The United States of America As Represented by the Secretary of

United States of America, As Represented by the Secretary of Agriculture

Jeffrey P Velten

John J Burke

Melvin John Oliver

Patrick J O'Mahony

Melvin J Oliver

Transgenic cells may be selected using temperature sensitive marker proteins. In this method, a population of host cells are transformed with a foreign DNA construct which includes at least one first nucleic acid coding sequence and a second nucleic acid sequence encoding a temperature sensitive marker protein, wherein each of the first and second nucleic acid coding sequences are operatively linked to gene expression control sequences. Suitable temperature sensitive marker proteins which may be used herein include heat shock proteins, heat shock transcription factors, cold regulated proteins (COR), or cold regulated protein transcription factors. Following transformation, the population of cells are cultured under temperature conditions wherein growth of non-transformed cells is suppressed or prevented while growth of cells transformed with the DNA construct is supported or promoted. Thus, survival and/or significant growth of a cell is an indication that the cell has been successfully transformed with the DNA construct. Those cells are considered presumptively positive transformants, and may be recovered.

Selection procedure for identifying transgenic cells, embryos, and plants without the use of antibiotics

A medium is disclosed that supports pollen germination and pollen tube growth in the presence of Agrobacterium, the medium comprising agarose, sucrose, NO.sub.3, MnSO.sub.4, H.sub.3 BO.sub.3, MgSO.sub.4 and gibberellic acid. A method is disclosed for the genetic transformation of plants and lines by a pollen-based Agrobacterium-mediated transformation. The method comprises the steps of obtaining pollen from a first plant, applying a lawn of Agrobacteria to a solid pollen culture medium, the Agrobacterium comprising at least one heterologous gene sequence capable of being transferred to a plant cell, applying the pollen to the solid medium, allowing the pollen to germinate and grow on the medium, whereby the Agrobacterium attaches to the pollen tube or mediates transfer of the heterologous gene sequence to the germinating pollen to obtain transgenic pollen, applying the transgenic pollen to the stigma of a second plant capable of being fertilized by the pollen of the first plant, thereby fertilizing the second plant, obtaining transgenic seed from the second plant and germinating the transgenic seed to obtain a transgenic plant. The method is particularly suited for use with so-called 'dry stigma' pollen.

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Lubbock, TX, United States of America

Jeffrey P Velten