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Anniversary large-scale maize transformation method

A large-scale, corn technology, applied in horticultural methods, botanical equipment and methods, introduction of foreign genetic material using vectors, etc., can solve the problems of hindering the development of transgenic corn, unable to screen out a single plant, low transformation rate, etc. Large-scale transformation, easy plant regeneration, and high regeneration efficiency

Inactive Publication Date: 2013-06-26
北京金冠丰生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the above reasons, the scale of transgenic maize in my country has been relatively small at present, and the transformation rate is relatively low, so that it is impossible to screen out single plants with excellent target traits, which seriously hinders the development of transgenic maize towards industrialization.

Method used

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  • Anniversary large-scale maize transformation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0056] Example 1 Large-scale transformation of young maize embryos mediated by Agrobacterium tumefaciens

[0057] 1. Planting of batch material in the field

[0058] The corn receptor material is sown in batches during the corn growing season, usually every 4-7 days. When the temperature is low in early spring, methods such as raising seedlings, laying plastic film, and erecting small sheds can be used to promote the growth of corn.

[0059] Strictly bagging, self-crossing or sister-crossing before corn loosening, control the number of pollinated plants to roughly the same number every day.

[0060] Pollination is 9-13 days, when the young embryo grows to 1.5-2.0mm, take the ear and peel the embryo, and shorten the time between getting the ear and peeling the embryo as much as possible.

[0061] 2. Preparation of Agrobacterium tumefaciens (containing the gene of interest)

[0062] (1) Preparation of Competent Agrobacterium: Pick a single colony of Agrobacterium tumefaciens ...

Embodiment 2

[0080] Example 2 Large-scale transformation of maize coleoptile node callus mediated by Agrobacterium tumefaciens

[0081] 1. Seed sterilization

[0082] First, clean up the sundries in the corn seeds, select the full and complete seeds, and then put them in a container and wash them with distilled water 3 times, during which time they shake vigorously to clean up the residues on the attached surface. Soak it in 75% alcohol for 3 minutes on the ultra-clean table, then soak it in 5.25% sodium hypochlorite for 20 minutes, and finally rinse it with sterilized water for 5-6 times.

[0083] 2. Germination culture

[0084] Place the sterilized seeds in the germination medium, and pay attention to horizontally inoculate the embryos of the seeds in the germination medium. In the culture room (16h / d light, light intensity 80-100μE / m 2 / s, 26-28℃) for 7-10 days.

[0085] The above germination medium is the following substances and final concentrations added to the MS basic medium: m...

Embodiment 3

[0120] Example 3 PCR detection of transgenic corn

[0121] 1. Extraction of DNA from maize leaves (CTAB method)

[0122] Cut the 2-3cm long transgenic corn plant leaves and cut them into pieces, put them into a 2.0ml centrifuge tube, add a steel ball; place the centrifuge tubes with the leaves and steel balls in the centrifuge tube rack dedicated to the sampler in order (8×5), soak the whole in a fresh-keeping box filled with liquid nitrogen for 1-2min; put the frozen centrifuge tube rack into the sampler, and sample at 1200 rpm for 20s; add 600μL CTAB extract ( 65°C preheating), 65°C water bath for 30min, take out and invert 1-2 times in the middle; add an equal volume (600uL) of chloroform:isoamyl alcohol (24:1) extract, close the lid tightly, mix up and down, and let stand 10-15min. Centrifuge at 11,000 rpm for 5 minutes until the phases are clearly separated; absorb 500 μL of the supernatant, transfer it to a new 1.5 mL sterilized centrifuge tube, add 2 / 3 volume of isopr...

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Abstract

The invention relates to an anniversary large-scale maize transformation method and belongs to the field of agricultural biotechnology and crop breeding. The anniversary large-scale maize transformation method mainly comprises the following steps of: (1) sowing maize acceptor materials in the maize growing reason in batches at an interval of 4-7 days; (2) selfing or carrying out sister cross, and controlling the number of plants pollinated each day to be approximately identical; (3) after pollinating for 9-12 days, stripping young embryos to carry out agrobacterium tumefaciens mediation conversion; (4) germinating maize seeds so as to obtain seedlings when the young embryos cannot be obtained; (5) intercepting coleoptile knots to induce calluses and subculturing; and (6) transforming calluses by adopting agrobacterium tumefaciens mediation conversion. The anniversary large-scale maize transformation method has the advantage of effectively overcoming the shortcomings of shortage of acceptor materials for maize transformation and high cost, anniversary large-scale transformation is easy to realize, and thus a large number of transgenosis seedlings are obtained.

Description

technical field [0001] The technical field to which the present invention belongs is the field of agricultural biotechnology and the field of crop breeding. Background technique [0002] Corn is one of the three most important food crops in the world, and it is also an important industrial raw material and bio-energy material. It plays a pivotal role in the food production of our country and the world. Among the many factors that increase the yield of corn, the role of new variety selection accounts for about 40%. With the rapid development of molecular biology technology, transgenic technology began to play an important role in the breeding of new maize varieties. [0003] Transgenic technology is based on the breeding objectives, the use of modern biotechnology means to isolate the target gene from the donor organism, through DNA recombination and genetic transformation into the recipient crops, after screening to obtain stable expression of transgenic lines, combined wit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 万向元方才臣吴锁伟赵虎基刘艳艳张丹凤肖中华徐媛媛王燕安学丽王超
Owner 北京金冠丰生物技术有限公司
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