Method for ultralow-temperature preservation of anti-bursaphelenchus-xylophilus pinus massoniana embryonic callus

A technology for cryopreservation of embryogenic callus, which is applied in horticultural methods, plant preservation, botanical equipment and methods, etc., can solve the problems of decreased embryogenicity, continuous accumulation of cytological changes, and genetic insufficiency of embryogenic callus. Stability and other issues

Pending Publication Date: 2019-07-26
NANJING FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] In the previous study, immature zygotic embryos of resistant P. massoniana were used as explants to induce stable proliferation of embryogenic callus. However, during the long-term proliferation process, cytological changes continued to accumulate due to continuous cell division, which eventually led to embryogenic callus. The callus is genetically unstable and the embryogenicity of the cells decreases

Method used

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  • Method for ultralow-temperature preservation of anti-bursaphelenchus-xylophilus pinus massoniana embryonic callus
  • Method for ultralow-temperature preservation of anti-bursaphelenchus-xylophilus pinus massoniana embryonic callus
  • Method for ultralow-temperature preservation of anti-bursaphelenchus-xylophilus pinus massoniana embryonic callus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Example 1 Effect of cryopreservation on cell activity of resistant Pine massoniana embryogenic callus

[0026] 1) Ultra-low temperature storage pretreatment

[0027] Take 0.5 g of resistant Pine massoniana callus that has propagated for 15 days, and place it in a proliferation medium (LP+1 mg / L2, 4-D+0.5 mg / L 6-BA+1 g / L inositol + 1g / L glutamine + 0.5g / L hydrolyzed casein + 6.24g / L agar) were pretreated for 12h, 24h, 36h, 48h respectively.

[0028] 2) cryopreservation

[0029] Place the pretreated embryogenic callus in a 1.8mL cryotube, add cryoprotectants of different proportions in Table 1 to the 1.8mL scale, tighten the lid and place it in a programmed cooling box, and put the cooling box Put it into a program cooling device for cooling treatment. After the temperature drops to 0°C, it is pre-cooled for 10 minutes, and then the temperature is lowered at a rate of 1°C / min. After the temperature drops to -80°C, it is maintained for 30 minutes. The freezing box is ta...

Embodiment 2

[0043] Embryogenic callus recovery growth assay after embodiment 2 cryopreservation

[0044] Thaw and wash the above-mentioned resistant Pine pine horse 7-6 and whole 172-1-1 embryogenic calli stored at cryopreservation for one month, and place them on the proliferation medium for recovery culture. Observe and count the status of embryogenic callus resuming growth, and place the resuming callus under a Zeiss stereo microscope to observe its microstructure.

[0045] The culture temperature is 23±2°C, and cultured in the dark. Proliferation medium is LP+1mg / L2, 4-D+0.5mg / L 6-BA+1g / L inositol+1g / L glutamine+0.5g / L hydrolyzed casein+6.24g / L agar.

[0046] Callus regrowth rate = number of recovery callus / number of thawed callus × 100%

[0047] Data analysis was processed by Excel 2007 and SPSS 19.0.

[0048] The results are shown in Table 4. The cryopreservation scheme was further screened by counting the callus regeneration rate and the time required for growth recovery. For...

Embodiment 3

[0054] Example 3 Observation on maturation and differentiation of embryogenic callus after cryopreservation

[0055] After the embryogenic callus of the thawed resistant Masson pine horse 7-6 cell line resumed growth, the thawed callus and the normal proliferating callus of the horse 7-6 cell line were placed on the maturation medium for differentiation Cultivate and observe callus differentiation ability.

[0056] The culture temperature is 23±2°C, and cultured in the dark. The maturation medium is LP+0.4mg / LABA+0.5mg / L GA+1g / L glutamine+1g / LAC+3.5g / L phytogel.

[0057] 1) Effects of cryopreservation on maturation of resistant pine embryogenic cells

[0058] Through mature culture, it was found that the normally proliferating callus had no obvious differentiation phenomenon on the mature medium, and the callus had been completely browned after 2 months of culture ( image 3 A). However, the thawed Ma 7-6 callus also had browning phenomenon after 2 months of mature culture...

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Abstract

The invention belongs to the technical field of tissue culture and discloses a method for ultralow-temperature preservation of anti-bursaphelenchus-xylophilus pinus massoniana embryonic callus. By proper pretreatment and adoption of a mixed cryoprotectant, the method is suitable for freeze preservation of the anti-bursaphelenchus-xylophilus pinus massoniana embryonic callus, cell viability of theembryonic callus unfrozen at 30-35 DEG C reaches 100% maximally, and the embryonic callus subjected to ultralow-temperature preservation has no evident difference from embryonic callus subjected to normal proliferation in appearance and microstructure and still has a function of differentiation for forming somatic embryos. An ultralow-temperature preservation system of the anti-bursaphelenchus-xylophilus pinus massoniana embryonic callus is established preliminarily, and technical references are provided for long-time embryonic maintaining of the resistant pinus massoniana embryonic callus.

Description

technical field [0001] The invention relates to the preservation of plant callus, in particular to a method for ultra-low temperature preservation of pine wood nematode-resistant embryogenic callus. Background technique [0002] Masson pine (Pinus massoniana Lamb.) is widely distributed in my country and is one of the most important timber tree species in my country. Pine wood nematode disease is a devastating pine tree disease caused by the pine wood nematode (Bursaphelenchus xylophilus), which has caused great harm to pine forests and the ecological environment in my country since its discovery. Some achievements have been made in the prevention and control of pine wood nematode at home and abroad, but disease-resistant breeding is one of the important ways to fundamentally solve pine wood nematode. At present, a preservation garden for candidate individuals of P. massoniana resistant to pine wood nematode has been established in Anhui, but the selected resistant P. masso...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N3/00A01H4/00
CPCA01H4/001A01H4/008A01N3/00
Inventor 吴小芹沈李元叶建仁陈婷婷
Owner NANJING FORESTRY UNIV
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