Haploid maize transformation

A haploid and double haploid technology, applied in the field of haploid maize transformation, can solve the problem that no one has explored site specificity

Inactive Publication Date: 2017-02-22
DOW AGROSCIENCES LLC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, stable site-specific (targeted) modification of selected positions in the maize haploid genome has still not been explored.

Method used

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  • Haploid maize transformation
  • Haploid maize transformation
  • Haploid maize transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0271] Example 1: Generation of microspore-derived haploid callus

[0272] Maize genotype 139 / 39-05 (U.S. Patent No. 5,306,864) was harvested from greenhouse-grown maize plants (approximately 5-6 weeks old) when the microspores were in the early binucleate stage of development (anthers approximately 3 mm long, bright, lustrous yellow) The pre-emergent tassel (Pre-emergent tassel). The tassels were wrapped in wet paper towels and aluminum foil and placed in an incubator set at 8°C for 7-14 days. Sterilized on surfaces (at 0.08% v / v Chlorox TM 15 minutes, followed by rinsing with sterile water), the anthers were aseptically separated, and placed in a liquid "anther culture medium" (N6 salt and Vitamins, 60 g / L sucrose, 5 g / L activated carbon, 500 mg / L casein hydrolyzate, 0.1 mg / L TIBA, adjusted to pH 5.8) on the surface and incubated at 28°C in the dark. Microspore-derived embryo-like structures that appeared between 14-28 days were transferred to "callus medium" (MS salts an...

Embodiment 2

[0274] Example 2: Ploidy determination of callus

[0275] In order to measure the cell ploidy level, 1 g of the callus prepared in Example 1 was transferred to sterile Petri TM Dishes (Fisher Scientific, St. Louis, MO). Pass through ice-cold Gailbraith buffer (0.01M MgSO) filtered in 1-2mL 4 , 0.005M KCl, 0.0005M HEPES, 1mg / mL DTT) and "MMG Medium" (4mM MES [pH 6.0], 0.6M Mannitol, 15mM MgCl 2 ) and 0.25% Triton X-100 TM The callus was minced with a single-edged razor blade in the presence of the nucleus to release it. Rinse the Petri with another 2 mL of buffer TM dish, which was combined with the initial nuclear extract to give a final slurry volume of approximately 5 mL. The crude nuclear extract was then gently homogenized by transferring to a glass tissue homogenizer, pumping the plunger up and down several times. Then, the homogenate was filtered through a tea strainer, 40 μm Steri-flip TM (Millipore; Billerica, Massachusetts, USA) the resulting filtrate was aspir...

Embodiment 3

[0276] Example 3: Constructs for targeted genome modification in protoplasts isolated from haploid callus

[0277] For targeted genome modification, the zinc finger nuclease (ZFN) construct pDAB111879 (depicted in figure 1 middle). Expression of the ZFN is driven by the maize ubi1 promoter and terminated with the maize per5 3'UTR. This expression cassette contains a "T2A" construct comprising two zinc finger monomer domains (zmPPL_1360-r23a1 and zmPPL_1360-30a1), encoded and expressed by a single coding region. The expressed transcript contains a T2A stutter signal (Mattion et al., 1996, J. Virol., 70:8124-7) to introduce a ribosomal stutter, which releases the first polypeptide during translation, and the designed To produce equimolar amounts of the first and second polypeptides upon further translation. The opaque2 nuclear localization sequence (NLS) was included in both ZF monomers for nuclear targeting. Both NLS-ZF domain fusions possess binding specificities for the u...

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Abstract

Disclosed are methods for transformation of an androgenic-derived, haploid cell line with a site-specific nuclease. In some embodiments, the androgenic-derived, haploid cell line is a maize microspore-derived plant tissue culture. In addition, the disclosure provides a method for modifying, e.g., by mutating or targeting and integrating donor DNA into, a specific locus of a haploid or dihaploid tissue genome. The disclosure further provides methods for regenerating a whole plant from the haploid or dihaploid tissue that contains either the mutation at a specific genomic locus or a donor DNA integrated within a specific genomic locus may be obtained from the subject disclosure.

Description

[0001] field of invention [0002] The present disclosure relates generally to plants, plant tissues and cell lines, regenerated plants, and methods for introducing and / or rearranging nucleic acids therein. Methods are provided for transformation (including site-specific insertion) of exogenous DNA and gene editing in maize male-derived haploid cells. [0003] Background of the invention [0004] Transgenic maize production typically involves delivery of the transgene by Agrobacterium co-cultivation or microparticle bombardment into diploid somatic tissues, such as the scutellum region of immature zygotic embryos. These procedures usually result in integrated transgenes in primary transformants (T 0 ) in "semi-merge" and in T 1 Plant generations segregate. Transferring transgenes into other genetic backgrounds requires introgression through backcrossing. Thus, such methods of delivering a transgene into the genome of a diploid somatic tissue or cell and transferring the tra...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H5/00C12N15/82C12N15/87
CPCC12N15/8213
Inventor J·F·佩达利诺T·L·斯特兰奇J·P·塞缪尔R·C·布卢M·A·辛普森
Owner DOW AGROSCIENCES LLC
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