. Embodiment 7 Hemin is prepared into solutions of various concentrations (0.1, 1.0 and 10.0 P M) f r carrying out hemin irrigation experiment in Shanghai greenhouse melon planting area, the melon species is XIBOLUOTUO, and the experimental area is lOOm'. Hemin treatment is carried out for continuously 60 days, 1-3 time each day, The result sho s that hemin treatment can significantly promote increase of melon yield, and also improve fruit quality. Compared with the control group (CK), the hemin treatments wi the aforementioned concentrations increase unit yield by 21.5%, 37.1%, and 16.4%a respectively, and increase fruit soluble solid matter by 7.2%, 12,7%, and - 1.2% (Tab e 8). Embodiment 8 Tomato rootstock species of LS-89 is provided by Jiangsu Academy of agricultur 1 sciences. Tomato rootstock seedling culture is carried out in 50-cell tray according lo routine method. Average day/night temperature is 25.6'C/15.8'C. After grown to 8-le f and chinese endive, the seedling is transferred to light incubator with light intensity< f 300 , mol * m- 2 s', and radiation cycle of 12 hr/12hr, pretreated at day/night temperature of 15'C/10'C for 2 days, then treated at day/night temperature of 10C/5. During treatment process, sampling is carried out once every three days at 10 seedling s per species per time, and the process is repeated for three times. In addition Hemin s prepared into solutions of different concentrations (0.1, 1.0 and 10.0 JA M). Compard with the control group (CK), the cold damage indexes of the tomato rootstock treated y the aforementioned concentrations of hemin solutions for 12 days are reduced by 217 7o, 13 38.4%, and 20.2% respectively (Table 9). Embodiment 9 High quality rice species "WUYUGENG No.7" widely cultured in Jiangsu province is adopted, ripe plump rice seeds are selected, got husk removed, soaked with 75% ethan 1 for 30sec, sterilized with 20% NaClO 4 for 30min, cleaned with sterile water for 7 8 times, soaked in sterile water for 4-5hr, and blown for 30min on sterile filter paper; 1l e embryo is inoculated on induction culture medium, rice yellow callus is induced after 3 weeks, and then adopted for root culture after inoculated on differentiation medium f r a certain time, hemin is added into the root induction medium simultaneously, and the final concentrations of hemin are 0.1, 1.0 and 10.0 P M respectively. The result sho s that compared with the control group, the rice callus treated by hemin has root inducir g rate improved by 20.9%, 28.4%, and 5.7% respectively. At the same time, treatments of the first two concentrations significantly accelerate the rice callus root induction by 1 9 days and 3.7 days respectively (Table 10). Embodiment 10 Hemin is prepared into solutions of different concentrations. (0.1, 1.0, and 10.0 Mi), and mixed with "MIELINGHUANG (lOg/acre)" compound pest killer into a mixed solution. Spray treatment using hemin solutions of different concentrations is adopted o study inhibition action of Bt transgenic cotton to growth of cotton bollworm larva, ar d the experiment species is pest resistant species "BAOLINGMIAN 328" of Monsanto, USA. The test result of resistance for third-stage larva shows that after taken 32B le f treated by HmO.1, Hml.0, and Hm10.0 for 3 days, the larvas have significantly reduced food consumption/weight ratio, relative growth rate, and efficiency in converting ingested food into body matter in the treatment concentration dependent form compared with those taken control cotton (CK, SIMIAN No. 3). The above result shows that Bt transgenic cotton treated by hemin has enhanced inhibition action on cotton bollwon 1 larva (Table 11), which shows hemin can be effectively used as pest killer enhancer. Embodiment 11 The experiment site has taken place in Huaning county Yunnan province, th experiment species is YUNYAN 87, and the prevention target is tobacco mosaic virus (TMV). The control group is sprayed with clean water, hemin is prepared into solution of different concentrations (0.1, 1.0, and 10.0 P M), and they are named as CK, HmO. , 14 Hml.0, and Hm10.0 respectively. First dose is applied at seedling resuscition time aftar seedling transplantation, then the agent is applied once every 8 days, and the treatme t is repeated for three times. All tobacco seedlings in the zone are investigated. Plant disease severity is divided into 0-4 grades, total seedling number and number cf diseased seedlings of each grade for each treatment are recorded respectively, and thea the disease index and prevention effect are calculated accordingly. Tobacco seedlin disease damage conditions for each treatment are investigated respectively on the 8 , 16"' and 24"' days after agent application. Farmland result shows that Hm treatme t solutions of different concentrations can effectively improve tobacco resistance to TM in which Hin 0.1 treatment has the best effect, which has prevention effect up to 76.100 at 24' day (Table 12), and significantly increased flavone compounds content (data n t listed). Embodiment 12 Hemin is prepared into solutions with concentrations of 0.1, 1.0, and 10.0 11 M. Hemi 1 application experiment is carried out in pea planting area in GAOMI city an WEIFANG city of Shandong Province, the pea species is common vetch 881 provide by Shandong Academy of Agricultural Sciences, with experiment area of I OOm'. Th result shows that SOD activity of seedling leaf has irritation induction effect to lo concentration Hg, and is improved by 12.8% under HgCl 2 concentration of 10 j M, the 1 is further increased along with increase of heroin concentration after addition of Hemi 1 (0.1 and 1.0 " M). Additionally, compared with Hg treatment alone (control group, superoxide anion generation rate, malonaldehyde content and cell membran permeability are reduced along with increase of hemin concentration within certai range, in which 1.0 1 M hemin treatment (Hml.0) has the best effect (Table 13). Embodiment 13 0.11MNaCl 0.6% agar solution is prepared, and filled in 100ml glass beaker, and each treatment is repeated for 3 times. After agar solution is cooled and solidified "WUYUGENG No.3" rice seeds soaked respectively in 0.1, 1.0, and 10.01u M hemi (Hm, Fluka, all including CaCl2 100 P M) solutions for 48hr are randomly selected, an inserted into the agar, 12 seeds are placed into each beaker, and then the beakers ar placed into turnover box, and cultivated in artificial climate container (temperature 30'O and humidity 70%). After salt stress for 5 days, the growth of rice seedlings i significantly inhibited, which is represented by reduced growth rate, lower seedlin 15 height, less root induction number, and shorter root length; while inhibition effect of s It stress to rice seedling growth is alleviated to different degrees after seed soakii g treatment with 0.1 P M and 1.0 p M hemin solutions, and seedling height increase, le f fonnation, and root growth and elongation can be ameliorated; but 10.0 i2 MH n treatment has less significant effect. For example, dry weight of overground part at d underground part and root crown of seedling after 1.0 t MHm treatment are increased by 48.1%, 25.8%, and 17.8% respectively compared with those of the control group. Cn the other hand, seeds treated by 1.0 P MHm has significantly reduced mass weigl t, which hints that low concentration Hm also can speed up degradation of rice se d storage substances, and the above result shows that low concentration Hm treatment ce n improve saline tolerance of the rice seedlings. Embodiment 14 The second leaf of three-leaf stage wheat (YANGMAI 158) is provided, pretreated wi h 0.01 and 0.1 p M hemin and distilled water (control group) in dark at 25C for 24hr, then treated with 1 Omg/L Paraquat or 150mM H20 2 in dark at 250C for 3hr. It is four d that the control group shows significant oxidation damage while leaf treated with hem n has less damage or no damage. At the same time, chlorophyll contents of sample s treated by 0.01 and 0.1 P M hemin are higher than that of the control group by 12.15 o and 26.10%. Activities of CAT, POD, APX, and SOD in wheat leaf are significant increased, and MDA content is also significantly reduced, which show that hem n pretreatment has effectively increased oxidation resistance of wheat leaf, and hemin cn be used as protector of excessive active oxygen generation induction type weed kille s like Paraquat and Diquat. Embodiment 15 Tomato seeds are soaked in 0.5% NaClO for 20min, and cultured in water for 3 da s after washing. The seeds are transferred to culturing dish (60mm diameter) with filtr paper and 4m1 of different treatment solutions after their roots grow to 2-3mm, and further cultivated at radiation cycle of 14hr/1 Ohr(L:D), 25 + l'C, and with light intensity of 200 P mol - m 2 + s-. After five day treatment, taproot length and lateral root numb r are measured (lateral root length >lmm is used as related index standard). The resu t shows that hemin with concentration of 1-40 P M can induce generation of tomat lateral root, and has significant root induction acceleration effect compared with clear a water treatment (control group, CK); particularly tomato treated by 1.0 P M hemin, th 16 lateral root is two times number of the control group; after hemin concentration is abo e 50 9 M, hemin has significant inhibition effect on lateral root growth. Embodiment 16 "YULU" peach fruit is selected as experiment material, which is collected fro Fenghua City Zhejiang Province at fruit prime color white conversion stage (ripeness about 70%). The fruits are transported to lab, fruits with uniform size are selected for treatment, and the fruits are treated at 60kg per batch. The treatment comprises ( ) soaking the fruits with water for 5min as control group; (2) soaking the fruits with 1.0 t M hemin for 5min as treatment group. The fruits are stored at 200C after treatment, at d fruit ripening process and related physiological indexes are periodically measured. T e result shows that hemin treatment can delay peach fruit aging process, reduce t e increasing rate of fruit cell permeability, improve fruit hardness by 30% compared wi h the control group, delay ethylene release, meanwhile, significantly inhibit ACC synthetase activity and ACC accumulation (P<0.05), and besides, inhibit activity of electron transmission chain, so as to effectively reduce generation of active oxygen. TI e above result shows that certain low concentration hemin can be used as fruit stora e preservative to prolong shelf life of the fruit. Embodiment 17 When CHANGAN No. 10 Chinese chestnut male inflorescence grows 7-10 pieces (tl e longest inflorescence is about 5-6cm), mixed solution of 1,000 P M hemin and CaCl 2 s sprayed, the fruiting rate is increased by 48-202%, and yield is increased by 165%; aft r treated for 60 days, staminate flower has reduced number and shortened length, so as o make young fruit (also called secondary fruit) formed after primary harvesting normal y developed in advance to form yield; chlorophyll content in leaf is increased by mo e than 26.7%, and photosynthesis intensity is increased from 7.19 to 17.11 1 i m)1 CO2m-2.s', so as to increase photosynthesis production capability; the generation rat s of the photosynthesis products like soluble total sugar, sucrose, and starch in the leaf a e increased by 59.0%, 240%, and 93% respectively compared with those of the contr l group (sprayed with water only). Embodiment 18 Hemin is used to control vegetable flower gender to increase yield. At four-leaf stage >f autumn greenhouse cucumber species "JINYOU No. I 1", 1,000 4 M hemin is sprayed 17 on leaf surface, which can significantly increase pistillate flower number, and improve seedling disease and cold resistance. Owing to the increase of pistillate flower, ear y stage yield and total yield are increased by 43% respectively. At 3 or 4-leaf stage of pumpkin, 1,000 L' M hemin solution is sprayed on the plants once every 10-15 days for totally 3 times, which can increase pistillate flower number, then accelerate ripening Iy 10-20 days, and total yield is increased by 30%. Table 1 influence of hemin with different concentrations on the germination rate and root length of wheat seed under osmotic stress (* and ** respectively represent that the improvement reaches significance levels P<0.05 or P<0.0 I compared with treatment (P)) Treatmen Germination rate (%)/root length (cm) t Day Day 2 Day 3 Day4 Day CK not tested 93.7+3.79/ 95.7±2.89/ 96.7±2.08/ 98.3±0.58/ same 2.88±0.66 7.00±0.99 7.44±0.78 8.88±0.81 below P 34.0±3.61/ 54.0±1.73/ 57.3±5.52/ 59.0±1.00/ - 1.35±0.42 3.17±1.03 5.54+0.81 6.33±0.72 P+HmO.1 - 38.0±3.46/ 53.3±3.06/ 55.7±5.52/ 57.7±1.58/ 1.40±0.37 3.38±0.98 6.13±0.82 7.09±0.81 P+Hml.0 - 45.3±1.53**/ 57.0±1.00*/ 58.3±0.58/ 58.7±0.58/ 1.98±0.17* 3.84±0.94 6.51+0.76 7.84±0.86 P+Hml0. - 41.0±4.58*/ 51.7±5.13/ 55.7+3.21/ 58.0±2.00/ 0 1.31±0.32 3,55±0.87 5.76±0.74 7.60±0.98 Table 2 influence of CO donor hemnin and its synthesize inhibitor ZnPPIX, and scavenger 1-b on the degradation of stored substance and amnylase activity of wheat sec in germination process after osmotic stress for 2 days (represents difference reaches significance level P<0.05 compared with treatment (P ) Tetm Degradation of stored substance and amylase activity (1 0 2 mg/g DW) rcte Soluble sugar Reducing sugar Starch content Amylase content -- content activity CK 1.71±0.04 1.57±0.06 2.09±0.03 32.97±0.03 P 0.91±0,05 0.83±0. 10 3.54±0. 11 24.00±0.34 P+ . 1.51±0.04* 1.20±0.04* 2.53±0.06* 40.01±0.35*" 59.01.00 Il-mI .0 2.03±0.10 1.74±0.12 1.95±0.11 31.08±0.14 ZnppIX5 1.35±0.16 1.23±0.09 2.76±0.01 31.80±0.21 11Ibl.0 1.56±0.03 1.35-0.04 3.14±0.12 18.52±0.31 P+Hm70.0 ±ZnPPI)( 0.89±0.02 0.79±0.00 3.9510.21* 28.49±0.45 P+Hml.0 1.04±0.09* 0.93±0.00* 3.81±0.00* 29.68±0.26 18 +Hbl.0 Table 3 influence of two-day hemin treatment on the rice seed germination under sally stress Treatment Germination vigor Germination rate Germination index (%) (%) (%) CK 28.4±0.83 67.3+1.24 52.8±2.06 Hm10.0 27.2±1.30 72.6±0.47* 60.1±1.32* Hml.0 45.2±1.67** 89.7±0.94** 79.4±2.56** HmO.1 39.6±0.84** 83.3±0.56** 71.2± 1.6** * and ** respectively represent that the improvement reaches significance levels P<0. 5 or P<0.01 compared with CK, Table 4 and 5 are the same as Table 3 Table 4 influence of two-day hemin treatment on the rice seedling growth under salt stress Treatment Root number Root length (cm) Bud length (cm) CK 3.49±0.23 2.16±0.12 2.65±0.26 Hm1O.0 4.08±0.78 2.21+0.27 2.50±0.11 Ilml.0 5.03±0.81* 2.62±0.11* 3.020.17* HmO.1 4.31:0.86 2.42+0.26 2.89±0.18 Table 5 influence of two-day hemin treatment on the amylase activity, and contents o reducing sugar and soluble total sugar of rice seed in germination process under salt stress Treatment Amylase activity Reducing sugar Soluble total sugar (I 0 2 mg/g DW) content (I Omg/g content (1 Omg/g DW) DW) CK 11.09±0.55 4.64±0.54 7.74±0.84 Hm1 0.0 12.13+0.81 5.96±0.89 10.32±1.08** Hml.0 18.63±0.93** 7.29+0.88* 14.12±1.02** HmO.1 13.02±0.85* 5.79±0.63 10.61+0.53** Table 6 influence of 1.0gM hemin on the antioxidase activity of rice leaf (A) and rool system (B) after salt stress of 2 days SOD activity U-g-l DW, others pimol-min-l -g-1 DW) (A) Treatment CAT APX POD SOD -Hm 56.16±5.36 2.68±0.44 19.43±0.81 70.1 4.6 +Im 204.13±12.93** 3.91±0.57* 52.47±4.28** 125.1±6.4** (B) 19 Treatment APX POD SOD -Hrm 2.78+0.14 81.45±0.65 44.0±2.2 +Hm 6.96±0.28** 170.13±11.17** 108.1±5.8** * and ** respectively represent improvement reaches significance levels P<0.05 r P<0.01, compared with treatment -Hm Table 7 influence of hemin with different concentrations on the life of cut Chinese ros "YINGXING" growing in vase Treatment Life in vase (day) Increase compared with control group (%) CK 6.1±0.1 HmO.1 5.4±0.2 -11.5 HmO.01 10.2±0.1** 67.2 HmO.001 8.6±0.3** 41.0 SA 8.2±0.3** 34.4 HmO.01+SA 10.5+0.1** 72.1 Im0.001+SA 9.0±0.2** 47.5 ** represents that improvement reaches significance levels of P<0.01 compared with control group (CK) Table 8 influence of hemin treatment with different concentrations on the yield and quality of melon Yield (kg/666.7m 2 ) Soluble solid matter % CK 1120 16.6 H-mo.1 1361 17.8 Hml.0 1535 18.7 Hm10.0 1304 16.4 Table 9 influence of hemin treatment with different concentrations on the cold damage of tomato root stock Treatment day Cold damage index % Ck Hm0.1 Hml.0 Hm10.0 0 0 0 0 0 3 2.3 2.0 1.6 1.9 6 10.0 8.9 6.9 8.8 9 13.1 10.6 8.1 11.4 12 20.3 15.9 12.5 16.2 Table 10 influence of hemin with different concentrations on the root induction of ric yellow callus Treatment Root induction rate Average root Root formation length cm time (day) 20 CK 70.4 3.8 15.1 Hm10.0 74.4 4.3 14.2 H-ml.0 90.4 5.6 11.4 Hm0.1 85.1 5.2 13.2 Table 11 inhibition action of Bt transgenic cotton 32B treated with hemin with differe t concentrations on the growth of cotton bollworm larva Food Reduct . Food consul ion Relati PGR Girth ECI Treatm consumpti mption Weigh rate of e reducti grow reducti ent on reducti t ratio weight growth on rate on (mg/larva) on rate ratio rate rate(% E rate(%) (% (%) (PR ) CK 198.0 - 2.54 - 0.28 - 24.12 HmlO 128.6 35.1 1.68 33.9 0.17 39.3 14.00 42.0 Hml.0 151.4 23.5 2.02 20.5 0.23 17.9 18.35 23.9 HmO.1 184.0 7.1 2.36 7.1 0.26 7.1 20.70 14.2 Table 12 influence of hemin treatment with different concentrations on the tobacco mosaic virus (TMV) resistance of farmfield tobacco 8 days 16 days 24 days Treatment Treatment Treatment Treatment Disease and Disease and Disease and index prevention index prevention index prevention effect (%) effect (%) effect (%) CK 7.95 - 12.35 - 18.65 HmlO.0 7.55 5.03 10.24 17.1 11.33 39.2 Hml.0 6.23 21.6 6.41 48.1 6.50 65.1 Hm0.1 3.21 59.6 4.12 66.6 4.45 76.1 Table 13 influence of 3-day hemin treatment with different concentrations on the oxidation resistance of pea seedling leaf under Hg stress Treatment Ck Hm0.1 Hml.0 Hm1O.0 SOD activity/% 100 122.2±9.1 142.3±5.2 126.9±9.2 superoxide anion generation rate % 100 84.3+2.0 65.3±4.2 90.2t1.7 malonaldehyde/% 100 87.4±5.3 64.7±3.2 91.2±4.3 cell membrane permeability /% 100 82.1+0.9 72.1±5.1 91.3+7.0 *superoxide anion generation rate, malonaldehyde content and cell membrane permeability adopt control group (CK, Hg treatment alone) as 100%. 21

Claims (7)

1. A plant growth regulator containing herini, wherein the plant growth regulator contains hemin, and effective final concentration of hernin is 0.001-1,000 P M after the regulator is prepared into solution.

2. The plant growth regulator containing henin as claimed in claim 1, wherein the plant growth regulator further contains Ca 24 with final concentration of 0-1,000 P M, salicylic acid with final concentration of 0-1,000 iL M, or other agricultural chemicals with final concentration of 0-1,000 v M after prepared into solution.

3. The plant growth regulator containing hemin as claimed in claim 1 or 2 wherein the regulator can induce plant to gradually release CO gas with total concentration no more than 1,000pprn in vivo.

4. An application of the plant growth regulator containing hemin as claimed it claim I or 2, wherein irrigating, spraying, immersing or soaking plants, and plan seeds or organs with the plant growth regulator containing hemin, or directly addin the regulator into culture medium containing the said plant materials, to improv various agronomic traits or metabolic functions and biotic or abiotic stress tolerance or resistance, and continuing the treatment for 1-60 days, 1-3 times per day.

5. The application of the plant growth regulator containing hemin as claimed it claim 4, wherein the said plants, and plant tissue and seeds, including monocotyledons, dicotyledons, or gymnosperms, and plants, and plant tissue anc seeds of genetically modified organisms (GMO) cultivated in farmland and greer house; the various abiotic stresses comprise salt damage, osmotic stress, drought anc waterlogging stress, cold damage, heat shock, UV radiation, ozone, trauma, meta toxication, and excessive or error use of weed killer, and the biotic stresses include various pests and pathogenic bacteria infection; the said various agronomic traits, metabolic functions, and biotic/abiotic stress tolerance/resistance refer to that during various development and production periods 22 of the seeding, germination, root induction, growth, transplanting, stein cutting flowering, fruit formation or ripening, and tissue culture and under various natural o artificial biotic and abiotic stresses, including, alpha/beta amylase activity is induce< to alleviate various stresses of inhibition on seed germination, improve see( germination rate under normal conditions, break hibernation, delay aging process prolong shelf life, induce lateral root and adventitious root, improve variou antioxidase gene expression and synthesis, increase enzyme activities, decreas running rate of various active oxygen generation systems, delay generation of lipi peroxide products, induce stoma closure of the plant blades, keep greenness, increas dry/fresh weight and elongation/growth rate of the overground and underground plan organs, increase crop yield and improve quality, enhance plant resistances to disease pest, and heavy metals, and induce synthesis of various protective substances lik proline, flavones, and phytoalexin.

6. A method of regulating plant growth by using the hemin, wherein: (l)solution preparation of the plant growth regulator containing hemin: th solution contains hemin with effective final concentration of 0.001-1,000 A M; ( 2 )irrigating, spraying, immersing or soaking plants, and plant seeds or organ with the solution of the plant growth regulator containing hemin, or directly addin the solution into culture medium containing the said plant materials, to improv various agronomic traits or metabolic functions and biotic or abiotic stress tolerance or resistance, then continuing the treatment for 1-60 days, 1-3 times per day.

7. The method of regulating plant growth by using hemin as claimed in claim 6, wherein the prepared solution containing hemin also contains Ca' with final concentration of 0-1,000 M, salicylic acid with final concentration of 0-1,000 P M, or other agricultural chemical with final concentration of 0-1,000 11 M. 23