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Method for continuously culturing high frequency regeneration plants through cereal crop single plant source microspores

A technology for microspore culture and plant regeneration, applied in the biological field, to achieve the effect of shortening the time for microspore culture

Active Publication Date: 2016-02-24
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Using physical and chemical measures to mutagenize the microspores derived from a single plant, a single plant (or single seed) can be obtained from a single plant (or a single seed) and a homozygous regenerated plant with completely consistent genetic background, which is an ideal material for genetic mutation analysis. Using microspores derived from a single plant of barley and other cereal crops to carry out mutagenesis and culture

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0086] Greenhouse planting of embodiment 1 barley single plant donor material

[0087] Three individual plants (numbers 6-3-4, 6-3-7, 6-3-23) of the barley variety "Hua 30" line 6-3 were obtained from microspore culture regeneration of "Hua 30" The homozygous line 6-3 was planted in an artificial climate chamber under the conditions of plant cultivation: light 2000 lx, 12h / d, humidity 70%, and temperature setting of light 22°C / darkness 18°C. The continuous tillering and booting is promoted through water and fertilizer regulation, the tillering period of the plant is prolonged, and finally the free microspore culture can be obtained by taking materials for more than 6 consecutive months.

Embodiment 2

[0088] Example 2 Drawing and pretreatment of barley single plant donor material

[0089] Select the barley ears whose small flowers and microspores in the middle are in the early-middle stage of mononucleation, cut off the leaves (keep about 1.5cm from the base of the upper two leaves), wrap the young ears with clean wet gauze, put them in a fresh-keeping bag to moisturize, and mark the material Date and quantity, etc., put them in a refrigerator at 4°C for 2 to 4 weeks of low-temperature pretreatment.

Embodiment 3

[0090] Example 3 Barley Single Plant Source Microspore Free Culture

[0091] Free microspores from flower buds were taken from the low-temperature-treated materials, and the methods for freeing microspores were as follows: Lu Ruiju et al. (2001) and Lu et al. (2008). During inoculation, the tassel disinfection and sterilization methods refer to Guo Guimei et al. (2014). Each test tube connects the flower buds of 4 spikes, adds 12ml extract (mannitol 60g / L, adds 1.1g / LCaCl 2 , 0.976g / L2-(N-morpholine) ethanesulfonic acid (MES) and 20mg / L colchicine, pH5.8), with a high-speed disperser ultra-speed rotary cutting. Filter the spin-cut suspension with a 300-mesh sieve, centrifuge the filtrate at a low speed of 700 rpm for 5 min, repeat 3 times, and collect the microspores. Collected microspores use extract (mannitol 60g / L, add 1.1g / LCaCl 2 , 0.976g / L 2-(N-morpholine) ethanesulfonic acid (MES) and 20mg / L colchicine, pH5.8) were pretreated at 25°C in the dark for 2 days, and then ...

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Abstract

The invention relates to a method for continuously culturing high frequency regeneration plants through cereal crop single plant source microspores, particularly to a culture method of the cereal crop single plant source microspores and the method for culturing and obtaining the regeneration plants through the cereal crop single plant source microspores. The method comprises the following steps: separating the microspores from young spikes of a single plant of the cereal crop; carrying out callus induction and differentiation steps on the separated microspores, wherein the step of separating the microspores from the young spikes of the cereal crop comprises the steps of continuously obtaining young spikes of which the middle floret microspores are in the mononuclear early-middle period from the same cereal crop plant which is planted indoors and continuously tillers and ears; carrying out the separation, callus induction and differentiation steps on the young spike obtained each time.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for continuously cultivating and high-frequency regeneration of microspores from a single plant of cereal crops. Background technique [0002] Microspore in vitro culture technology is an important haploid breeding technology developed at present. Free microspore culture has been carried out in various plants, but the establishment of high-frequency regeneration system is limited to barley, rape, tobacco, wheat and other crops. Lu Ruiju (Lu Ruiju, Optimization of Barley Free Microspore Culture Technology and Establishment of Haploid Salt Tolerance and Low Nitrogen Stress Screening System[D], Nanjing Agricultural University, 2012) and others used field planted barley (Hordeumvulgare L.) as materials. However, due to the complex field environment and the influence of weather, the growth conditions of donor plants are difficult to control, and the donor materials for microspore ...

Claims

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Application Information

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IPC IPC(8): A01H4/00
CPCA01H4/00A01H4/001
Inventor 郭桂梅刘成洪何婷黄剑华陆瑞菊
Owner SHANGHAI ACAD OF AGRI SCI
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