Cryopreservation and recovery culture methods for dioscorea alata protocrom-like bodies
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A cryopreservation and protocorm-like technology, which is applied in the field of plant cell engineering, can solve the problems that the construction of large potato protocorms has not been reported, and achieve stable and reliable cryopreservation methods, reduce damage, and restore good growth conditions
Inactive Publication Date: 2017-04-19
SHANGHAI AGROBIOLOGICAL GENE CENT
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Problems solved by technology
At present, most of the relevant reports on similar plants focus on the cryopreservation of embryogenic cells and shoot tips of potato, sweet potato, yam and other crops. There is no report on the construction of cryopreservation technology system for large potato protocorms.
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Embodiment 1
[0027] Embodiment 1, the acquisition of big potato sterile seedlings and the induction of protocorm-like
[0028] refer to figure 1 and figure 2 As shown, take large potato seedlings with jointed stems, rinse them repeatedly under running water for about 1 hour, disinfect with 70% alcohol for 1 min, 0.1% mercury chloride for 10 min, rinse with sterile water several times, and inoculate in the 2mg·L -1 BA, 0.5mg·L -1 Axillary buds were induced on MS medium with NAA. The culture temperature was 25±2°C, and the light was 12 h d -1 , light intensity 36µmolm -2 the s -1 . Axillary buds were excised and inoculated in 0.1 mg·L -1 NAA was subcultured on MS medium. Subculture every 3 months. Take the aseptic tissue-cultured seedlings of large potato that have been subcultured for more than 3 months, cut the stem section with axillary buds about 1 cm above, and inoculate them with BA1mol·L -1 , NAA 0.2mol·L -1 ,GA 3 0.5mol·L -1 MS medium, wherein, the MS medium contains...
Embodiment 2
[0029] Embodiment 2, the gradient pre-cultivation of large potato class protocorms
[0030] Get the stem section of the protocorm with class class gained in embodiment 1 and change into containing calcium chloride 0.1mol L -1, sucrose 0.3mol L -1 And abscisic acid 0.5-5mg·L -1 After culturing on the culture medium for 3-7 days, transfer to the medium containing calcium chloride 0.1mol·L -1 , sucrose 0.7mol L -1 And abscisic acid 0.5-5mg·L -1 The medium was pre-incubated for 16 hours in the dark.
Embodiment 3
[0031] Embodiment 3, the device fixing of big potato class protocorm
[0032] refer to image 3 As shown, the steps for fixing the protocorm to the loading strip are as follows: peel off the protocorm of 2-3 mm under the dissecting microscope, dip the protocorm in 2-4% calcium ion-free sodium alginate solution with tweezers, Then place them neatly on 5mm×20mm metal mesh loading strips, with 5-10 protocorms on each loading strip. Then immerse the loading strip with protocorm-like 0.1-0.3% calcium chloride, 0.8mol·L -1 Sucrose and 2mol·L -1 Glycerol MS liquid medium, after 10-30min fixation, put the loading strip on sterile filter paper to absorb excess liquid.
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Abstract
The invention relates to cryopreservation and recovery culture methods for dioscorea alata protocrom-like bodies, and belongs to the field of plant cell engineering. The method comprises the cryopreservation steps: carrying out aseptic seedling induction and subculture, carrying out protocrom-like body induction, carrying out gradient domestication culture, stripping the protocorm-like bodies, fixing the protocorm-like bodies to loading bars, then placing the loading bars in a pretreatment solution, treating, transferring the treated loading bars into a vitrification protective agent, carrying out ice bath treatment, then quickly putting into liquid nitrogen, and preserving. The method comprises the recovery culture steps: taking out the loading bars with the protocrom-like bodies from the liquid nitrogen, putting the loading bars into a 1 / 2MS liquid culture medium, thawing and washing, adsorbing to dry with filter paper, then placing in a 1 / 2MS culture medium without hormones, carrying out dark culture, stripping the protocrom-like bodies from the loading bars one by one under an anatomical lens, transferring the protocrom-like bodies to a culture medium containing BA and GA3, carrying out dark culture, regenerating, then transferring into a culture medium containing BA and NAA, and carrying out illumination culture. The method has the advantages that a large number of materials which can be preserved and regenerated easily can be obtained by using the protocrom-like bodies as the material for cryopreservation of dioscorea alata resources.
Description
technical field [0001] The invention relates to a method for ultra-low temperature preservation of large potato protocorms. Simultaneously, the invention also relates to a recovery culture method for large potato protocorms after cryopreservation, belonging to the technical field of plant cell engineering. Background technique [0002] Big potato ( Dioscorea alata ) is a vine of the genus Dioscorea in the Dioscoreaceae family. It is an economic crop with both medicine, food and feed and a promising energy crop. However, due to its seldom or no flowering, low seed setting rate, long seed dormancy period and low germination rate, etc., the germplasm resources are degraded, so the protection of the genetic diversity of Dapotamus is urgent. At present, the protection measures for big potato mainly include germplasm gardens preserved by seed stems and preserved by plant tissue culture. Field preservation is easily affected by natural disasters, resulting in germplasm variation ...
Claims
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