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Monocotyledon transgenic method for invading growing points of seed buds minimally and fully

A technology for monocotyledonous plants and growing points, which is applied in plant genetic improvement, botanical equipment and methods, biochemical equipment and methods, etc., can solve the problems of poor transformation effect, insufficient conditions, and heavy biological damage to growing points, etc. To achieve the effect of stable conversion effect, simple and convenient operation, and low cost

Inactive Publication Date: 2013-01-16
HEBEI ACADEMY OF AGRI & FORESTRY SCI INST OF GENETICS & PHYSIOLOGY
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AI Technical Summary

Problems solved by technology

[0007] (1) Transformation is carried out after vernalization, the timing of transformation is too late, which reduces the effective coverage of transformation, and provides insufficient conditions for Agrobacterium-mediated transformation in terms of trauma, and the biological damage is serious
[0008] (2) The operation is difficult, the scale is not easy to grasp, and the work efficiency is not high
[0009] In some reports, various needles were used to injure the growth point of the transformed object, but the diameter of the needles used is generally thick [African Journal for Biotechnology, 2011, 10(5):740-750], and some even reach 710 μm [Journal of bioscience and bioengineering, 2005, 100(4): 391-397; 2006, 102(3): 162-170], causing heavy biological damage to growth points, but not enough trauma for transformation
In some reports, a few strokes with a scalpel blade resulted in insufficient trauma for transformation, resulting in poor transformation results; there are also many reports that use Agrobacterium for transformation without any wound treatment, and the transformation effect is difficult to guarantee
[0010] In short, many patents or reports that focus on the growth point as the transformation object still do not leave the "in vitro culture" link, and the resistance screening strategy used is not scientific enough

Method used

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  • Monocotyledon transgenic method for invading growing points of seed buds minimally and fully
  • Monocotyledon transgenic method for invading growing points of seed buds minimally and fully
  • Monocotyledon transgenic method for invading growing points of seed buds minimally and fully

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Embodiment 1: Utilize minimally invasive brush to transform the experiment of winter wheat seed bud growth point

[0061] (1) Materials and methods

[0062] Wheat variety: "Shi 4185".

[0063] Agrobacterium strain: EHA105.

[0064] Exogenous Gene: gus + npt-II , constructed in pCAMBIA2201 plasmid.

[0065] Pick a single colony, transfer it to 50ml LB liquid medium containing 50mg / L kanamycin and 40mg / L rifampicin, and culture it at 28°C and 220rpm until the bacterial liquid OD 600 =0.6, 4000rpm, 5min centrifugation to collect the bacterial cells, discard the supernatant and add 1 / 2 of the LB liquid medium volume of the infection base solution (containing 100μmol / L AS, 100mg / L F68, 400 / L MES, 1 / 2 10 MS salt, 10g / L glucose, 40g / L maltose, pH 5.6) and shake well to prepare Agrobacterium-mediated transformation solution.

[0066] Select 90 plump and tidy seeds, soak them at 25°C for 7 hours, perform routine disinfection, wash them with sterile water, place them in ...

Embodiment 2

[0071] Embodiment 2: Utilize minimally invasive brush to transform the experiment of different genotype wheat seed bud growth points

[0072] (1) Materials and methods

[0073] Wheat varieties: "Jinhe 9123", "China Spring", "Bobwhite".

[0074] Agrobacterium strain: C58C1

[0075] Exogenous Gene: gus + npt-II , constructed in pCAMBIA2201 plasmid.

[0076] Pick a single colony, transfer it to 50ml LB liquid medium containing 50mg / l kanamycin + 40mg / l rifampicin, and culture it at 28°C and 220 rpm until the bacterial liquid OD600 =0.5, 4000rpm, 5min centrifugation to collect the bacterial cells, discard the supernatant and add 1 / 5 of the LB liquid medium volume of the infection base solution (containing 100μmol / L AS, 100mg / L F68, 400 / L MES, 1 / 10MS salt, 10g / L glucose, 40g / L maltose, pH 5.6) and shake well to prepare Agrobacterium-mediated transformation solution.

[0077] Qualified seeds of three wheat genotypes were respectively selected, soaked at 25°C for 10 h, routi...

Embodiment 3

[0085] Embodiment 3: Utilize minimally invasive brush to transform the experiment of rice seed bud growth point

[0086] (1) Materials and methods

[0087] Rice variety: "Longdao 10".

[0088] Agrobacterium strain: EHA105.

[0089] Exogenous Gene: gus + bar , constructed in pCAMBIA3301 plasmid.

[0090] Pick a single colony, transfer it to 50ml LB liquid medium containing 50mg / l kanamycin and 40mg / l rifampicin, and culture it at 28°C and 220 rpm until the bacterial liquid OD 600 =0.6, 4000rpm, 5min centrifugation to collect the bacterial cells, discard the supernatant and resuspend in 1 / 2 of the LB liquid medium volume of the infection base solution (containing 100μmol / L AS, 100mg / L F68, 400mg / L MES, 1 / 10 MS salt, 10g / L glucose, 40g / L maltose, pH 5.6) and shake well to prepare the transformation solution.

[0091] Select 120 plump and neat seeds, remove the seed husk, put them in a 90mm diameter petri dish after high temperature sterilization with 2 layers of filter p...

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Abstract

The invention relates to a monocotyledon transgenic method for invading growing points of seed buds minimally and fully. According to the technical key point, the monocotyledon transgenic method comprises the following steps of: germinating seeds for 1 to 2 days, and when buds extend to be 0.2 to 2 centimeters long, removing coleoptile to expose the growing points; pricking and brushing the growing points by using a minimally-invasive brush of which the diameter of a single brush hair is 4 to 20 micrometers, the exposed length is 0.5 to 3 millimeters and the number of the brush hairs is 100 to 5,000 and which is dipped with agrobacterium mediated conversion liquid to perform full minimally-invasive conversion; after performing co-culture, developing further to form seedlings, promoting the development of spikes and grains, and harvesting in a plant division mode; and performing identification on T1 generation. The monocotyledon transgenic method has the advantages that tissue culture, the limit of genotypes and resistance screening are avoided, the method is easy and convenient to operate and easy to realize in large scale and is suitable for all monocotyledons which bear seeds. By the monocotyledon transgenic method, the transformation effects that the genetic transformation rate of wheat is 49 percent, the genetic transformation rate of paddy is 66.3 percent and the genetic transformation rate of corn is 100 percent are achieved.

Description

technical field [0001] The invention relates to a monocotyledonous plant transgenic method which fully and minimally injures the growth point of seed buds, and is suitable for all monocotyledonous plants bearing seeds. Background technique [0002] Among many plant transgenic methods, the Agrobacterium-mediated method is the most widely recognized. It has the advantages of higher fertility of the obtained transgenic plants, integration of foreign genes in single copy or low copy in recipients, transfer of large fragments of DNA, and the like. However, most of the current Agrobacterium-mediated transgenic technologies are inseparable from tissue culture, so there are limitations of genotype, complicated operation, selection of resistance markers, long cycle, low transformation efficiency, and inconsequential transformation results. outstanding issues such as stability. In particular, transgenic monocotyledonous plants such as wheat, rice, and corn are constrained by genotyp...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
CPCC12N15/8205C12N15/8207
Inventor 王海波董福双吕孟雨张艳敏任志恒杨帆梁新潮左文博石学萍张欢欢高义平赵和徐显孙果忠柴建芳刘永伟朱金永韩秋芬张强马辉杰王占武关军锋
Owner HEBEI ACADEMY OF AGRI & FORESTRY SCI INST OF GENETICS & PHYSIOLOGY
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