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Technique for epicotyl of cotton culturing in vitro regenerated plant, and decapitated epicotyl explant

A technology of in vitro culture and culture method, which is applied in the field of in vitro culture of cotton epicotyls to directly produce and regenerate plants with multiple buds, which can solve the problems of explant browning, low rooting efficiency, and high deformity rate, and achieve the goal of transplanting Easy to survive, strong regenerated buds, and high rooting rate

Inactive Publication Date: 2009-07-29
THE INST OF MICROBIOLOGY XINJIANG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0014] 1. At present, plants are mainly regenerated by inducing callus to differentiate into somatic embryos, which is limited by genotype, and has a high deformity rate and a long culture period;
[0015] 2. Among the many methods of using explants to directly induce bud generation to regenerate plants, it is either limited by genotype, or the bud generation efficiency is not high, or the explants are severely browned, or the rooting efficiency is low and depends on genotype, or Regenerated plants take a relatively long time to obtain

Method used

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  • Technique for epicotyl of cotton culturing in vitro regenerated plant, and decapitated epicotyl explant
  • Technique for epicotyl of cotton culturing in vitro regenerated plant, and decapitated epicotyl explant
  • Technique for epicotyl of cotton culturing in vitro regenerated plant, and decapitated epicotyl explant

Examples

Experimental program
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Effect test

Embodiment 1

[0047] Example 1: The method of in vitro culture of sea-island cotton epicotyls for direct multi-bud generation and regenerated plants and decapitated epicotyl explants

[0048] The seeds of sea-island cotton variety Xinhai No. 19 were treated with sulfuric acid to obtain light seeds. The seeds were first surface sterilized with 70% ethanol for 30 sec, followed by 15% hydrogen peroxide (H 2 o 2 ) aqueous solution for 2hr., then rinsed with sterilized water for 6 times and soaked in sterilized water, drained after 12-36hr., squeezed out the seed coat, inoculated the seed kernel on 1 / 2MS solid medium and cultured in a dark place On the 1st to 3rd day, the cotyledon and the hypocotyl and radicle 4-6 mm below the cotyledon node were cut off and discarded, and the remaining part was the epicotyl explant. Cut a gap of 0.3-0.7 mm longitudinally at the top of the epicotyl explant, avoid splitting the hypocotyl, and then insert the multi-bud induction medium (MS salt+B) with the 4-6 ...

Embodiment 2

[0049] Example 2: The method of in vitro culture of upland cotton epicotyls for direct multi-bud regeneration and decapitated epicotyl explants

[0050] The seeds of Xinluzao 17, an early-maturing upland cotton variety, were treated with sulfuric acid to obtain light seeds. The seeds were first surface sterilized with 70% ethanol for 30 sec, followed by 15% hydrogen peroxide (H 2 o 2 ) aqueous solution for 2hr., then rinse with sterilized water for 5 times and soak in sterilized water. After 36-48hr., drain the water and squeeze out the seed coat, inoculate the seed kernel on 1 / 2MS solid medium and culture in a dark place On 2-5 days, cut off and discard the cotyledons and the hypocotyls and radicles 6-10 mm below the nodes of the cotyledons, and the remaining part is the epicotyl explant. Cut a gap of 0.7-1.3 mm longitudinally at the top of the epicotyl explant, avoid splitting the hypocotyl, and then insert the multi-bud induction medium (MS salt+B) with the 6-10 mm hypoco...

Embodiment 3

[0051] Example 3: The method of in vitro culture of Xintiansi No. 1 epicotyl and direct multi-bud regeneration plant and decapitated epicotyl explant

[0052] Gently crush the seed coat of Xintiansi No. 1 seed to remove the kernel, first sterilize the surface with 70% ethanol for 30 sec, then sterilize with 0.1% mercuric chloride aqueous solution for 10 min., and then rinse with sterilized water for 6 times. Kernels were inoculated on 1 / 2MS solid medium and cultured under light. Cut off the cotyledons and hypocotyls and radicles 6-10mm below the cotyledon nodes from the aseptic seedlings that had been cultured for 5-7 days. For the epicotyl explants, the upper embryo was treated with a small gap, and then transferred to the medium for inducing multiple shoots (MS salt+B 5Organic + 6-BA 0.1 ~ 0.5mg / l + NAA 0.5 ~ 1.5mg / l + glucose 30g / l), after 20 days of cultivation, multiple buds can be seen. After culturing the epicotyl explants that did not produce multiple buds for 15 days...

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Abstract

The invention discloses an epicotyl explant (epicotyl explant) of a cotton in vitro culture and direct multi-bud generation regenerated plant and a cultivation method for obtaining a complete regenerated plant by using the explant. The invention also discloses a decapitated epicotyl explant. The present invention utilizes epicotyl explants and fresh epicotyl explants to establish a technique for directly inducing multi-bud generation, elongation and rooting that does not depend on genotype, simple and fast cotton in vitro culture regeneration plant technology, Through the optimal screening of medium formula and carbon source, the degree of browning of explants is reduced to the lightest, the maximum number of buds in one explant is ≥ 3, and the rooting rate of regenerated shoots is above 90%, obtaining a plant with complete roots, stems and leaves. It only takes 3 to 4 months to regenerate plants, and the rate of deformed seedlings is low, which lays the foundation for the genetic improvement of cotton varieties used in production.

Description

field of invention [0001] The invention relates to the fields of plant tissue culture and biotechnology. Specifically, the present invention relates to a method for cultivating cotton epicotyls in vitro and direct multi-bud generation and regenerated plants. Background technique [0002] Tracing back to the methods of in vitro culture and regeneration of plants reported on cotton, the main method is to induce differentiation of somatic embryos through callus to regenerate plants, which can easily cause high deformity rate and long culture period, and only It is suitable for Kezi cotton or wild cotton, and it is invalid for the main cultivated cotton varieties. Despite the commercial success of genetically modified cotton, genetic transformation and regeneration of cotton remains challenging compared to other crops. These issues are mainly genotype-dependent regeneration, efficiency of somatic embryogenesis, and somatic variation. An important advance in cotton biotechnolog...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A01H4/00C12N5/04
Inventor 郝秀英孙立军欧阳立恒段肖霞代兴荣王新勇李雪源谢迪佳毛鸿才闫建庆
Owner THE INST OF MICROBIOLOGY XINJIANG ACADEMY OF AGRI SCI
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