Hormone-free corn callus regeneration method

A callus, hormone-free technology, applied in the field of plant tissue culture, can solve the problems of increased frequency of maize genome variation, bound genetic engineering breeding technology, and high deformity rate of regenerated plants, so as to improve the fertility rate of plants and make them easy to transplant and survive , the effect of saving experimental cost

Inactive Publication Date: 2013-12-18
CROP RES INST GUANGDONG ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] This kind of regeneration method not only has many steps and takes a long time, but also often causes the following adverse consequences: 1) Inducing growth on a medium containing hormones for a long time, the frequency of genome variation in maize increases, and the deformity rate of regenerated plants is high, and even caused by hormone poisoning. 2) This method of regenerating plants first and then rooting takes a long time to root, often causes root expansion, few root hairs, and low rooting rate, which affects later field growth
The application of genetic engineering breeding technology in maize breeding production is severely restricted by the existing regeneration plant regeneration technology

Method used

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  • Hormone-free corn callus regeneration method
  • Hormone-free corn callus regeneration method
  • Hormone-free corn callus regeneration method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1: Effect of sucrose content in regeneration medium on differentiation and regeneration of super sweet corn inbred line 1132

[0027] (1) Select the super-sweet corn inbred line 1132, which is crisp and grainy and grows well. Injured tissues were transferred to embryoid body induction medium containing 20,000 mg / L, 40,000 mg / L, 60,000 mg / L, and 80,000 mg / L of sucrose, respectively. The composition of the embryoid body induction medium is: MS macroelement (that is, the macroelement in MS medium) + MS trace element (that is, the trace element in MS medium) + inositol 250mg / L + thiamine hydrochloride (vitamin B1) 3.5mg / L+pyridoxine niacin (vitamin B6) 1.0mg / L+niacin 0.5mg / L+glycine 4.0mg / L+sucrose (concentrations are 20,000mg / L, 40,000mg / L, 60,000mg / L and 80,000mg / L)+proline 300mg / L+hydrolyzed casein 100mg / L+MES500mg / L+plant gel 2500mg / L, pH5.8. The culture temperature is 24-26°C. When the culture reaches the third week (20th day), it is observed that on the med...

Embodiment 2

[0030] Embodiment 2: Carry out regeneration culture with super sweet corn inbred line 1132 callus

[0031] (1) Select 72 pieces of crisp, granular callus with good growth, and transfer them to culture dishes poured with embryoid body induction medium, and place 6 calli on each dish (10 cm in diameter). damage tissue. The composition of the embryoid body induction medium is: MS macroelements + MS trace elements + inositol 250mg / L + thiamine hydrochloride (vitamin B1) 3.5mg / L + nicotinic acid pyridoxine (vitamin B6) 1.0mg / L + nicotinic acid 0.5 mg / L+glycine 4.0mg / L+sucrose 60,000mg / L+proline 300mg / L+hydrolyzed casein 100mg / L+MES500mg / L+plant gel 2500mg / L, pH 5.8, culture temperature 25°C, cultured for 3 weeks The embryoid body was fully induced on the callus, and the shape of the embryoid body was as follows: figure 1 as shown in a.

[0032] (2) Transfer the 72 embryoid bodies obtained in step (1) to glass culture bottles containing regeneration medium to cultivate regenerate...

Embodiment 3

[0034] Embodiment 3: Carry out regeneration culture with HiII callus

[0035] (1) Select crisp, granular HiII with good growth (has been described in "Ma Yunxia et al. Agrobacterium-mediated genetic transformation of glyphosate-resistant gene (sxglr-11) in maize, Shanxi Agricultural Science, 2009, 6" 90 pieces of calli from public) were transferred to culture dishes filled with embryoid body induction medium, and 6 pieces of calli were placed on each culture dish (10 cm in diameter). The composition of the embryoid body induction medium is: MS macroelements + MS trace elements + inositol 250mg / L + thiamine hydrochloride (vitamin B1) 3.5mg / L + nicotinic acid pyridoxine (vitamin B6) 1.0mg / L + nicotinic acid 0.5 mg / L+glycine 4.0mg / L+sucrose 60,000mg / L+proline 300mg / L+hydrolyzed casein 100mg / L+MES500mg / L+plant gel 2500mg / L, pH5.8, culture temperature 25℃, culture for 3 weeks (20 days), embryoid bodies were fully induced on all calli, and the morphology of embryoid bodies was as f...

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Abstract

The invention discloses a hormone-free corn callus regeneration method. The hormone-free corn callus regeneration method comprises the following steps: culturing granular corn calluses with good growth vigor on a hormone-free embryoid induction culture medium at 24-26 DEG C for 20-23 days, and inducing to generate an embryoid; transferring the embryoid to a hormone-free regeneration culture medium, wherein the culturing temperature is 24-26 DEG C, the illumination intensity is 12000lux-18000lux and the illumination time each day is 16h; culturing until roots and buds are generated to obtain a regenerated plant; and then continually culturing the regenerated plant for 6-7 days to raise strong seedlings under the following conditions that the temperature is 26-28 DEG C, the illumination intensity is 24000lux-30000lux and the illumination time each day is 16h. According to the hormone-free corn callus regeneration method, the osmotic pressure of a regeneration culture phase is regulated and controlled by changing the use amount of sucrose so as to realize the efficient regeneration of the calluses, and the whole process does not need plant hormones. The method not only saves the experiment cost, but also avoids poisoning tissue materials due to application of hormones for a long time.

Description

technical field [0001] The invention belongs to the technical field of plant tissue culture, in particular to a hormone-free corn callus regeneration method. Background technique [0002] The establishment of efficient regeneration system is the key to genetic engineering breeding of maize. Existing maize regeneration technologies all rely on auxin and cytokinin, these plant hormones mainly include naphthaleneacetic acid (NAA), 6-benzyl adenine (6-BA), indolebutyric acid (IBA), abscisic acid (ABA )Wait. The existing maize regeneration method is usually: first induce crisp, granular and vigorously growing callus on the induction medium containing auxin, and transfer these calli to the medium containing NAA (0.1 ~ 0.3mg / L)+6-BA (2~4mg / L) or other hormone combinations to induce plant regeneration, and then transfer the regenerated plants to NAA (0.1~0.3mg / L) or NAA (0.1~0.3mg / L) L) + IBA (0.3-0.5mg / L) rooting medium to induce root regeneration; there are also some methods f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 祁喜涛胡建广吴景
Owner CROP RES INST GUANGDONG ACAD OF AGRI SCI
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