Somatic embryo in-vitro regeneration method applied to genetic transformation and seed ball rapid propagation of Lilium pumilumDC. Fisch

A technology of genetic transformation and somatic embryos, applied in botany equipment and methods, plant regeneration, horticultural methods, etc., can solve problems such as thin and weak stems, and achieve the effects of increased germination rate, high regeneration efficiency, and good preservation effect

Inactive Publication Date: 2014-11-05
SHENYANG AGRI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, the stalks of Lilium fine-leaved plants are relatively thin, and the ornamental traits need to be further improved.

Method used

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  • Somatic embryo in-vitro regeneration method applied to genetic transformation and seed ball rapid propagation of Lilium pumilumDC. Fisch
  • Somatic embryo in-vitro regeneration method applied to genetic transformation and seed ball rapid propagation of Lilium pumilumDC. Fisch
  • Somatic embryo in-vitro regeneration method applied to genetic transformation and seed ball rapid propagation of Lilium pumilumDC. Fisch

Examples

Experimental program
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Effect test

example 1

[0036] Example 1. In Vitro Regeneration Technology of Lilium microphylla Somatic Embryos

[0037] 1. Non-embryogenic callus induction stage (explant dedifferentiation stage)

[0038] Select aseptic seedlings of Lilium microphyllum with a small bulb diameter of 1-1.2 cm, cut the leaves into 0.5 cm long segments, inoculate them on the dedifferentiation medium MS+BA 1.0 mg·L with the front facing up -1 +NAA 0.5 mg L -1 (ie BA / NAA is 2), light culture. Contains 30 g·L of sucrose -1 , agar (bar) 7 g·L -1 , pH 5.8, autoclaved at 121°C for 20 minutes. The temperature of the culture room is 25±1°C, and an airtight sealing film is used to ensure that the humidity in the tissue culture bottle reaches 80-90%, with 16 hours of light and 8 hours of darkness every day, and the light intensity is 36 μmol m -2 ·s -1 .

[0039] In the experiment, it was found that if the scales were used as explants, the direct regeneration of adventitious buds would occur. However, adventitious buds d...

example 2

[0057] Example two, identification of non-embryogenic callus and embryogenic callus

[0058]Observation by stereomicroscope showed that non-embryogenic callus had smooth surface, while embryogenic callus had tumor-like structure. Observation of paraffin sections showed that there were obvious differences between the two types of callus, and there was no obvious cell structure in the non-embryogenic callus (see appendix Figure 13 ), while the embryogenic callus is mostly of external origin, the internal cell structure is obvious, and most of them are spherical. From the inner layer to the outer layer, the cells gradually become smaller and more closely arranged (see appendix Figure 14 ).

example 3

[0059] Example 3. Validation of the Efficiency of the Somatic Embryogenesis Pathway

[0060] Although the somatic embryo regeneration system has the advantages of single-cell origin and high reproduction coefficient, due to the relative difficulty of induction, only a few species of lilies at home and abroad can obtain somatic embryos, and most of them are cut flower varieties. Many wild lilies are rarely reported. At present, the research on the in vitro regeneration of Lilium tenifolia mainly focuses on the organogenesis pathway. When the scales of Lilium tenifolia (taken from adult bulbs, not sterile seedlings) are used as explants for adventitious bud induction, the induction coefficient is 3- 4.8; using lily lily filaments as explants, the callus induction rate is 40%, and when the callus undergoes adventitious bud differentiation, the differentiation coefficient is 10.8 (Jin Shumei et al., 2006; Ge Beibo et al., 2010); in other In the research on somatic embryogenesis o...

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Abstract

The invention provides a somatic embryo in-vitro regeneration method applied to genetic transformation and seed ball rapid propagation of Lilium pumilumDC. Fisch. The method comprises the following process steps: preparing Lilium pumilumDC. Fisch aseptic seedling leaves, inoculating the prepared seedling leaves into a culture medium (MS+BA1.0mg.L<-1>+NAA0.5mg.L<-1>), and culturing the inoculated seedling leaves for 45 days to obtain non-embryonic calli; transferring the non-embryonic calli into a culture medium (MS+BA0.5mg.L<-1>+NAA1.0mg.L<-1>), culturing the non-embryonic calli for 45 days to obtain embryonic calli, transferring the embryonic calli into a culture medium (MS+NAA0.5mg.L<-1>), adjusting the embryonic proportion, and alternatively using the two culture media in sequence to obtain an embryonic callus culture which has a high embryonic proportion and a low germination rate; and transferring the embryonic callus culture into a culture medium (MS), wherein more than 21 somatic embryos can be formed in the embryonic callus culture per cubic centimeter. According to the somatic embryo in-vitro regeneration method, an aseptic seedling is taken as an explant, so that the consumption of lily stocks is lowered; the embryonic calli have high induction rates and good preserving effects, can be distinguished by naked eyes, and are good receptors of genetic transformation; and compared with the conventional method, the somatic embryo in-vitro regeneration method has the advantages that the callus induction rate and seedling coefficient of an established somatic embryo regeneration system are increased by 40 percent and 3-4 times respectively.

Description

technical field [0001] The invention belongs to the research field of in vitro culture and genetic breeding of bulbous flowers, in particular to the technical field of in vitro regeneration of somatic embryos for genetic transformation and bulb breeding of lily lily. Background technique [0002] Somatic embryo is an embryoid body developed from a single cell. Propagation in this way has the advantages of less material, high genetic stability, low mutation rate and high regeneration rate, which can effectively reduce the heavy screening work of transgenic plants in the later stage , is considered to have broad application prospects in the fields of genetic engineering, somatic cell fusion, cell mutant induction, artificial seed preparation, rescue of hybrid zygote embryos, germplasm preservation, and rapid propagation of seedlings. It has become a research field in genetic engineering such as genetic transformation in recent years. hotspots. [0003] Lily Lily ( Lilium pum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01H4/00
Inventor 孙红梅张静
Owner SHENYANG AGRI UNIV
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