Methods for producing genetically modified plants, plant materials and plant products produced thereby

Abstract

Methods for producing genetically modified plants, particularly woody plants, and most particularly plants of the Eucalyptus and Pinus species, involve transformation of target plant material with a desired genetic construct and regeneration of the transformed plant material using an adventitious shoot bud system. The methods provide a high transformation efficiency and substantially reduce the duration of the transformation and regeneration protocols. Stem segments of a target plant are transformed using Agrobacterium-mediated techniques, and adventitious shoot buds are regenerated from the Agrobacterium-infected stem segments. Preferred culture media, including selection media, and improved plant culture techniques are disclosed.

We claim:  
     
         1 . A method for producing genetically modified plant material of the Eucalyptus or Pinus species, comprising: 
 culturing nodal stem segments of a target plant selected from the Eucalyptus and Pinus species;    transforming the stem segments with a genetic construct by incubating the nodal stem segments with an Agrobacterium culture transformed with the genetic construct;    promoting regeneration of adventitious shoot buds from the transformed stem segments;    selecting transformed adventitious shoot buds; and    regenerating transformed plant material from the transformed adventitious shoot buds.    
     
     
         2 . A method according to  claim 1 , wherein the stem segments are derived from micropropagated shoot cultures.  
     
     
         3 . A method according to  claim 1 , wherein the target plant stem segments are pretreated in a multiplication medium and then transferred to a shoot elongation medium.  
     
     
         4 . A method according to  claim 1 , wherein the stem segments are wounded prior to incubation with the Agrobacterium culture.  
     
     
         5 . A method according to  claim 1 , additionally comprising excising adventitious shoots from the stem segments prior to selection of the transformed adventitious shoot buds.  
     
     
         6 . A method according to  claim 1 , wherein the genetic construct comprises a selection marker that confers resistance to a selection agent, the method additionally comprising selecting transformed adventitious shoot buds by exposing the adventitious shoot buds to a first selection medium having a first concentration of a selection agent and subsequently exposing the adventitious shoot buds surviving exposure to the first selection medium to a second selection medium having a second concentration of a selection agent greater than the first concentration.  
     
     
         7 . A method according to  claim 6 , wherein the first and second selection media comprise kanamycin.  
     
     
         8 . A method according to  claim 7 , wherein the first selection medium has a concentration of kanamycin less than or equal to 50 mg/l, and the second selection medium has a concentration of kanamycin greater than 50 mg/l.  
     
     
         9 . A method according to  claim 1 , wherein the genetic construct comprises genetic material that is homologous to the genome of the target plant.  
     
     
         10 . A method according to  claim 1 , wherein the genetic construct comprises genetic material that is heterologous to the genome of the target plant.  
     
     
         11 . A method according to  claim 1 , wherein the genetic construct comprises genetic material that affects one of the following phenotypic properties of the target plant: insect tolerance; disease resistance; herbicide tolerance; sterility; rooting ability; temperature tolerance; drought tolerance; salinity tolerance; wood properties; and growth rates.  
     
     
         12 . A method according to  claim 1 , wherein the genetic construct comprises genetic material encoding a polypeptide of interest or a functional portion of a polypeptide of interest.  
     
     
         13 . A method according to  claim 1 , wherein the genetic construct comprises an antisense copy of a gene or a portion of a gene encoding a polypeptide of interest or a functional portion of a polypeptide of interest.  
     
     
         14 . A method according to  claim 1 , additionally comprising exposing the stem segments to a reduced pressure atmosphere during incubation with the Agrobacterium culture.  
     
     
         15 . A method according to  claim 1 , wherein the genetic construct comprises at least one promoter region.  
     
     
         16 . A method according to  claim 1 , wherein the genetic construct comprises more than one promoter region.  
     
     
         17 . A method according to  claim 1 , additionally comprising promoting axillary shoot growth prior to transforming.  
     
     
         18 . A method according to  claim 1 , comprising regenerating transformed plant material by transferring the transformed adventitious shoot buds to a rooting medium and forming plantlets.  
     
     
         19 . A method according to  claim 18 , additionally comprising transferring the plantlets to a planting medium and growing the plantlets to form mature, genetically modified plants.  
     
     
         20 . Genetically modified plants produced according to the method of  claim 19 .  
     
     
         21 . Plant materials and plants derived from the genetically modified plants of  claim 20 .  
     
     
         22 . Plant products derived from the genetically modified plants of  claim 20 .  
     
     
         23 . Genetically modified plant materials produced according to the method of  claim 1.