Method for transforming exogenous gene by using maize coleoptile joint induced calluses

A callus, foreign gene technology, applied in horticultural methods, botanical equipment and methods, introduction of foreign genetic material using vectors, etc., can solve the problems of genotype limitation, low transformation rate, difficulty in regeneration, etc. The effect of efficiency, strong transformation ability and convenient material acquisition

Inactive Publication Date: 2013-07-10
北京金冠丰生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the genotype of the recipient material used for the transformation of immature embryos is severely restricted, and the material is easily restricted by seasons and greenhouse conditions. It cannot be used for continuous high-throughput corn transformation, which has great limitations in industrial applications.
Among the materials of non-immature embryo types, although it is easy to transform and regenerate directly with the shoot apical meristem, and it is not limited by the genotype, the transformation rate is low, and most of the regenerated plants obtained are chimeras, and the offspring need to do a lot of screening work
However, it is difficult to induce embryogenic callus and regeneration by using mature embryos, leaves, shoot tips and other materials.

Method used

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  • Method for transforming exogenous gene by using maize coleoptile joint induced calluses
  • Method for transforming exogenous gene by using maize coleoptile joint induced calluses
  • Method for transforming exogenous gene by using maize coleoptile joint induced calluses

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Induction of Corn Seed Coleoptile Node Callus

[0048] 1. Seed sterilization

[0049] 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 in 75% alcohol for 3 minutes on the ultra-clean bench, then soak in 5.25% sodium hypochlorite for 20 minutes, and finally rinse with sterilized water for 5-6 times.

[0050] 2. Germination culture

[0051] 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, temperature 26-28℃) for 7-10 days.

[0052]The above germination medium is MS + maltose 40g / L + enzyme hydrolyzed casein 0.1g / L + glutamine 0.5g / L + MES (2-morpholineethanesulfonic acid) 2g / L + MgCl 2 6H2...

Embodiment 2

[0062] Example 2 Transformation and regeneration of coleoptile node callus

[0063] 1. Agrobacterium Preparation

[0064] (1) Liquid Agrobacterium preparation method

[0065] Streak activated strains in LB solid medium. The petri dish was sealed with Parafilm, and placed upside down in a 28-degree incubator for 2 days; the petri dish with Agrobacterium was placed in the refrigerator; in the afternoon of two days before infecting the explant, a single colony of Agrobacterium was picked, Inoculate in a 250ml flask containing 25ml LB liquid medium (the antibiotics corresponding to the plasmid / strain should be added to the LB liquid medium). Place the flask in a shaker at 150rpm and culture overnight at 26 degrees in the dark; the next morning, dilute Agrobacterium at a concentration of 1:5 (that is, add 10ml of bacterial solution to 40ml of fresh LB liquid medium with antibiotics) ; Shake the bacteria until the afternoon of the same day, divide 50ml of the bacterial solution i...

Embodiment 3

[0086] Example 3 PCR detection of transgenic corn

[0087] 1. Extraction of DNA from maize leaves (CTAB method)

[0088] 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 a method for transforming an exogenous gene by using maize coleoptile joint induced calluses, which belongs to the field of agricultural biotechnology and crop breeding. The method mainly comprises the following steps of: (1) placing sterilized maize seeds in a sprouting culture medium for cultivation, and germinating to obtain seedlings; (2) cutting off the coleoptile joints from the seedlings to induce the calluses; (3) carrying out subculture propagation on the calluses, and introducing the exogenous gene by adopting an agrobacterium tumefaciens mediated method; (4) obtaining kanamycin-resistant calluses by 3-4 times of screening; (5) respectively carrying out embryoid induction and differentiation to obtain transgenosis regeneration seedlings; and (6) carrying out PCR (Polymerase Chain Reaction) detection on the regeneration seedlings to check whether the transgenosis is introduced. According to the method, acceptor materials are free of limitation of seasons and convenient to obtain and have strong regeneration capacity; and the large-scale genetic transformation of the maize is easy to realize.

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 five major crops in the world, and it plays an important role in the grain production of our country and even 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, bio-high technologies such as transgenic technology and molecular marker-assisted breeding technology have begun to play an important role in the selection of new corn varieties. Breeders at home and abroad attach great importance to it. [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 scree...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H4/00A01H5/00
Inventor 万向元吴锁伟赵虎基方才臣张丹凤肖中华刘艳艳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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