The United States of America as Represented by the Secretary of

The United States of America as Represented by the Department of

Bruce E Haissig

Jack R Gaines

David A Boyles

Charles H Michler

Glen Giacoletto

Methods of in vitro culturing of tree tissue are disclosed in which high frequency tissue regeneration is achieved thereby increasing the likelihood of regenerating somaclonal variants having desirable traits. These methods include: culturing with a ratio of naphthaleneacetic acid and benzyladenine, or of benzyladenine and zeatin, in a tissue regeneration medium, the ratio being selected to induce a high frequency of tissue regeneration; culturing on a tissue regeneration medium having a high auxin concentration with a relatively low cytokinin concentration for a short period of time, e.g. up to ten days, to regenerate a high number of morphogenic callus cells; and, regenerating somatic embyros using a medium comprising 2,4-dichlorophenoxyacetic acid, benzyladenine, sucrose and glutamine, for high frequency regeneration of emboyogenic calli which are then developed into somatic embryos. These high frequency tissue regeneration methods are used to develop plants having tolerance to toxic chemicals by combining the methods with in vitro exposure to toxic chemical thereby to challenge somaclonal variants with the toxic chemical to test for and recover tolerant variants.

Methods of high frequency tissue regeneration, regeneration of

Aryl esters and thioesters of indole-3-alkanoic acids, such as indole-3-acetaic acid and indole-3-butyric acid, are prepared by chemical reaction of the acid with a phenol, substituted phenol, or thiophenol in a tertiary amine solvent. A phosphorus or sulfur halide in a suitable solvent is added dropwise to the cold amine solution. The reaction produced is separated and purified by standard methods. Phenyl-indole-3-thiolobutyrate as well as other aryl esters of indole-3-alkanoic acids is effective as an auxin plant growth regulator and adventitious root initiator.

Auxin compositions of phenyl thioesters of indole-3-alkanoic acids and

Aryl esters and substituted aryl esters of indole-3-alkanoic acids, such as indole-3-acetic and indole-3-butyric acids, are prepared by a chemical reaction mixture of the acid, a phenol, thiophenol or substituted phenol, a halogenated alkylpyridinium salt, and a tertiary amine in an organic solvent, stirring the mixture for a period of time, and isolating and purifying the product by standard procedures. An aryl amine or substituted aryl amine is substituted for phenol in the reaction mixture when the N-phenyl or N-substituted phenyl indolyl-3-acetamide or butyramide is the desired product. N-phenyl or N-substituted phenyl indolyl-3-acetamides are particularly effective as auxin plant growth regulators and adventitions root initiators.

Auxin compositions of N-phenyl and N-chloro phenyl indolyl-3-alkylene

Aryl esters of auxins, such as phenyl indole-3-butyrate, are found to significantly improve the initiation of rooting of cuttings is difficult-to-root species, such as pinus.

Growing community of inventors

Rhinelander, WI, United States of America

Bruce E Haissig