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D-amino acid selection for soybean

一种大豆、丝氨酸的技术，应用在基因工程、植物基因改良、重组DNA技术等方向，能够解决低再生效率等问题

Inactive Publication Date: 2009-04-08
BASF PLANT SCI GMBH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Although many of the problems associated with the transformation of soybean have been overcome by the methods described in the art, there is still a significant need for improvement, since all methods known to date have only low to moderate transformation and especially regeneration efficiencies

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0643] Example 1: Sterilization and Germination of Soybean Seeds

[0644] Almost any seed of any soybean variety can be used in the methods of the invention. Several soybean cultivars, including Jack, Williams 82, and Resnik, are suitable for soybean transformation. Soybean seeds were sterilized in a chamber with chlorine gas generated by the dropwise addition of 3.5 ml of 12N HCl to 100 ml of bleach (5.25% sodium hypochlorite) in a desiccator with a tightly capped bottle cap. After 24 to 48 hours in the chamber, the seeds were removed and approximately 18 to 20 seeds were placed on solid GM medium in a 25 x 100 mm dish with or without 5 μM 6-benzylaminopurine (BAP ). Seedlings without BAP were more elongated, with root development, especially secondary and lateral root formation. BAP strengthens the seedlings by forming shorter, stubby seedlings.

[0645] Seven-day-old seedlings grown under light (>100 μM / m2s) at 25°C were used for explant material for the three explant t...

Embodiment 2

[0646] Example 2: Growth and preparation of Agrobacterium cultures

[0647] Prepare Agrobacterium cultures by streaking Agrobacterium carrying the desired binary vector (e.g., A. tumefaciens or A. rhizogenes) onto solid YEP growth medium and incubating at 25°C , until colonies appear (approximately 2 days). Different selection compounds will be used for selection of A. tumefaciens and A. rhizogenes in YEP solid and liquid media, depending on the selectable marker gene present on the Ti or Ri plasmid, binary vector and bacterial chromosome. A variety of Agrobacterium strains are available for transformation methods.

[0648] After roughly 2 days, pick a single colony (use a sterilized toothpick), inoculate 50ml of liquid YEP containing antibiotics, and shake at 175rpm (25°C) until an OD of 0.8-1.0 is reached 600 (roughly 2 days). A working glycerol stock (15%) was prepared for transformation and 1 ml of the Agrobacterium stock was aliquoted into 1.5 ml Eppendorf tubes and st...

Embodiment 3

[0650] Example 3: Explant preparation and co-cultivation (inoculation)

[0651] Seedlings have elongated epicotyls by at least 0.5 cm by this time, but usually between 0.5 and 2 cm. Elongated epicotyls up to 4 cm long have been used successfully. Explants are then prepared which:

[0652] i) with or without some roots,

[0653] ii) With part, one or two cotyledons, all pre-formed leaves are removed, including the apical meristem, using a sharp scalpel to cut down and damage the nodes located in the first set of leaves.

[0654] Cutting at the nodes not only induces Agrobacterium infection, but also disperses the axillary meristem cells and destroys pre-formed shoots. After injury and preparation, the explants were removed from the dish and subsequently co-incubated with the liquid CCM / Agrobacterium mixture for 30 minutes. The explants were then removed from the liquid medium and placed on top of sterile filter paper on 15 x 100 mm plates with solid co-cultivation medium. ...

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Abstract

The present invention relates to improved methods and means for transformation of soybean (Glycine max) based on a D-alanine and / or D-serine selection.

Description

technical field [0001] The present invention relates to an improved method for the transformation of soybean (Glycinemax) based on D-alanine and / or D-serine selection. Background of the invention [0002] Soybean (Glycine max) belongs to the family Fabaceae (Leguminosae). Soy is thought to have originated in China. Wild-type soybeans are vine-growing in nature, which may explain why soybeans were originally introduced to the United States as a hay crop. For the United States, introductions from China, Manchuria, Korea, and Japan were important in developing the breed. Modern breeding efforts to improve agronomic traits such as more erect growth, reduced lodging and increased seed size were major factors in the development of soybean as an important crop worldwide. The area and proportion of crops harvested for grain has grown steadily and soybeans are now the leading world commodity. [0003] The cultivated soybean has high commercial value in the world. Worldwide, more...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H1/00C07K14/415C12N9/02C12N9/88
CPCC12N15/821
Inventor D·阿里亚斯S·普里斯L·格里斯特M·程H·洪L·贝尔纳尔P·奥尔霍夫特H-S·宋L·曼金
Owner BASF PLANT SCI GMBH
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