Methods and compositions for the introduction of molecules into cells

Abstract

The present invention is directed to the introduction of molecules, including nucleic acids, carbohydrates, plant growth regulators and peptides into cells and tissues. The present invention is also directed to media and methods for enhancing embryogenic callus production of elite lines of soybean.

What is claimed is:  
     
         1 . A method for the introduction of molecules into a cell, comprising: 
 (a) providing a solution containing said molecules;    (b) producing aerosol droplets comprising said molecules;    (c) accelerating said aerosol droplets toward said cell; and    (d) impacting said cell with said accelerated aerosol droplets.    
     
     
         2 . The method of    claim 1   , wherein said molecules are selected from the group consisting of carbohydrates, nucleotide sequences, plant growth regulators, peptides, and combinations thereof.  
     
     
         3 . The method of    claim 2   , wherein said molecules comprise carbohydrates.  
     
     
         4 . The method of    claim 2   , wherein said molecules comprise nucleotide sequences.  
     
     
         5 . The method of    claim 2   , wherein said molecules comprise plant growth regulators.  
     
     
         6 . The method of    claim 2   , wherein said molecules comprise peptides.  
     
     
         7 . The method of    claim 1   , wherein said cell is selected from the group consisting of plant cell, animal cell, and bacterial cell.  
     
     
         8 . The method of    claim 7   , wherein said cell is a plant cell.  
     
     
         9 . The method of    claim 8   , wherein said plant cell is a monocotyledonous plant cell.  
     
     
         10 . The method of    claim 9   , wherein said monocotyledonous plant cell is a corn cell.  
     
     
         11 . The method of    claim 8   , wherein said plant cell is dicotyledonous plant cell.  
     
     
         12 . The method of    claim 11   , wherein said dicotyledonous cell is a soybean cell.  
     
     
         13 . The method of    claim 1   , wherein said aerosol droplets which impact said cell comprise aerosol droplets which are less than 0.1 micron in diameter.  
     
     
         14 . The method of    claim 1   , wherein said aerosol droplets are continuously targeted toward said cell.  
     
     
         15 . The method of    claim 1   , further comprising the placement of said cell on a target surface the linear and rotational movement of which can be separately controlled.  
     
     
         16 . The method of    claim 4   , wherein said nucleotide sequences comprise a vector.  
     
     
         17 . The method of    claim 16   , wherein said vector is selected from the group consisting of pBARGUS, pRBTBAR, pBARGFP, pSB12BARAHAS, pNPTAHAS, and combinations thereof.  
     
     
         18 . The method of    claim 1   , wherein said aerosol droplets are produced by a micro-flow nebulizer.  
     
     
         19 . The method of    claim 8   , wherein said molecules comprise nucleotide sequences, and said method further comprises transforming said plant cell with said nucleotide sequences and regenerating a transgenic plant from said transformed plant cell.  
     
     
         20 . The method of    claim 19   , further comprising obtaining progeny of said transgenic plant, wherein said progeny comprises said nucleotide sequence.  
     
     
         21 . The transgenic plant produced by the method of    claim 19   , wherein said transgenic plant is a monocot.  
     
     
         22 . The transgenic monocot plant of    claim 21   , wherein said monocot is a corn plant.  
     
     
         23 . The transgenic plant produced by the method of    claim 19   , wherein said transgenic plant is a dicot.  
     
     
         24 . The transgenic dicot plant of    claim 23   , wherein said dicot is a s oybean plant.  
     
     
         25 . The progeny of the transgenic plant of    claim 21   .  
     
     
         26 . The progeny of the transgenic plant of    claim 22   .  
     
     
         27 . The progeny of the transgenic plant of    claim 23   .  
     
     
         28 . The progeny of the transgenic plant of    claim 24   .  
     
     
         29 . Cells obtained from the progeny of    claim 20   .  
     
     
         30 . A method of enhancing embryogenic callus production from embryos of elite soybean lines which comprises culturing said embryos on a medium comprising phytic acid, wherein the concentration of said phytic acid is between about 1 gm/I to less than 3000 mg/I.  
     
     
         31 . A method of enhancing embryogenic callus production from embryos of elite soybean lines which comprises culturing said embryos on a medium comprising coconut water, wherein the concentration of said coconut water is between about 3% and about 6% by volume of said medium.  
     
     
         32 . The method of    claim 31   , wherein said medium further comprises myoinositol and wherein the concentration of said myoinositol is about 1 g/I to about 10 g/I.  
     
     
         33 . The method of    claim 32   , wherein said medium further comprises inorganic phosphorous.  
     
     
         34 . The method of    claim 33   , wherein said inorganic phosphorous is in the form of KH 2 PO 4  and wherein the concentration of said KH 2 PO 4  is between about 500 mg/I and about 1000 mg/I.  
     
     
         35 . The method of    claim 32   , wherein said medium further comprises phytic acid and wherein the concentration of said phytic acid is about 1 mg/I to less than about 3000 mg/I.  
     
     
         36 . The method of    claim 33   , wherein said medium further comprises phytic acid and wherein the concentration of said phytic acid is about 1 mg/I to less than about 3000 mg/I.  
     
     
         37 . The method of    claim 12   , further comprising culturing said soybean cell prior to impact using the method of    claim 30   .  
     
     
         38 . The method of    claim 12   , further comprising culturing said soybean cell prior to impact using the method of    claim 31   .  
     
     
         39 . The method of    claim 12   , further comprising culturing said soybean cell prior to impact using the method of    claim 32   .  
     
     
         40 . The method of    claim 12   , further comprising culturing said soybean cell prior to impact using the method of    claim 33   .  
     
     
         41 . The method of    claim 12   , further comprising culturing said soybean cell prior to impact using the method of    claim 35   .  
     
     
         42 . The method of    claim 12   , further comprising culturing said soybean cell prior to impact using the method of    claim 36   .  
     
     
         43 . The method of    claim 12   , further comprising exposing the soybean cell to about 1 gm/I phytic acid for about 3 to about 10 days before impacting said soybean cell.  
     
     
         44 . An aerosol beam apparatus, comprising: 
 a vacuum chamber having a nozzle and adapted to contain a target to be impacted;    an entrainment housing having an interior communicating with said vacuum chamber;    a pressurized gas supply;    a nebulizer located within said entrainment housing and having a nebulizer orifice;    an sample material supply conduit communicating with said nebulizer and an sample material supply for supplying sample material;    a nebulizer conduit communicating with said pressurized gas source and said nebulizer; and    an entrainment tube communicating with said pressurized gas source and said interior of said entrainment housing;    wherein a pressurized gas from said pressurized gas supply travels through said nebulizer conduit to said nebulizer and said sample material is carried by said pressurized gas through said nebulizer orifice, whereupon said pressurized gas and said sample material travel through said interior of said entrainment housing and enter said vacuum chamber through a nozzle and impact said target while said pressurized gas also travels through said interior of said entrainment tube to said entrainment housing and a resulting pressurized entrainment gas flows in a guiding manner through said entrainment housing substantially parallel to and around said pressurized gas and said sample material.    
     
     
         45 . The aerosol beam apparatus of    claim 44   , wherein said vacuum chamber is evacuated to a partial vacuum of about 26 to about 30 inches of Mercury.  
     
     
         46 . The aerosol beam apparatus of    claim 44   , wherein said vacuum chamber further includes a movable stage for supporting said target.  
     
     
         47 . The aerosol beam apparatus of    claim 44   , wherein said pressurized gas supply is a pressurized helium supply.  
     
     
         48 . The aerosol beam apparatus of    claim 44   , wherein said pressurized gas supply further includes a filter.  
     
     
         49 . The aerosol beam apparatus of    claim 44   , wherein said pressurized gas supply further includes at least one pressure regulator.  
     
     
         50 . The aerosol beam apparatus of    claim 44   , wherein said pressurized gas supply further includes a pressure regulator before said nebulizer conduit and a pressure regulator before said entrainment tube.  
     
     
         51 . The aerosol beam apparatus of    claim 44   , wherein said entrainment housing further includes a nucleospot.  
     
     
         52 . The aerosol beam apparatus of    claim 44   , wherein said entrainment housing further includes a temperature controller for controlling a gas temperature in said entrainment housing.  
     
     
         53 . The aerosol beam apparatus of    claim 44   , wherein said entrainment housing further includes a temperature controller that maintains a gas temperature in said entrainment housing in a range of about 32 degrees to about 80 degrees Centigrade.  
     
     
         54 . The aerosol beam apparatus of    claim 44   , wherein said nozzle has an orifice with a diameter of about 200 microns to about 500 microns.