PLANT BIOREGULATORY COMPOSITION AND METHOD USING (BENZYL SUBSTITUTED) TRIALKYLAMINE ETHER COMPOUNDS

Abstract: The present invention is directed to a new matter of composition in the form of chemical compounds and to methods for enhancing plant growth and the properties exhibited by plants treated with the compounds in accordance with this method. In particular, application of the compounds results in an increase in sugar content, essential oils and proteins along with an increase in total plant biomass. Fruits harvested from treated plants exhibit an accelerated biochemical and structural maturity. Citrus fruits typically show a reduced peel thickness and exhibit significant improvements in vitamin C, carotenoid, and essential oil contents. The methods of the present invention produce balanced improvements in the structural, biochemical, and sensory quality of Citrus fruits. Mixtures of the compounds may be applied. Certain mixtures are known to have synergistic effects. The compounds or mixtures thereof may also be applied in vitro.

Description: PLANT BIOREGULATORY COMPOSITION AND METHOD USING (BENZYL SUBS ITOTED) ETHER CXWPOONDS Field of Invention The present invention is directed to a new matter of composition in the form of chemical compounds and to methods for enhancing plant growth and properties associated with plants that have been treated with the compounds. Application of the compounds results in an increase in sugar content, essential oils, proteins and an increase in total plant biomass. Fruits harvested from treated plants exhibit an accelerated biochemical and structural maturity. Mature fruits typically exhibit increased pigment accumulation, increased essential oil accumulation, and reduced peel thickness. The methods include application of individual bioregulator compounds, mixtures thereof, and in vitro application of individual compounds and mixtures thereof. Certain mixtures of the bioregulator compounds exhibit synergistic effects. The new compounds function as plant bioregulators and thus enhance plant growth in accordance with the method of the invention. Background of the Invention Developments in agriculture have produced chemical compounds and methods for their application which function as plant bioregulators and thus serve to enhance one or more properties exhibited by the treated plant. For example, United States Patent Number 3,671,219 discloses a guartenary ammonium compound which when applied to plants enhances the sugar content of sugar cane. United States Patent Number 4,204,859 discloses that the addition of certain phenoxytrialkylamines enhance the hydrocarbon production of rubber in plants. United States Patent Number 4,159,903 discloses a method for increasing polyisoprene production in rubber producing plants such as Guayule. United States Patent Number 3,833,350 discloses that carotenoid accumulation in plants can be increased according to a method comprised of applying compounds including (halogenated phenoxy) trialkyla ines. United States Patent Numbers 3,864,501," 3,911,148, and 3,911,152 disclose a method for increasing the carotenoid pigments of fruits and vegetables which comprises the application of compounds including (methyl phenoxy) trialkylamines. United States Patent Number 4,797,153 discloses a method for increasing total plant biomass and individual plant constituents such as sugar, protein, lipid, and essential oils wherein certain substituted phenoxytrialkylamines and substituted phenylthiotrialkyl amines, or dialkylmorpholium halides are applied to plants. The compounds are applied in bioregulatory amounts to plant seeds, plant seedlings, or plant buds at the early stage of plant development, or to trees a week before or after flower bud swell. It has since been shown that the application of these compounds in bioregulatory amounts facilitates the assimilation of carbon dioxide in the photosynthetic pathway of green plants, thereby increasing the carbon atoms available for synthesis of total biomass and individual plant constituents. Summary of the Invention The present invention is directed towards a new class of (benzyl substituted) trialkylamine ether compounds which when applied to plants (either individually or in mixtures) in regulatory amounts increase important plant constituents, increase total plant biomass, and increase the rate of plant growth, and reduce the time to crop maturity. Pigment accumulation in plant leaves and mature fruits is increased. In Citrus crops, the fruits harvested from treated trees exhibit a reduced peel thickness. The compounds are applied to the plants in bioregulatory amounts - that is, an amount sufficient to increase plant biomass and accelerate growth but insufficient to harm the plant. The compounds of the present invention are selected from the group of chemical compounds having the structure: wherein X is either oxygen or sulfur, R 1 and R 2 are lower alkyl groups containing 1 to 6 carbon atoms each of identical or dissimilar structure, n. and n 2 are integers from 1 to 6, with n 1 and n 2 being independent of each other, j and R 4 are independently hydrogen, chlorine, bromine, fluorine, lower alkyl compounds containing 1 to 6 carbon atoms, lower alkoxy containing 1 to 6 carbon atoms, or condensed mono- and polycyclic aromatic ring systems, and wherein: if R 3 and R 4 are 3,5-substituents, then the lower alkyl or alkoxy group must contain 1 to 6 carbon atoms; and wherein: if R 3 is hydrogen, then R 4 must be a 4-substituent, with the proviso that R 4 is other than hydrogen; or b) an acid addition salt of the compounds defined above. It has been found that the application of the compounds of the invention or mixtures thereof causes the treated plants to form and store valuable plant constituents over untreated plants. The plants which have been treated with the bioregulatory compounds of the invention have greater biomass than untreated plants resulting in increased crop production per unit area. Moreover, field studies have been conducted in which compounds of the present invention have been compared to the bioregulator compounds disclosed in United States Patent Number 4,797,153, specifically 3,4-dichlorophenoxy triethyla ine (3,4-DCPTA) and 2,4-dichlorophenoxytriethylamine (2,4-DCPTA). It was determined that a compound of the present invention known as N,N-diethylaminoethyl (4- methylbenzyl) ether (MBTA) is generally more effective as a plant bioregulator than the bioregulator compounds disclosed in the *153 patent. That is, MBTA treated plants exhibit a greater increase in total plant biomass and valuable plant constituents relative to DCPTA. A second compound of the invention, N,N-diethylaminoethyl 3,4- dichlorobenzyl ether (DCBTA) performs comparably as a bioregulator with respect to the DCPTA. Thus, the compounds of the present invention exhibit a structure-activity correlation at least comparable and even superior to the disclosed prior art and thus represent an advance in the state of the art of bioregulator applications. Moreover, plants treated with mixtures of the compounds disclosed herein exhibit enhanced metabolic activity in forming and storing valuable plant constituents and increased plant-biomass when compared to plants treated with individual bioregulator agents, which is an unpredicted and unexpected result. The mixtures of the present invention exhibit a greater than additive effect when they are combined as bioregulatory agents and yield synergistic results relative to plants treated with individual bioregulator agents. We have further discovered that application of a mixture of DCBTA and MBTA to plants effects an unpredicted and unexpected enhancement of plant metabolic activity in forming and storing valuable plant constituents and in increasing plant biomass with respect to similar treatments of individual bioregulator agents, including the DCPTA disclosed in United States Patent No. 4,797,153 and the MBTA and DCBTA disclosed herein. The mixture of MBTA and DCBTA, when applied to plants, results in a greater than additive bioregulatory effect when compared to treatments of the aforementioned individual bioregulator agents. This greater than additive effect, or synergistic effect, further advances the state of the art. In many cases the invention increases the growth rate of the treated plant relative to untreated plants, resulting in accelerated maturation. Moreover, accelerated and increased growth make likely the possibility that growing cycles will be shorter while yielding a harvest equivalent or superior to that of untreated plants. Such a harvest would be greater since the treated plants exhibit increased biomass. The present invention is also directed to a method of in vitro application of the bioregulator compounds and mixtures thereof as disclosed above. One particular aspect of in vitro applications concerns the usage of compounds and mixtures as growth inducing agents for orchid seed germination and germinated orchid seed (protocorm) . The addition of the bioregulator compounds of the present invention to an aseptic culture medium significantly enhances orchid protocorm growth and substantially reduces the period of time for which they are cultured to produce mature, blooming plants. Detailed Description of the Preferred Embodiment The benefits of the invention are obtained by applying any of the following compounds, or mixtures thereof, to leaves, to plant seeds, seedling plant buds, immature fruits, or vegetative propagules. • ■ Mixtures" as used herein, refers to a combination of at least any two compounds encompassed by this disclosure. Examples, by way of illustration and not limitation, of compounds that can be used in the process of the invention are: A. N,N-dialkylaminoalkyl 2,4-substituted benzyl ethers wherein the 2,4-substituents are independently chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, methoxy, ethoxy, propoxy, butoxy, pentoxy or hexoxy, and wherein the alkyl and dialkyl groups are independently either methyl, ethyl, propyl, butyl or pentyl or isomers thereof. B. N,N-dialkylaminoalkyl 3,5-substituted benzyl ethers wherein the 3 ,5-substituents are independently chloro, bromo, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, propoxy, butoxy, pentoxy or hexoxy, and wherein the alkyl and dialkyl groups are the same as those in A. C. N,N-dialkylaminoalkyl 3,4-substituted benzyl ethers wherein the 3,4-substituents are independently chloro, bro o, iodo, methyl, ethyl, propyl, butyl, pentyl, hexyl, methoxy, ethoxy, proproxy, butoxy, pentoxy or hexoxy, and wherein the alkyl and dialkyl groups are the same as those in A. D. N,N-dialkylaminoalkyl 4-substituted benzyl ether wherein the 4-substituent is either methyl, ethyl, propyl, butyl, pentyl, hexyl, methoxy, ethoxy, propoxy, butoxy, pentoxy or hexoxy, and wherein the alkyl and dialkyl groups are the same as those in A. E. N,N-dialkyla ~ ti ~ roalkyl (substituted naphthyl) ether wherein the alkyl and dialkyl groups are the same as those in A. The preferred compounds of the present invention as set forth in groups A through E are those where n. is 1 and n 2 is 2, X is oxygen, the N,N- dialkyl groups are diethyl, the alkyl group is ethyl, and the benzyl substituents are 2,4-dichloro; 3,4-dichloro; 3-5-diisopropyl; 3,5,-ditertiary butyl; 3,4-dimethyl; 3,4-dimethoxy; 3-methyl, 4- methyl, 4-chloro or 3,4-naphthoxy. It has been found that two particular compounds are especially preferred in that plants treated with either of these compounds exhibit significant improvements in total plant biomass and individual plant constituents, and in particular compare favorably to the bioregulator compounds disclosed in the United States Patent 4,797,153. These compounds are N,N-diethylaminoethyl 3,4-dichlorobenzyl ether (DCBTA) and N,N-diethylaminoethyl 4-methylbenzyl ether (MBTA) . It has been found that a mixture of these two compounds is also preferred in that plants treated with this mixture exhibit significant improvements in total plant biomass and individual plant constituents with respect to plants treated with individual bioregulator compounds. Mixtures are preferably comprised of equal amounts (1:1, w/w) of each compound. However, use of unequal amounts of two or more bioregulators in mixture form does not limit the scope of the invention. Various acid addition salts of the above compounds can be readily produced. For example, by adding acid to the compounds of the invention, the following acid addition salts are formed: Wherein the molecular constituents are as set forth above, and wherein A is the anion derived from the acid added to the amine to form a salt. Mixtures of acid addition salts can also be used as bioregulatory agents. In order to achieve increase in total biomass yield, enhancement of individual plant constituents or increase in rate of plant growth, the compounds or mixtures thereof must be first applied to the plant at an early stage of development. That is, immediately prior to, or at the time when cell differentiation and plant growth are great. If application is made at a late stage of development, some increase in yield or plant constituents may occur but not the significant increase which occurs when treatment is earlier. As a practical matter, treatment is made to the seed; to the post-emergent seedling plant, that is, to the plant at or prior to the full expansion of the fourth set of primary leaves, such as at the cotyledon, true leaf, two- leaf or four-leaf stage; or to trees during flower bud swell or a week before or after. For plants which are not grown from seed or do not produce flower buds such as vegetatively propagated plants like sugarcane,•application should be at the developmental growth stages equivalent to the ones mentioned above. Since growth of the plant or tree dilutes the concentration of the bioregulatory application due to increase in plant biomass resulting in a biomass dilution effect, it may be desirable to apply more than one application subsequent to the initial one. Subsequent applications should be made before completion of cell differentiation of the growing plant or when applied to a growing tree before the completion of cell differentiation of the growing fruit. Generally, where the compounds or mixtures thereof are applied to the seeds, the concentration is about 0.001 to 0.3 mg of active ingredient per seed. Application is conveniently made by dissolving the compound to be used in water at a concentration of 0.1 to 50 parts per million (ppm) in the diluent and soaking the seeds for 2 to 6 hours. Other means of treatment of seeds such as encapsulation of the seeds with the compounds by conventional methods are encompassed by the invention. When compounds or mixtures thereof are applied to the seedling, that is at the cotyledon, true leaf, two-leaf or four-leaf stages and the like, the treatment is about 0.001 mg to 0.3 mg active ingredient per plant. This can be accomplished by using a treatment rate of about 0.1 to 200 ppm and preferably 5 to 120 ppm. Use of treatment rates of 300 ppm or greater on young seedlings or young plants, in other words, prior to the full expansion of the fourth set of primary leaves, will either not cause increases in biomass contemplated by the invention or in many cases, may have a phytotoxic effect on the plant causing it to have stunted growth. Treatment of perennial trees requires a greater amount of the bioregulator compound or mixture due to the greater mass of the tree. Generally, about one to four grams active Ingredient per tree is applied using a treatment rate of 0.1 to 500 ppm of bioregulatory compound. The compounds or mixtures of the invention may be applied to the plant in any convenient manner. For example, after being dissolved in water, the compound or mixture can be sprayed onto the branches and leaves of the plant. Other application techniques known to the skilled artisan may be employed. Appropriate wetting agents such as Triton X-100 (polyethylene glycol p-isooctylphenylether made by J. T. Baker) , ORTHO X-77 (a mixture of fatty acids, fatty alcohols and isopropanol made by Chevron Chemical Company) , Sweep 4F (chlorothalonil from Diamond Shamrock Company) and the like may be added to the aqueous solution to aid in plant treatment. Appropriate penetrating agents such as B- cyclodextrin (B-(heptamer)-cyclodextrin made by Takeda Chemical Industries, Ltd.) or Tween 80 (polyoxyethylene (20) sorbitan monooleate, available from E. Merck, Darmstadt Germany) may be added to the aqueous solution to increase penetration of the bioregulatory compound. Solutions of bioregulator and appropriate wetting agent may be adjusted to an acidic pH (pH 4 to 5) prior to plant application. With respect to pH, "the applicant has found the following conditions to be preferable. When ungerminated seeds are hydrated in solutions of pH 5 to 7, seedling growth was optimized (solutions contained 10 ppm MBTA 0.1% Tween 80(%). Seedling root growth was significantly enhanced by MBTA within test solutions of pH 3 to 6. Hypocotyl growth was enhanced by MBTA within test solutions of pH 4 to 5. The results suggest that incubation of seeds in pH 3 to 5 solutions may facilitate the transport of protonated bioregulator into plant tissues. When the compounds or mixtures thereof are used in vitro, any number of isotonic, buffered, nutrient media containing mineral salts as macronutrients and icronutrients, hormones, vitamins and supplements may be utilized in accordance with the present invention so long as they are capable of supporting propagation of the subject plant. For example germination of orchid seeds and orchid protocorm on Hill's seed germination medium, available from Gallup and Sterling Laboratories, Santa Barbara, CA. , was shown to be acceptable. In order to achieve beneficial results with in vitro applications, the compounds or mixtures of compounds should be introduced into the nutrient media at the earliest stages of plant development. Most preferably, this will be after sterilization of the medium and before or contemporaneous with seed or propagule sowing. It should be understood that application of the bioregulators is best made prior to solidification of the agar support. If application is made later some increase in yield or plant constituents may occur but not the significant increase which occurs if treatment is earlier. Since the growth of the plant or propagule dilutes the concentration of the chemical mixture due to increase in plant biomass resulting in a biomass dilution effect, it may be desirable to apply more than one application subsequent to the initial one. Subsequent applications should be made before completion of cell differentiation of the growing plant or when- applied to a growing propagule before the completion of cell differentiation of the growing fruit. Generally, where the mixtures are applied to the seeds in vitro, application is conveniently made by dissolving the compounds or mixture to be used in water at a concentration of 0.1 to 50 parts per billion (ppb) and introducing the compounds to the sterilized culture medium. It is preferred that if a mixture is used then the mixture can be comprised of equal amounts of each bioregulator agent in the mixture. Treatment of perennial trees propagated in vitro requires a greater amount of the bioregulator mixture due to the greater mass of the tree. Generally, about 0.01 to 10 mg total active ingredients per tree is applied using a treatment rate of 1 to 100 ppb of bioregulatory mixtures. However, we have found that bioregulatory effects result from applications as low as 0.01 ppb. When applied in accordance with the method of the invention the compounds or mixtures of the invention substantially increase total biomass, enhance the amount of some or all plant constituents and in many cases increase the rate of growth in green plants over untreated plants as long as constituents such as water and light necessary for plant growth are present in the required amount. Using the method of invention, seed treatment of radish resulted in a greatly enhanced root and leaf development at crop harvest as compared with controls. Seed or foliar treatment of petunia, verbena, aster, and other ornamental.crops increased root development, secondary branching and increased bud count per plant. Treated ornamental crops typically flower sooner and have a greatly improved aesthetic appeal. Treatment of Citrus trees causes the fruit to mature faster, to bear an increased fruit yield, to increase the Vitamin C content and to produce fruits with an increased essential oil content. USDA color score values of juice recovered from treated Citrus is superior due to an increased pigment content. Thus, the method of the invention finds use on any green plant where increased rate of growth, biomass or the like is desired. The method is particularly valuable for use on plants which produce food, vitamins, nutrients, fiber, or energy; or on plants where commercial production is limited due to low plant yield when grown without bioregulators. The composition and method can be used on annual or perennial plants, such as seasonal row crops, vineyards, orchards, and all ornamental or horticultural plants. For example, treated Hamlin, Valencia, and Pineapple sweet orange trees show an accelerated ripening of fruit. When compared with untreated controls, treatment of Citrus trees produces mature fruits that have an increased brix, essential oil, and vitamin C contents. Juice color is enhanced by chemical treatment. Thus, the method of the invention produces significant improvements in the nutritional and sensory qualities of Citrus products and reduces the time to harvest of mature Citrus fruits. Similar reductions in the days to crop maturation have been observed in a variety of ornamental crop plants (aster, verbena, petunia, pansy) . EXAMPLE 1 Two year old grafted trees of *Okitsu-wase' Satsuma mandarin as well as two year old "Kara" and "Kinnow" grafted trees were planted into 25 liter pots and were maintained under 40% saran cover in Pasadena, California. The following tertiary amines were prepared as 100 ppm solutions (pH 5.0) in 0.5% Tween 80 (v/v) : N,N-diethylaminoethyl 3,4-dichlorophenylether (DCPTA) (a/k/a (3,4-dichlorophenoxy) trialkylamine) N,N-diethylaminoethyl 3,4-dichlorobenzylether (DCBTA) N,N-diethylaminoethyl hexanoate (HTA) The bioregulator solutions were applied to foliage runoff in a single application. Each treatment group contained two trees. Control trees were sprayed to foliage runoff using 0.5% Tween 80 (v/v) . At the time of bioregulator application, Satsuma fruit diameters ranged from 0.8 to 1.4 cm. During fruit development, " the trees were fertilized every 14 days using a 20N-20P-20K soluble fertilizer and the trees received a monthly side-dressing of Ironite. Mature Satsuma fruits were harvested at months after bioregulator application. In each bioregulator treatment group, mature fruits from the two replicate trees were combined for fruit quality analysis. For analysis, five fruits of approximately 50 to 55 mm in diameter were chosen from each treatment group. Total fruit fresh weight was determined. Fruits were cut in half and peel thickness was determined. Fruits were juiced by hand. The combined juice and pulp were pressed through a 0.5 mm sieve and the final juice volume and juice fresh weight were determined. Peel fresh weight after juicing and pulp fresh weight were determined. Table 1 Letters within colians indicate separations according to Dincan's Multiple range test.5% level. ND = not detenmined. SUBSTITUTESHEET EXAMPLE 2 The coordinated improvement of peel structure and juice composition of citrus fruits is measured. Citrus trees were maintained as: a. Mature orchard trees of , 01inda / Valencia sweet orange located at the Agricultural Experiment Station, University of California, Riverside, California; and b. Orchard trees of Hamlin and Pineapple sweet orange located in Arvin, California. a. Riverside Field Planting: Bioregulator treatments occurred in a 24-tree block of *01inda' Valencia orange. The trees were 10 years old and ha been skirted. Each treatment group contained three trees. Chemical treatment groups consisted of: Control; DCPTA-50 ppm DCPTA-100 ppm; DCBTA-50 ppm; DCBTA-100 ppm; MBTA-50 ppm; MBTA 100 ppm; MBTA-200 ppm. All solutions (pH 5.0) contained 0.5% Tween 80 (v/v) . Bioregulator solutions were applied as a single application. Each tree received 4 liters of bioregulator solution that was applied as evenly as possible to the entire foliage canopy. At the time of bioregulator treatment, fruit sizes ranged from 2 to 3.5 cm in fruit diameter. Fruits were harvested in December, six months aft chemical treatment. ns, *, L, Q Not significant or significant at P=0.05(*> according to linear (L) or quadratic (Q) models Fruit diameters and ml fruit/fresh weight remain comparable among controls and all treatment groups (Table 2) . Chemical treatments show a reduction in peel thickness relative to controls, while fruit diameters are comparable fo all groups including controls. MBTA-50 treated citrus exhibi a significant increase in Brix. When compared with control juice samples, chemical treatment significantly increased the Vitamin C content of Valencia sweet orange. Among all treatments, MBTA-200 ppm treatment resulted in the largest numerical increase in Vitamin C accumulation in mature fruits (Table 2) . Bioregulator application to fruiting Λ 01inda' Valencia trees significantly enhanced the flavedo carotenoid SUBSTITUTE SHEET accumulation of fruits that were harvested 6 months after chemical treatment (Figures la, lb, lc) . Fruits harvested from 50 ppm DCPTA, 50 ppm DCBTA, and 50 ppm MBTA-treated tre generally showed the most uniform improvements in flavedo carotenoid development (Figure la) when compared with contro fruits. Sectioned fruits (Figure lc) from control and bioregulator-treated trees visually showed similar endocarp carotenoid development. However, fruits harvested from bioregulator-treated 01inda' trees showed significant reductions in peel thickness (Figure lc and Table 2) . b. San Joaguin alley Field Trials: Bioregulator treatments were applied to commercial orchard stock of Pineapple, Hamlin, and Valencia sweet orang Each bioregulator treatment represents one tree for each cultivar. The trees were 12 to 15 years old and have not be skirted. Experimental trees are internal plantings within a 500 tree block. Chemical treatments were applied as a singl foliar application and consisted of: Control; DCPTA-lOOppm; DCBTA-100; MBTA-lOOppm. All solutions (pH 5.0) contained 0. Tween 80 (v/v) . Approximately 5 liters of solution were applied to each tree and the foliage canopy was covered as evenly as possible. At the time of chemical treatment, frui sizes ranged from 1.5 to 3 cm in diameter. Mature fruits of Hamlin and Pineapple orange were harvested as part of a December harvest 6 months after chemical treatment. Fruits within all treatment groups had attained uniform peel color. Medium-sized, canopy fruits were chosen for harvest. The dat below represents eight fruits/sample. Table 3 Letters within columns indicate mean separations according to Duncan's multiple range lest, 5% level. DCBTA shows a marked increase in juice recovery/fresh fruit weight in Hamlin and Pineapple fruits. For both Hamlin and Pineapple sweet orange, MBTA treatment shows significant improvement in brix and vitamin C content when compared with control fruits. These results indicate that MBTA-treatment resulted in sweeter fruits with an improved nutritional quality. Application of DCBTA resulted in the best juice recovery in both Hamlin and Pineapple fruits. However, MBTA foliar- application resulted in the largest numerical increase in bri and vitamin C contents when compared with the values of controls. Improved juice recovery was generally related to a SUBSΠTIΠΈ SHEET reduction in peel thickness (Tables 1, 2, and 3). However, chemical treatment had no significant effect on final fruit size or fruit shape. EXAMPLE 3 Four substituted tertiary amines (3,4-DCPTA, 3,4-DCBTA, (2,4-DCBTA), and MBTA) were synthesized and purified accordin to the methods of Echols, Maier, Poling, and Sterling, 1981, New bioregulators of Gibberellin Biosynthesis in Gibberella Fuiikuroi, Phytochemistry 20:433-437; Poling, Hsu, Yokoyama, 1977 Structure Activity Relationships of Chemical Inducers of Carotenoid Biosynthesis. Phytochemistry 14:1933, respectively. Radish seeds (Raphanus sativus L. cv. Scarlet turnip white tipped) were supplied by Ferry Morse Seed Co. , Modesto CA. Seeds were soaked for 6 hrs at 22°C in 0.1, 1.0, 10.0, 50.0, and lOO.O ppm bioregulator solutions. All bioregulator solutions (pH 5.0) contained 0.1% Tween 80. Control seed lot were soaked for 6 hrs at 22"C in 0.1% Tween 80. Seeds were planted immediately after chemical treatment. All plants wer greenhouse grown using a standardized radish as described previously. Keithly, J.H., H. Kobayashi, H. Yokoyama, and H.W. Gausman 1991 Promotive Effects of Tertiary Amine Bioregulators on Radish (Raphanus sativus) Growth and Development. PGRSA Quarterly 19(3): 182-187. The growth enhancing properties of the newly synthesized tertiary amine analogs showed significant differences in a standardized radish growth test (Table 4) . The growth of nontreated controls and of DCPTA-treated plants were used as reference plant growth systems. The growth of DCBTA-treated plants were numerically and statistically similar to the growth of DCPTA-treated plants. When compared to the taproot growth of DCPTA-treated plants, the order of compound effectiveness appears to be as follows: MBTA > 3,4-DCBTA = DCPTA » 2,4-DCBTA Table 4 Enhanced Leaf and Taproot Growth of Radish by Tertiary-aaine Bioregulators Bioregulator Cone Leaf Dry Leaf Area Root Dry Root Die Root to Abbreviation PP~, wt g da * - Shoot Ratio Control 1.04 2.64 0.82 22.89 0.79 NS, 0*, 0**, L* Not significant or significant at P=0.05(*) or P-0.0K**) according to Linear (L) o Quadratic(Q) aodels. * Siot detemiπed SUBSTITUTE SHEET EXAMPLE 4 Mesophyll chloroplast development during leaf expansion has been shown to regulate the amount of photosynthate available for vegetative crop growth and reproductive plant development. Fruit set and crop yield are often determined b the amounts of partitioned photosynthate that are available during early fruit growth. The effects of DCPTA, DCBTA, and MBTA on chlorophyll accumulation and Rubisco activity in mature leaves of Valencia, Pineapple, and Hamlin sweet orange is shown herein. Chemical treatments were performed when a majority of th trees had started a vegetative growth cycle (growth flush) during April, 1990. Bioregulator solutions (pH 5.0) containe 0.5% Tween 80. Citrus cultivars were divided into treatment groups that contained three trees per treatment. Foliar applications of bioregulator were performed using a trigger- action hand sprayer. Controls received a foliar application of 0.5% Tween 80. Solutions were applied to the point of foliage runoff. After chemical treatment, trees were arrang as a completely randomized block. Leaf growth analysis was performed at 6 to 8 weeks afte chemical treatment. Individual leaves were harvested from 3 vegetatively similar branches within each treatment group fo leaf morphology analysis. Leaves were numbered basipetally from the first visible leaf at the apical meristem. For eac leaf, leaf area (dm 2 ) , leaf blade length at the midvein, and leaf fresh weight was determined. Specific leaf weights (SL g fresh weight/dm 2 leaf area) were calculated from leaf fresh weight and leaf area data. When compared with controls, foliar application of DCPT DCBTA, and MBTA to Valencia, Pineapple, and Hamlin sweet orange significantly increased the SLW of mature leaves that were harvested at 6 to 8 weeks after chemical treatment (Tabl 5) . Among the bioregulator treatment groups, SLW was numerically similar. Bioregulator application significantl (P=0.05) increased Chi accumulation in mature orange leaves when compared with that of controls. When compared with controls, bioregulator treatment resulted in generally improved total carotenoid accumulation in mature leaves. Rubisco activity was measured in a wide range of leaf ages, and the most reliable enzyme activities were obtained from leaf numbers 15 to 18 basipetally from the apical meristem. Bioregulator treatment appeared to increase the CCS of sweet orange (Table 6) . In all orange cultivars, the soluble protein to Chi ratio was increased significantly (P=0.05) in all chemical treatment groups when compared with controls. Within the chemical treatment groups, the soluble protein to Chi ratios of 50ppm treatments often appeared to be superior to lOOppm treatments. When compared with controls, the observed improvements in leaf soluble protein to Chi ratios within the chemical treatment groups supported a significant (P=0.05) increased Rubisco activity per mg Chi (Table 6). Within all treatment groups among all cultivars, 50ppm MBTA application appeared to be one of the most useful chemical treatments. Rubisco activity was not determined in all treatment groups due to the limited amount of experimental material. The in vitro Rubisco analysis suggests that MBTA may be very effective chemical regulator of chloroplast development. TABLE 5 Specific leaf weight (g fresh weight/dm ). Determined on leaf numbers 15 to 18 numbered basipetally from the apical meristem. Letters within columns indicate significant differences (cultivars analyzed separately) according to Duncan's multiple range test, 5% level. SUBSTITUTE SHEET TABLE 6 Enhanced soluble protein accumulation and Rubisco activity of sweet orange leaves by tertiary-amine bioregulators Soluble Protein To Total Activated Rubisco Activity Treatment Ch1 ratio Activity/mg protein Activitv/mg CM VALENCIA Control 41.09d DCPTA-50 44.93b DCPTA-100 44.08c DCBTA-50 45.30ab DCBTA-100 44.26c MBTA-50 46.12a MBTA-100 44.90b PINEAPPLE Control 44.29c DCPTA-50 45.40a DCPTA-100 DCBTA-50 44.90b DCBTA-100 44.52c MBTA-50 45.49a MBTA-100 HAMLIN Control 41.33d DCPTA-50 45.06c DCPTA-100 44.67c DCBTA-50 47.88a DCBTA-100 46.87b MBTA-50 46.41b MBTA-100 2mg protein/(mg Chi) in Citrus leaf chloroplast preparations YRubisco activity = mg C0,/h. Letters within columns indicate mean separations according to Duncan's multiple range test, 5 level. SUBSTITUTE SHEET EXAMPLE 5 Orchard trees of % Olinda' Valencia orange were maintaine at the Agricultural Experiment Station, University of California, Riverside, California. Twenty four trees were treated with tertiary amine bioregulators as foliage treatments. Each treatment group contained three trees. The randomized complete block experimental design contained the following bioregulator treatments: Control; DCPTA-50 ppm; DCPTA-lOO ppm; DCBTA-50 ppm; DCBTA-100 ppm; MBTA-50 ppm; MBTA 100 ppm; and MBTA-200 ppm. All bioregulator solutions (pH 5.0) contained 0.1% Tween 80 (v/v). Fruits were harvested at 181 days, 215 days, and 259 days after bioregulator treatment (DAT) . Sized fruits were analyzed for peel thickness, juice recovery, juice brix, and total peel pigment accumulation. Each fruit sample contained eight randomly chosen fruits. Compared with controls, fruits harvested from bioregulator-treated *01inda' trees showed improved peel development and juice accumulation during fruit maturation (Table 7) . All fruits harvested from bioregulator-treated trees showed a general reduction in peel thickness as compar with the peel development of controls. Among the three frui harvests, total soluble solids (brix) accumulation in treate fruits was increased significantly as compared with controls (Figure 2) . Fruits harvested from bioregulator-treated trees showed significant improvements in peel pigment accumulation when compared with controls (Table 8) . Among all treatments, chlorophyll content was inversely related to total carotenoid content. The biological activities of DCBTA and MBTA on carotenoid accumulation appeared greater than that of DCPTA. The brix and carotenoid accumulation of 01inda' fruits harvested from bioregulator-treated trees indicates that chemical treatment has reduced the days to fruit harvest by approximately 40 days when compared with controls. Among all treatments, improved juice recovery is related to a reduction in peel thickness. Of the test bioregulators, MBTA appears t have the greatest biological activity on carotenoid accumulation. At 259 DAT, fruits harvested from 50 ppm MBTA- treated trees showed a 68% increase in carotenoid content, a 10% increase in brix, and an 8% increase in juice recovery when compared with the values of controls. TABLE 7 Enhanced juice recovery and reduced peel thickness of Valencia sweet orange by tertiary-amine bioregulators Letters within colu ns indicate aean separations according to Duncan's Multiple range test, 5X level. SUBSTITUTE SHEET TABLE 8 Enhanced pigaent accuaulation of Valencia sweet orange by tertiary-aaine bioregulators Total Chlorophylls Control DCPTA-50 DCPTA-100 DCBTA-50 DCBTA-100 HBTA-50 MBTA-100 NBTA-200 Total Carotenods Control DCPTA-50 DCPTA-100 DCBTA-50 DCBTΛ-100 MBTA-50 MBTA-100 MBTA-200 Letters within columns indicate mean separations according to Duncan's multiple range test, 5% level. SUBSTITUTE SHEET EXAMPLE 6 At Republic Groves of Hardee County, Florida five year- old trees of Hamlin Sweet Orange were treated with the following individual compounds and mixtures in the following concentrations: DCBTA 10, 50, 100 ppm MBTA 10, 50, 100 ppm MBTA/DCBTA mixture l, 10, 50 ppm of each compound All bioregulator solutions contained 0.1% Tween 80 (w/v) . A trees received a single foliage spray application of chemica as a complete canopy spray. Fruit sizes ranged from 9 to 12 mm in diameter at time of chemical treatment. Control trees received a single application of 0.1% Tween 80. Each treatment consisted of five trees. Mature fruits were harvested from each treatment at 6 months after chemical treatment. Application of a mixture of MBTA and DCBTA to Hamlin sweet orange trees at the beginning of fruit growth significantly enhanced the juice content (Table 9) and juice quality (Figure 3 a-d) of mature fruits when compared with t values of controls. Among all treatments, chemical improvements of peel development and juice content (Table 1) were greatest within the MIX 10/10 treatment. Among the mixture treatments, the MIX 1/1 treatment showed the largest numerical improvements in juice quality (Figure 3) . In general, the MIX 1/1 treatment showed the largest numerical improvements in juice brix, juice BAR, and juice vitamin C content, when the chemical treatments contained less than 50 ppm bioregulator. Mixture (MIX 1/1 and MIX 10/10) treatments showed improvements in juice color when compared with 10 ppm MBTA and 10 ppm DCBTA treatments. These results suggest that the biological activities of MIX treatments, when used in low concentrations, are increased relative to the bioactivity of a single chemical treatment. Thus, the use of a mixture of MBTA and DCBTA as foliage treatments allows less chemical to be applied per tree, when compared with single chemical treatments. Table 9. Promotive effects of tertiary amine bioregulators on fruit development * - ψ w-miin sweet orange. Fruit Composition (%) ""Mixture of DCBTA & MBTA, (1/1 , /v) SUBSTITUTE SHEET EXAMPLE 7 The propagation of epiphytic orchids requires plant cultivation in vitro. Arditti, J. 1977. Clonal propagation of Orchids by Means of Tissue Culture-A Manual. In: J. Arditti, (ed) , Orchid Biology, Reviews and Perspectives, pp. 203-293. Cornell Univ. Press, Ithaca, New York. Orchid seeds and meristem propagations e cultured on aseptic, artificial media for up to 3 years before the seedling plants are grown for 3 to 5 years to produce mature, blooming plants. Compared with controls, application of DCPTA to seedling phalaenopsis orchids during routine seedling transfer from aseptic growth in vitro to greenhouse- culture significantly enhanced plant growth and reduced the time to flowering. In addition, seedling death after transplanting was reduced significantly within the DCPTA treatment groups as compared with controls. In this example, the growth promoting effects of MBTA, DCBTA, DCPTA, and a mixture of MBTA/DCBTA on seed germination and protocorm development in vitro of Brassolaeliocattleya orchid are examined. Protocor (germinated seed) development represents the initial growth stage of orchid plant development in vitro. Leaf and root meristems differentiate from the unspecialized protocorm cells to produce a functional orchid seedling. All orchid seeds were sown on sterilized Hill's seed germination medium obtained from Gallup and Stribling Laboratories, Santa Barbara, CA. The solid-support medium contained a mixture of buffered mineral salts, auxin, cytokinin, amino acids, organic acids, and agar. The medium (34 g/liter) was solubilized in distilled water and was adjusted to a Ph. of 5. All orchid cultures were grown in 65 X 65 X 100 mm plastic vessels (Magenta Corporation, Chicago, IL) that contained 100 ml seed germination medium. All culture vessels were autoclaved for 10 min at 15 psi. After medium sterilization 10 ppb MBTA, 10 ppb DCBTA, 10 ppb DCPTA, and a 10 + 10 ppb mixture of MBTA and DCBTA were filter-sterilized and were added to the medium (5 ml/vessel) before the agar-support solidified. The bioregulator solutions were prepared in distilled water. Experimental controls received 5 ml aliquots of filter- sterilized water. All treatment groups contained three replicate vessels. All orchid seed transfers were performed under sterile conditions. Dry seeds of Brassolaeliocattleya X Ruben's Verde (Blc. Green Heart 'Imperial Jade' x Blc. Lester McDonald 'Kelly' AM/AOS) were surface sterilized for 20 minutes. The seeds were then sown, without rinsing, on the bioregulator-amended media. Seeds were distributed evenly on the medium surface with gentle shaking. Seeds were germinated at 23°C under continuous illumination (75 μE m "2 s *1 ) using two wide-spectrum fluorescent lamps. Days to seed germination were recorded for each bioregulator treatment. Sixty days after seed sowing, protocorms were harvested from each vessel and the total protocorm fresh weight was determined. Fresh weight and diameters for 50 protocorms were determined. Each group of 50 protocorms was extracted into 100% acetone and the chlorophyll and total carotenoids contents were quantified. Orchid seed germination and protocorm development was enhanced significantly by the addition of tertiary amine bioregulators to the aseptic culture medium (Table 11) . During seed sowing, visut. ly equal amounts of seeds were plated into each vessel. Seeds were observed to germinate 13 days after sowing among the control, DCPTA- amended, and DCBTA-amended cultures. However, seeds plated on the MBTA/DCBTA mixture and MBTA-amended cultures were observed to germinate 10 days after seed sowing. Compared with controls, protocorm fresh weight was increased significantly (P = 0.05) by the addition of bioregulators to the in vitro culture medium (Table 11) . Chlorophyll a, chlorophyll b, and total carotenoid contents of mature protocorms grown on tertiary amine bioregulator-amended media (TAB-media) were increased significantly compared with controls. However, the chlorophyll a to b ratios of all treatment groups were statistically similar. Of the compounds that were tested, the MBTA and MBTA/DCBTA mixture treatments showed the largest numerical improvements in protocorm fresh weight and pigment accumulation when compared with controls. This study indicates that orchid protocorm growth in vitro is enhanced significantly by the addition of tertiary amine bioregulators to an aseptic culture medium (Table 11) . Among the compounds that were tested, treatments that contained MBTA showed the greatest improvements in seed germination, protocorm growth, and total pigment accumulation when compared with controls. The chlorophyll a to b ratios of control and all chemical treatments were numerically similar. These results suggest that chloroplast size (volume) or chloroplast number per cell was enhanced in response to tertiary amine- reatment, rather than a specific enhancement of chlorophyll a or chlorophyll b biosynthesis. The potentially enhanced chloroplast size of orchid protocorms grown in TAB-media is significant, since the total chloroplast volume per cell generally determines the photosynthetic carbon fixation rate and cell growth rate of plants. Table 10 Protocorm fresh Pigment content ( ■ ig/g fresh wt) Treatment 2 wt (g x 50) Chla Chlb Chla/b Cartot DCPTA, N,N-diethylaminoethyl 3,4-dichlorophenylether DCBTA, N,N-diethylaminoethyl 3,4-dichlorobenzylether MBTA, N,N-diethylaminoethyl 4-methylbenzylether SUBSTITUTE SHEET

Claims: We Claim: 1) A new ' matter of composition comprised of chemical compositions of the formula: Wherein X is either oxygen or sulfur, R, and R-, are lower alkyl containing 1 to 6 carbon atoms, n, and n 2 being integers from 1 to 6 each, n 1 and n 2 being independent of each other; R j and R 4 are independently hydrogen, chlorine, fluorine bromine, iodine, lower alkyl containing 1 to 6 carbon atoms, lower alkoxy compounds containing 1 to 6 carbon atoms, or condensed mono and polycyclic aromatic ring systems, and wherein: if R 3 and R A are 3,5,-substituents, then the lower alkyl or alkoxy group must contain 3 to 6 carbon atoms; and wherein: if. R j is hydrogen, then R 4 must be a 4-substituent, with the proviso that R 4 is other than hydrogen; or an acid salt thereof. 2) The matter of composition as set forth in claim 1 wherein the compound is N,N-diethylaminoethyl dichlorobenzyl ether or an acid salt thereof. 3) The method of claim 1 wherein the compound is N,N- diethylaminoethyl 4-methylbenzyl ether or an acid salt thereof. 4) A mixture of at least two of the compounds set forth in claims 1-3. 5) A synergistic mixture of the compounds set forth in claims 1-4. 6) A method for enhancing plant growth comprised of the steps of applying to a plant a compound or mixtures thereof selected as set forth in claims 1-5. 7) A method for enhancing plant growth comprised of the steps of applying to a plant a compound selected from the matters of composition or mixtures set forth in claims 1- 5, said compound being applied to the plant immediately prior to or at a time when cell differentiation and growth of the plant or flower buds are great, that is, to seeds, to plant seedlings, or to trees during flower bud initiation, bud swell, or during a period of exponential vegetative growth, said compounds being applied in an amount enhancing plant growth but not harming the plant and being applied in an amount of approximately 0.001 to 0.3 mg active ingredient per plant seedling or about 1 to 10 grams active ingredient per tree, said enhancing of plant growth consisting of an increase in photosynthesis, total plant biomass and plant constituents selected from the group consisting of protein, lipid, sugar, and essential oil. 8) The method as set forth in claims 6-7 wherein the plant is propagated in vitro. 9) The method of claims 6-8 wherein the enhancing of plant growth further consists of an increased pigment accumulation, total soluble solids, vitamin, nutrient, or juice contents of fruit harvested from treated plants relative to untreated controls. 10) The method of claims 6-8 wherein the plant is a cereal grain. 11) The method of claims 6-8 wherein the plant is a legume. 12) The method of claim 6-8 wherein the plant is a citrus tree. 13) The method of claim 12 wherein the fruit of the treated citrus tree exhibits a reduced peel thickness relative to untreated citrus. 14) The method of claims 6-8 wherein the plant is a vegetable plant. 15) The method of claims 6-8 wherein the compound is applied as an aqueous dispersion. 16) The method of claims 6-8 wherein the compound is applied to the plant more than once. 17) The method as set forth in claims 6-8 wherein the plant is derived from an annual or perennial agronomic crop plant, a woody plant tissue, or from a plant grown for ornamental purposes. 18) The method of claims 6-8 wherein the enhancing of plant growth further consists of an accelerated structural maturation of the plant and reduction of days to crop harvest relative to untreated plants.

Inventors: GAUSMAN HAROLD W, KEITHLY JAMES H, YOKOYAMA HENRY, GAUSMAN, HAROLD, W., KEITHLY, JAMES, H., YOKOYAMA, HENRY
Applicants: TROPICANA PROD INC, US AGRICULTURE, THE UNITED STATES OF AMERICA AS REPRESENTED BY THE, TROPICANA PRODUCTS, INC.
Application year: 1993
Main classification: A01N33
IPC codes: A01N33, C07C217, C07C323