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Methods and compositions for improving plant traits

一种植物、植物根的技术，应用在用于改良植物性状及组合物领域，能够解决途径不清楚等问题

Active Publication Date: 2020-02-14
PIVOT BIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although tremendous progress has been made in understanding the development of nitrogen-fixing symbiotic relationships between rhizobia and legumes, the pathways for harnessing this knowledge to induce nitrogen-fixing nodules on non-legume crops remain unclear
At the same time, the challenge of providing adequate supplemental nitrogen sources, such as in fertilizers, will continue to increase with the growing need for increased food production

Method used

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  • Methods and compositions for improving plant traits
  • Methods and compositions for improving plant traits
  • Methods and compositions for improving plant traits

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0351] Example 1: Isolation of microorganisms from plant tissue

[0352] Topsoil was obtained from various agricultural regions in central California. Twenty plots of soil with different textural characteristics were collected, including heavy clay, peat clay loam, silty clay and sandy loam. Seeds of various field corn, sweet corn, conventional corn and tomato were planted in each soil as shown in Table 1.

[0353]

[0354] Table 1: Types and varieties of crops grown in soils with different characteristics

[0355] Plants were pulled out after 2-4 weeks of growth, and excess soil on the root surfaces was removed with deionized water. After removing the soil, surface-sterilize the plant with bleach and rinse vigorously in sterile water. Cleaned 1 cm root segments were excised from plants and placed in phosphate-buffered saline solution containing 3 mm steel beads. A slurry was generated by vigorously shaking the solution with a Qiagen TissueLyser II.

[0356] Root and...

Embodiment 2

[0358] Example 2: Characterization of isolated microorganisms

[0359] Sequencing, analysis and phylogenetic characterization

[0360] 16S rDNA sequencing with the 515f-806r primer set was used to generate preliminary phylogenetic characterization of isolated and candidate microorganisms (see eg Vernon et al.; BMC Microbiol. 2002 Dec 23; 2:39). As shown in Table 2, microorganisms include different genera including: Enterobacter, Burkholderia, Klebsiella, Bradyrhizobium, Lahnella, Xanthomonas genera, Raoultella, Pantoea, Pseudomonas, Brevundomonas, Agrobacterium, and Paenibacillus.

[0361]

[0362]

[0363] Table 2: Diversity of microorganisms isolated from tomato plants determined by deep 16S rDNA sequencing.

[0364] Subsequently, the genomes of 39 candidate microorganisms were sequenced using the Illumina Miseq platform. Genomic DNA from pure cultures was extracted using the QIAmp DNA Mini Kit (QIAGEN), and total DNA libraries for sequencing were prepared by a th...

Embodiment 3

[0413] Example 3: Mutagenesis of Candidate Microorganisms

[0414] λ-Red-mediated knockout

[0415] Several mutants of candidate microorganisms were generated using plasmid pKD46 or derivatives containing a kanamycin resistance marker (Datsenko et al., 2000; PNAS 97(12):6640-6645). Knockout cassettes were designed with 250 bp of homology flanking the target gene and generated by overlap extension PCR. Candidate microorganisms were transformed with pKD46, grown in the presence of arabinose to induce expression of the λ-Red machinery, ready for electroporation, and transformed with the knockout cassette to generate candidate mutagenized strains. As shown in Table 4, four candidate microorganisms and one laboratory strain Klebsiella oxytoca M5A1 were used to generate thirteen candidate mutants of the nitrogen fixation regulatory genes nifL, glnB and amtB.

[0416]

[0417] Table 4: List of individual knockout mutants generated by λ-Red mutagenesis

[0418] Oligonucleotide...

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Abstract

Methods and systems are provided for generating and utilizing a bacterial composition that comprises at least one genetically engineered bacterial strain that fixes atmospheric nitrogen in an agricultural system that has been fertilized with more than 20 lbs of Nitrogen per acre.

Description

[0001] cross reference [0002] This application requires U.S. Provisional Application 62 / 445,570 filed on January 12, 2017, U.S. Provisional Application 62 / 445,557 filed on January 12, 2017, U.S. Provisional Application 62 / 447,889 filed on January 18, 2017, 2017 Interests in U.S. Provisional Application 62 / 467,032 filed on March 3, U.S. Provisional Application 62 / 566,199 filed on September 29, 2017, and U.S. Provisional Application 62 / 577,147 filed on October 25, 2017, all of which are incorporated by reference Incorporated herein. [0003] Statement about sequence listing [0004] This application contains a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on January 3, 2018, named 47736-707_601_SL.txt, and is approximately 599kb in size. [0005] Statement Regarding Federally Funded Research [0006] This invention was completed with support from the U.S. gove...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C05F11/08C07K14/195C12N1/20C12N15/63A01N63/20
CPCC12N1/20A01H3/00C05F11/08C12N15/52A01N63/20C12R2001/025C12N1/205C12R2001/22C12R2001/01A01H6/4684C12N1/04C12N15/111C12N15/70C12N15/743C12R2001/065C12R2001/07
Inventor 卡斯滕·泰米阿尔文·塔姆希尔莎拉·布洛赫罗斯玛丽·克拉克艾米丽·董凯文·哈米尔道格拉斯·希金斯奥斯汀·戴维斯-理查德森
Owner PIVOT BIO
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