US 20210062203A1 IN ( 19 ) United States ( 12 ) Patent Application Publication ( 10 ) Pub . No .: US 2021/0062203 A1 ANAND et al . ( 43 ) Pub . Date : Mar. 4 , 2021 ( 54 ) METHODS FOR PLANT TRANSFORMATION Related U.S. Application Data ( 71 ) Applicant: PIONEER HI - BRED ( 60 ) Provisional application No. 62 / 641,733 , filed on Mar. INTERNATIONAL , INC . , Johnston, 12 , 2018 , provisional application No. 62 / 641,725 , IA (US ) filed on Mar. 12 , 2018 . ( 72 ) Inventors : Ajith ANAND , West Des Moines, IA Publication Classification ( US ) ; William James GORDON -KAMM , Urbandale , IA ( 51 ) Int. Ci . ( US ) ; George J. HOERSTER , Des C12N 15/82 ( 2006.01 ) Moines , IA ( US ) ; Keith S. LOWE , ( 52 ) U.S. CI . Johnston , IA ( US ) ; Kevin E. CPC C12N 15/8216 ( 2013.01 ) ; C12N 15/8207 MCBRIDE , Davis , CA (US ) ( 2013.01 ) ; C12N 15/8205 ( 2013.01 ) ( 73 ) Assignee : Pioneer Hi - Bred International , Inc. , Johnston , IA ( US ) ( 57 ) ABSTRACT ( 21 ) Appl. No .: 16 /979,324 The methods disclosed herein use a morphogenic gene or a repressor gene to provide a transgenic plant containing a ( 22 ) PCT Filed : Mar. 11 , 2019 single copy of a transgenic event wherein the morphogenic gene or the repressor gene does not integrate into the ( 86 ) PCT No .: PCT / US2019 / 021629 genome of the plant and the plant does not contain vector $ 371 ( c ) ( 1 ) , ( plasmid ) backbone. ( 2 ) Date : Sep. 9 , 2020 Specification includes a Sequence Listing . Patent Application Publication Mar. 4 , 2021 US 2021/0062203 A1 80 EmbryoFormation(9) SoxtatieofEmciency 40 30 WUS TAG - RFP Treatment Treatment FIG . 1 US 2021/0062203 A1 Mar. 4 , 2021 1 METHODS FOR PLANT TRANSFORMATION [ 0006 ] In an aspect , the disclosure provides a method of producing a transgenic plant having one copy of a trait gene CROSS - REFERENCE TO RELATED expression cassette comprising : contacting a plant cell with APPLICATIONS a T -DNA containing a trait gene expression cassette and a [ 0001 ] This application claims the benefit of PCT Appli T -DNA containing a morphogenic gene expression cassette; cation Serial Number PCT /US2019 /021629 , filed Mar. 11 , selecting a plant cell containing the trait gene expression 2019 , which claims the benefit of U.S. Provisional Patent cassette and no morphogenic gene expression cassette ; and Application Ser . No. 62 / 641,725 , filed Mar. 12 , 2018 and regenerating a transgenic plant from the plant cell . In a U.S. Provisional Patent Application Ser. No. 62 / 641,733 further aspect , contacting comprises simultaneously contact filed 12 Mar. 2018 , each of which are hereby incorporated ing a plant cell with a T - DNA containing a trait gene herein by reference in their entireties . expression cassette in a first bacterial strain and a T - DNA containing a morphogenic gene expression cassette in a FIELD OF THE DISCLOSURE second bacterial strain . In yet a further aspect , contacting comprises contacting a plant cell with one bacterial strain [ 0002 ] The present disclosure relates generally to the field containing a plasmid that comprises a trait gene expression of plant molecular biology, including genetic manipulation cassette within the T - DNA and a morphogenic gene expres of plants. More specifically , the present disclosure pertains sion cassette outside of the T - DNA . In yet another aspect , to rapid , high efficiency methods for producing a trans contacting comprises contacting a plant cell with the T - DNA formed plant. containing a trait gene expression cassette and the T -DNA containing a morphogenic gene expression cassette in one REFERENCE TO A SEQUENCE LISTING bacterial strain . In a further aspect , the morphogenic gene SUBMITTED AS A TEXT FILE VIA EFS - WEB expression cassette comprises : ( i ) a nucleotide sequence [ 0003 ] The official copy of the sequence listing is submit encoding a WUS / WOX homeobox polypeptide ; or ( ii ) a ted electronically via EFS - Web as an ASCII formatted nucleotide sequence encoding a Babyboom ( BBM ) poly sequence listing with a file named 7486 - US - PCT SeqList , peptide or an Ovule Development Protein 2 ( ODP2 ) poly created on Sep. 3 , 2020 , and having a size of 803,226 bytes peptide; or ( iii ) a combination of ( i ) and ( ii ) . In a further and is filed concurrently with the specification . The aspect , the nucleotide sequence encodes the WUS / WOX sequence listing contained in this ASCII formatted docu homeobox polypeptide. In a further aspect , the nucleotide ment is part of the specification and is herein incorporated by sequence encoding the WUS /WOX homeobox polypeptide reference in its entirety . is selected from 81 , WUS2 , WUS3 , WOX2A , WOX4 , WOX5 , and WOX9 . In a further aspect , the nucleotide BACKGROUND sequence encodes the Babyboom ( BBM ) polypeptide or the Ovule Development Protein 2 ( ODP2 ) polypeptide. In a [ 0004 ] The use of standard transformation and regenera further aspect , the nucleotide sequence encoding the Baby tion protocols is time consuming and inefficient, and nega boom ( BBM ) polypeptide is selected from BBM2 , BMN2, tively impacts transgenic product development timelines , and BMN3 or the Ovule Development Protein 2 ( ODP2 ) given that there is usually a seasonally limited " priority polypeptide is ODP2 . In a further aspect , the nucleotide development window " for making decisions regarding sequence encodes the WUS / WOX homeobox polypeptide which genetic constructs to prioritize for use in larger scale and the Babyboom ( BBM ) polypeptide or the Ovule Devel field work based on results obtained during initial research . opment Protein 2 ( ODP2 ) polypeptide. In a further aspect , The available standard methods of transformation and the nucleotide sequence encoding the WUS / WOX homeo regeneration have multiple drawbacks that limit the speed box polypeptide is selected from WUS1 , WUS2, WUS3 , and efficiency with which transgenic plants can be produced WOX2A , WOX4 , WOX5 , and WOX9 and the Babyboom and screened . For example, many standard methods of ( BBM ) polypeptide is selected from BBM2 , BMN2 , and transformation and regeneration require the use of high BMN3 or the Ovule Development Protein 2 ( ODP2 ) poly auxin or cytokinin levels and require steps involving either peptide is ODP2 . In a further aspect , the trait gene expres embryogenic callus formation or organogenesis , leading to sion cassette comprises: a trait gene selected from the group procedures that take many weeks before producing plants of a gene conferring pest resistance, a gene conferring for growth in a greenhouse setting following transformation . herbicide resistance , a gene conferring stress tolerance , a There remains a need for transformation methods that pro gene conferring drought resistance, a gene conferring nitro duce significantly higher transformation frequencies and gen use efficiency ( NUE ), a gene conferring disease resis significantly more quality events ( events containing one tance , and a gene conferring an ability to alter a metabolic copy of a trait gene expression cassette with no vector pathway. In a further aspect , the first bacterial strain and the ( plasmid ) backbone ) in multiple inbred lines using a variety second bacterial strain are the same bacterial strain . In a of starting tissue types, including transformed inbreds rep further aspect , the same bacterial strain is a Rhizobiales resenting a range of genetic diversities and having signifi selected from the group of a disarmed Agrobacteria, an cant commercial utility . Ochrobactrum bacteria , and a Rhizobiaceae bacteria . In a further aspect , the disarmed Agrobacteria is selected from SUMMARY the group of AGL - 1 , EHA105 , GV3101, LBA4404 , and [ 0005 ] The present disclosure comprises methods and LBA4404 THY- . In a further aspect , the Ochrobactrum compositions for producing transgenic events that contain bacteria is selected from Table 2. In a further aspect , the one copy of a trait gene . In a further aspect , the present Rhizobiaceae bacteria is selected from Table 3. In yet a disclosure provides a seed from the plant produced by the further aspect , the first bacterial strain and the second methods disclosed herein . bacterial strain are different bacterial strains . In a further US 2021/0062203 A1 Mar. 4 , 2021 2 aspect , the different bacterial strains are selected from : ( i ) a repressor - dCAS9 fusion having a guide RNA targeting the disarmed Agrobacteria and an Ochrobactrum bacteria; ( ii ) a promoter in the T - DNA . In a further aspect , the nucleotide disarmed Agrobacteria and a Rhizobiaceae bacteria; and sequence encodes the tetracycline repressor ( TETR ) repress ( iii ) a Rhizobiaceae bacteria and an Ochrobactrum bacteria . ing the promoter operably linked to the selectable marker in In a further aspect , the disarmed Agrobacteria is selected the T - DNA . In a further aspect , the nucleotide sequence from the group of AGL - 1 , EHA105 , GV3101, LBA4404 , encodes the ethametsulfuron repressor ( ESR ) repressing the and LBA4404 THY-. In a further aspect , the Ochrobactrum promoter operably linked to the selectable marker in the bacteria is selected from Table 2. In a further aspect , the T - DNA . In a further aspect , the nucleotide sequence encodes Rhizobiaceae bacteria is selected from Table 3. In a further the chlorsulfuron repressor ( CSR ) repressing the promoter aspect , the first bacterial strain and the second bacterial operably linked to the selectable marker in the T -DNA . In a strain are present in a 50:50 ratio . In a further aspect , the first