CN101796925A - In vitro conservation method of oriental hybrid lily germ plasm resource - Google Patents
In vitro conservation method of oriental hybrid lily germ plasm resource Download PDFInfo
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Abstract
The invention discloses an in vitro conservation method of an oriental hybrid lily germ plasm resource, belonging to the technical field of plant tissue culture and germ plasm resource conservation. The in vitro conservation method of the oriental lily germ plasm resource comprises the following steps of: preparing a culture medium; processing and sterilizing provenance bulbs; carrying out induction culture of bulb buds; carrying out propagation culture of the bulb buds; carrying out in vitro conservation and expansion culture of germ plasms; sprouting, refrigerating and planting in-vitro bulbs; detecting the genetic stability of the germ plasms, and the like. By improving the formula of the culture medium and the culture steps and method, the invention prolongs a transferring-culture period of the in vitro conservation to 12-15 months, more than 6 months longer than that of an MS culture medium, achieves high survival rate of 100 percent without generating genetic variation by detection, enhances the efficiency of the in vitro conservation, also obviously reduces the conservation cost and can be popularized in and applied to oriental hybrid lily breeding and seedball producing enterprises.
Description
Technical Field
The invention relates to the technical field of plant tissue culture rapid propagation and germplasm resource preservation, in particular to an in vitro preservation method for prolonging oriental lily germplasm.
Background
Oriental lily (Oriental hybrid lily) is a perennial ornamental plant and is mainly used for cut flower cultivation, and the production of cut flowers of lily in China is increased by more than 20% every year in recent years. The oriental lily has the characteristics of beautiful plant shape, excellent cut flower quality, gray mold resistance, insensitivity to photoperiod, good cold storage resistance of bulbs and the like, can realize annual production of lily cut flowers by combining facility cultivation by utilizing climate differences at different latitudes and altitudes in China, and has high production benefit, so the oriental lily has good market development prospect. At present, oriental lily bulbs in China mainly depend on foreign import, bottleneck factors limiting the production and development of the domestic lily bulbs are the lack of cultivation of excellent new varieties, wherein the collection, preservation and utilization of germplasm resources are delayed, so that the breeding original resource materials are degraded, and the method is one of important factors influencing the breeding process of new lily varieties in China at present. The traditional method for breeding and storing the lilies is characterized in that scale cuttage breeding is adopted, field planting is carried out every year, a large amount of manpower, material resources and financial resources are consumed, the bulbs are easy to infect fusarium, rhizoctonia, pythium and other fungal diseases and easily spread a plurality of virus diseases including lily asymptomatic virus, the propagation of the diseases and insect pests such as fungi, viruses and the like is easily caused through the asexual propagation for many years, the loss and the degeneration of the resources are caused, and the huge loss is brought to the utilization and the storage of the lilies germplasm resources.
At present, the preservation method of plant germplasm resources mainly comprises the methods of field planting preservation, in vitro low-temperature or ultralow-temperature preservation, tissue culture in vitro preservation and the like. Wherein, the in vitro preservation is not influenced by external environmental conditions, and the in vitro preservation has incomparable advantages compared with the field growth preservation. There are many reports about plant in vitro preservation technologies, for example, patent ZL200710164732.4 reports a method for preserving calla germplasm in vitro by reducing macroelements in a culture medium and adding mannitol and penicillin, CN200710019345.1 and CN200710019346.6 report a method for preserving chrysanthemum in vitro by adding abscisic acid and reducing macroelements in the culture medium respectively, and the invention name of the invention is "in vitro preservation method of dendrobium cultures" (application No. 200810058045.9) reports a method for preserving dendrobium test-tube seedlings in vitro by reducing MS concentration at 15-20 ℃. The invention discloses an ultra-low temperature preservation method for vegetative propagation flower lycoris radiata, which introduces a method for preserving plants in vitro in an ultra-low temperature mode, for example, the in vitro preservation period of the germplasm of the colorful calla can reach 12 months, and the in vitro preservation period of chrysanthemum can reach 10 months.
The colored calla, chrysanthemum and dendrobium related to the patent are stored by adopting tissue culture seedlings with leaves, but the method is simply applied and is not suitable for the germplasm storage of oriental lilies; the ultra-low temperature preservation method of the lycoris has the defects of strict preservation equipment conditions, high difficulty in recovery and culture after preservation and the like. Due to the particularity of the growth of the bulbs of the oriental lily, for example, callus is easy to generate at the incision part of the base plate of the bulbs, and for example, the necrosis of the bulb tissue is easily caused by improper method to influence the preservation effect of long-term culture; and the common in vitro preservation method is carried out under the conditions of 20 +/-2 ℃ and illumination, which can cause the outer-layer scales of the lily bulb buds to be differentiated into leaves, and the phenomena of aging and withering can occur after the lily bulb buds grow for a period of time, but when the lily bulb buds are cultured at the temperature of 13-15 ℃ or 5-10 ℃, the test tube bulbs can be released from dormancy to induce the differentiation and the leaf emergence of terminal buds, so that the phenomena of stem drawing and leaf reoccurrence in a test tube bulb bottle are caused, and the phenomena are not beneficial to the purposes of bulb nutrient accumulation and long-term preservation. At present, no report about the in vitro preservation of oriental lily germplasm resources is found.
Disclosure of Invention
The invention aims to provide a method for in vitro preservation of oriental lily germplasm resources, which has the advantages of good quality of preservation characteristics, long preservation period, self-regulation and self-extension, low cost and simple and convenient operation and is suitable for in vitro preservation of oriental lily germplasm resources, aiming at the defects of easy necrosis of bulb tissues, easy leaf differentiation, aging and withering of outer-layer scales, unfavorable nutrient accumulation of bulbs, short preservation period and the like in the existing in vitro preservation method of lily germplasm resources.
The purpose of the invention is realized by the following technical scheme:
the in vitro conservation method of oriental lily germplasm resources comprises the following steps:
(1) preparation of a culture medium: comprises the components of a basic culture medium and a culture medium in each stage of in vitro preservation, and the weight of each component in each liter of culture medium is as follows:
a) basic culture medium: wherein,
the induction culture medium is an MS culture medium with 30-50 g/L of cane sugar, 4-5 g/L of agar powder and pH of 5.6-5.8;
the proliferation culture medium is 3/5MS culture medium with 50-60 g/L of cane sugar, 4-5 g/L of agar powder and pH of 5.6-5.8;
the synchronous culture medium for in vitro preservation and test tube bulb expansion is 1/2MS culture medium with sucrose of 60-75 g/L, agar powder of 4.0-5.0 g/L and pH of 5.6-6.0;
b) induction medium I: MS +6-BA 1.0-2.0 mg/L + IBA 0.1-0.5 mg/L + AC 1.0-3.0 mg/L;
c) induction medium II: MS +6-BA 0.5-1.0 mg/L + IBA 0.1-0.5 mg/L;
d) proliferation culture medium: 3/5MS +6-BA 0.2-1.0 mg/L + IBA 0.1-0.5 mg/L;
e) in-vitro preservation and test tube bulb expansion synchronous culture medium: 1/2MS + 0.1-0.5 mg/L NAA + 0.1-10 mg/L PP 3331 + 1.0-5.0 g/L mannitol + 1.0-5.0 mg/L AC;
(2) seed bulb treatment and sterilization: treating the seed bulbs at 5 ℃ for 3-4 months to break the dormancy; wrapping the bulbs with a breathable plastic film, placing the bulbs into moist peat with the water content of 60-70%, and placing the peat in a 50 ℃ incubator for heat treatment for 70-90 min; peeling off the outer layer scale, taking seed bulb buds with the length of 4-6 cm in the scale, soaking the seed bulb buds in 1% of detergent liquid for 10min, washing the seed bulb buds with clear water, and sterilizing the seed bulb buds for later use;
(3) and (3) induction culture of bulb buds: taking a stem tip 0.2-0.3 mm away from a sterilized seed scale stem bud as an explant, inoculating the explant to an induction culture medium I, and culturing for 50-60 days under the environmental conditions of the temperature of 20 +/-1 ℃, the illumination for 12h/d and the illumination intensity of 1000-3000 Lx until the scale stem bud is induced; cutting off top leaves of the bulb bud, longitudinally cutting into 2 pieces, transferring to an induction culture medium I, and culturing for 60-90 days under the same environmental condition until cluster bulb buds are induced to form; placing the stem in an illumination incubator, performing cyclic heat treatment for 50-60 days by changing the grade every 3 days according to the sequence of 30 ℃, 35 ℃ and 38 ℃, stripping 0.2-0.3 mm of stem tips in the illumination incubator as explants, inoculating the explants in an induction culture medium II for secondary induction culture, culturing for 60-90 days under the same environmental condition until the buds are induced to form bulb buds, and then performing low-temperature treatment for 45-60 days at 6 ℃ to recover the growth activity of the bulb buds;
(4) multiplication culture of bulb buds: cutting off leaves and part of base tissues of the bulb buds after low-temperature treatment, longitudinally cutting the bulb buds into 4-6 blocks, inoculating the 4-6 blocks on a proliferation culture medium, and culturing the 4-6 blocks at 20 +/-1 ℃ in the dark for 60-90 days to form robust bulb buds;
(5) and (3) germplasm in vitro preservation and test tube bulb expansion synchronous culture: cutting off the top and partial base tissues of the bulb buds obtained in the step (4), longitudinally cutting into 4-6 pieces, inoculating the cut bulb buds into an in vitro preservation culture medium, and culturing at 20 +/-2 ℃ in the dark for 12-15 months until the bulb buds expand to form a test tube bulb with the diameter of 0.8-1.2 cm; namely, seedling emergence or cutting off the bulb root system of the test tube, longitudinally cutting the bulb root system into 4-6 pieces, inoculating the cut bulb root system in an in vitro preservation culture medium, and circularly preserving and expanding culture for 1-4 times under the same condition; after 4 times, the seeds need to be planted and recovered in the field and then preserved in vitro according to the steps until the requirement on the in vitro preservation period of the seeds is met;
(6) emergence and cold storage of the test tube bulb: washing off agar from the test tube bulb by using clean water, wrapping the test tube bulb in sterilized peat with the water content of 60-70%, pretreating for 10-20 days at 10-12 ℃, refrigerating for 50-60 days at 3-6 ℃ until the dormancy breaking period, and then planting or planting at a time selected within an extended storage period of 2-3 months at 1-3 ℃;
(7) planting: selecting a mountain area with an altitude of 1000-1900 m and a cool summer climate, planting the refrigerated bulbs obtained in the step (6) in a sterilized substrate in the late 4 th to middle 5 th months under the rain sheltering and insect prevention isolation conditions, and beginning to sprout, emerge and grow after 15-20 days;
(8) and (3) detecting the genetic stability of the germplasm: selecting plants which grow normally and vigorously and are detected to have no genetic variation through ISSR molecular marker, and culturing the plants into qualified oriental lily seedballs.
The stem bud sterilization treatment of the seed scales comprises the steps of soaking in 75% alcohol for 0.5-1.0 min, washing with sterile water for 6 times, sterilizing with 0.1% mercuric chloride solution for 15-20 min, washing with sterile water for 6 times, sterilizing with 10% sodium hypochlorite aqueous solution for 13-15 min, and washing with sterile water for 6 times.
The planting medium is peat: the perlite is prepared according to the volume ratio of 7: 3.
The invention has the beneficial effects that:
(1) according to the invention, an 1/2MS culture medium is adopted, and 60-75 g/L, NAA 0.1-0.5 mg/L, PP 3331-10 mg/L of sucrose, 1.0-5.0 g/L of mannitol and 1.0-5.0 mg/L of AC are added, compared with a conventional in vitro preservation method, the MS concentration is reduced in the culture medium, the sucrose concentration and the NAA concentration are improved, 6-BA is omitted, PP333, mannitol and AC are added, the callus growth is controlled, and the bulbar bud is directly induced; the in-vitro preservation of the bulb buds and the expanding culture of the test tube bulbs are combined into a whole, through the improvement of the formula and the method, the interval time of the transfer culture is prolonged to 12-15 months from about 6 months of the preservation of a common MS culture medium, the survival rate reaches 100%, the labor of the transfer culture is greatly saved, the in-vitro preservation efficiency of the test tube bulbs of the oriental lily is remarkably improved, and the in-vitro preservation efficiency is improved by 50-60%.
(2) In the invention, dark culture is adopted in the whole process of the step of in vitro preservation and culture, so that the leaf emergence rate of the bulb buds is 0, the leaf emergence rate of the bulb buds cultured by the commonly adopted illumination can reach more than 90 percent, the growth of the leaves causes the large consumption of nutrients, the expansion of the bulb in the test tube is not facilitated, the phenomenon of aging and death can occur after the leaves grow for a certain period, so that the bulb is required to be transferred for 1 time every 6 months, and the dark culture needs to be carried out for one time after 12-15 months because the nutrition of the culture medium is consumed; in addition, the energy consumption is obviously increased by illumination culture, so the method is favorable for controlling the growth of the bulb bud leaves, the transfer period is prolonged, and the cost of in vitro preservation is only 1/2 of the cost of the common illumination culture method.
(3) The test tube bulbs stored by the method have developed root systems, the weight of the test tube bulbs can reach more than 0.5 g/grain after the preservation period is finished (12-15 months), the number of the roots is more than 15 strips/grain, the low-temperature treatment is convenient, the test tube bulbs which break through dormancy are subjected to the low-temperature treatment at 3-6 ℃, the test tube bulbs can be immediately planted, the test tube bulbs can be centrally planted at an appropriate period according to the climate and soil conditions within the prolonged storage period of 2-3 months at 1-3 ℃, the cultivation management is convenient, the problem that the traditional rooting tissue culture seedlings cannot be delayed at any time after being taken out of a bottle, the survival rate of the planting is low is overcome, the survival rate of the planting can reach more than 95%, the number of the roots of the common rooting tissue culture seedlings is 5 strips/plant, and the survival rate is about 70%.
Detailed Description
The present invention is further illustrated in detail by the following examples, but the present invention is not limited thereto.
The Oriental lily seed source in the following examples is Oriental Hybrid 'Sibreia' variety bulb with the stem circumference of 14-16 cm.
Example 1: (Oriental lily germplasm resources in vitro preservation 1)
The method comprises the following steps:
(1) preparation of a culture medium: comprises the components of a basic culture medium and a culture medium in each stage of in vitro preservation, and the weight of each component in each liter of culture medium is as follows:
a) basic culture medium: wherein,
the induction culture medium is an MS culture medium with sucrose of 40g/L, agar powder of 4g/L and pH5.8;
the proliferation culture medium is 3/5MS culture medium with 50g/L of sucrose, 5g/L of agar powder and pH of 5.6;
in-vitro preservation and test tube bulb expansion synchronous culture medium is 1/2MS culture medium with 60g/L of cane sugar, 5.0g/L of agar powder and pH of 5.6;
b) induction medium I: MS +6-BA1.0mg/L + IBA0.1mg/L + AC 2.0 mg/L;
c) induction medium II: MS +6-BA1.0mg/L + IBA 0.25 mg/L;
d) proliferation culture medium: 3/5MS +6-BA1.0mg/L + IBA0.5mg/L;
e) in-vitro preservation and test tube bulb expansion synchronous culture medium: 1/2MS + NAA 0.1mg/L + PP 3331 mg/L + mannitol 1.0g/L + AC1.0 mg/L;
(2) seed bulb treatment and sterilization: treating the seed bulbs at 5 ℃ for 3-4 months to break the dormancy; wrapping the bulbs with a breathable plastic film, placing the bulbs into moist peat with the water content of 60-70%, and placing the peat in a 50 ℃ incubator for heat treatment for 70 min; peeling off outer-layer scales, taking seed scale stem buds with the length of 4-6 cm in the outer-layer scales, soaking the seed scale stem buds in 1% detergent liquid for 10min, washing the seed scale stem buds with water, soaking the seed scale stem buds in 75% alcohol for 0.5min, washing the seed scale stem buds with sterile water for 6 times, sterilizing the seed scale stem buds with 0.1% mercuric chloride solution for 15min, washing the seed scale stem buds with the sterile water for 6 times, sterilizing the seed scale stem buds with 10% sodium hypochlorite aqueous solution for 15min, washing the seed scale stem buds with the sterile water;
(3) and (3) induction culture of bulb buds: taking a stem tip 0.2-0.3 mm away from a seed scale stem bud after sterilization as an explant, inoculating the explant to an induction culture medium I, and culturing for 55 days under the environmental conditions of the temperature of 20 +/-1 ℃, the illumination of 12h/d and the illumination intensity of 2000Lx until the scale stem bud is induced; cutting off top leaves of the bulb bud, longitudinally cutting into 2 pieces, transferring to an induction culture medium I, and culturing for 60 days under the same environmental condition until cluster bulb buds are induced to form; placing the stem in an illumination incubator, performing cyclic heat treatment for 60 days by changing the grade every 3 days according to the sequence of 30 ℃, 35 ℃ and 38 ℃, stripping 0.2-0.3 mm of stem tips in the illumination incubator as explants, inoculating the explants in an induction culture medium II for secondary induction culture, culturing for 80 days under the same environmental condition until the bulb buds are induced to form, and then placing the bulbs at 6 ℃ for low-temperature treatment for 45 days to recover the growth activity of the bulb buds;
(4) multiplication culture of bulb buds: cutting off leaves and part of base tissues of the bulb buds after low-temperature treatment, longitudinally cutting the bulb buds into 4-6 blocks, inoculating the 4-6 blocks on a proliferation culture medium, and culturing the 4-6 blocks at the temperature of 20 +/-1 ℃ for 90 days in the dark until robust bulb buds are formed;
(5) and (3) germplasm in vitro preservation and test tube bulb expansion synchronous culture: cutting off the top and partial base tissues of the bulb buds obtained in the step (4), longitudinally cutting into 4-6 pieces, inoculating the cut bulb buds into an in vitro preservation culture medium, and culturing at 20 +/-2 ℃ in the dark for 12 months until the bulb buds expand to form a test tube bulb with the diameter of 0.8-1.2 cm; in order to prolong the storage life, cutting off the bulb root system of the test tube, longitudinally cutting the bulb root system into 4-6 pieces, inoculating the cut bulb root system into an in vitro preservation culture medium, and preserving and culturing again for 1 time under the same condition, so that the requirement on the in vitro storage life of the germplasm is met;
(6) emergence and cold storage of test tube bulbs: washing off agar from the test tube bulbs by using clean water, wrapping the test tube bulbs in sterilized peat with the water content of 60-70%, pretreating at 10 ℃ for 20 days, refrigerating at 6 ℃ for 50 days until the dormancy breaking period, and then planting the test tube bulbs or planting the test tube bulbs in a time-selective manner within an extended storage period of 2-3 months at 1-3 ℃;
(7) planting: selecting a mountain area with an altitude of 1000-1900 m and a cool summer climate, planting the refrigerated bulbs obtained in the step (6) in a sterilized substrate in the late 4 th to middle 5 th months under the rain sheltering and insect prevention isolation conditions, and beginning to sprout, emerge and grow after 15-20 days;
(8) and (3) detecting the genetic stability of the germplasm: selecting plants which grow normally and vigorously and are detected to have no genetic variation through ISSR molecular marker (see example 4 specifically), and culturing the plants into qualified oriental lily seedballs.
Example 2: (Oriental lily germplasm resources in vitro preservation 2)
In this example, the medium of step (1) was prepared as follows:
a) basic culture medium: wherein,
the induction culture medium is an MS culture medium with 30g/L of cane sugar, 4.5g/L of agar powder and pH of 5.6;
the proliferation culture medium is 3/5MS culture medium of 55g/L sucrose, 4.5g/L agar powder and pH5.7;
in-vitro preservation and test tube bulb expansion synchronous culture medium is 1/2MS culture medium with sucrose of 68g/L, agar powder of 4.5g/L and pH5.8;
b) induction medium I: MS +6-BA 2.0mg/L + IBA0.5mg/L + AC1.0 mg/L;
c) induction medium II: MS +6-BA 0.75mg/L + IBA0.1 mg/L;
d) proliferation culture medium: 3/5MS +6-BA0.2mg/L + IBA0.1mg/L;
e) in-vitro preservation and test tube bulb expansion synchronous culture medium: 1/2MS + NAA 0.5mg/L + PP 3335 mg/L + mannitol 5.0g/L + AC 5.0 mg/L;
treating and sterilizing seed bulbs: placing in 50 deg.C incubator for heat treatment for 80 min; the sterilization treatment comprises soaking in 75% alcohol for 1min, sterilizing with 0.1% mercuric chloride solution for 20min, and sterilizing with 10% sodium hypochlorite aqueous solution for 13 min; and (3) induction culture of the bulb buds: inoculating the explant to an induction culture medium I, and culturing for 60d under the environmental conditions of the temperature of 20 +/-1 ℃, the illumination of 12h/d and the illumination intensity of 1000 Lx; culturing under the same environmental conditions for 90 d; after the circulation heat treatment is carried out for 50 days, the explant is stripped and inoculated in an induction culture medium II, and after the explant is cultured for 60 days, the explant is put at 6 ℃ for low-temperature treatment for 60 days; and (4) multiplication culture of the bulb buds: culturing at 20 + -1 deg.C in dark for 75 days; step (5), germplasm in vitro preservation and test tube bulb expansion synchronous culture: culturing at 20 + -2 deg.C in dark for 13 months; in order to prolong the storage life, after 3 times of re-preservation and synchronous culture of test tube bulb expansion are carried out under the same condition, the requirement on the in vitro storage life of the germplasm is met; and (6) emergence and cold storage of the test tube bulbs: pretreating at 11 deg.C for 15 days, refrigerating at 4 deg.C for 55 days, and planting at selected time; the rest steps and the process are the same as the example 1.
Example 3: (Oriental lily germplasm resources in vitro preservation method 3)
In this example, the medium of step (1) was prepared as follows:
a) basic culture medium: wherein,
the induction culture medium is an MS culture medium with 50g/L of cane sugar, 5.0g/L of agar powder and pH of 5.7;
the proliferation culture medium is 3/5MS culture medium with sucrose 60g/L, agar powder 4.0g/L and pH5.8;
in-vitro preservation and test tube bulb expansion synchronous culture medium is 1/2MS culture medium with 75g/L of cane sugar, 4.0g/L of agar powder and pH of 6.0;
b) induction medium I: MS +6-BA 1.5mg/L + IBA0.3mg/L + AC 3.0 mg/L;
c) induction medium II: MS +6-BA0.5mg/L + IBA0.5 mg/L;
d) proliferation culture medium: 3/5MS +6-BA0.5mg/L + IBA0.3mg/L;
e) in-vitro preservation and test tube bulb expansion synchronous culture medium: 1/2MS + NAA 0.3mg/L + PP 33310 mg/L + mannitol 3.0g/L + AC 3.0 mg/L;
treating and sterilizing seed bulbs: placing in 50 deg.C incubator for heat treatment for 90 min; the sterilization treatment comprises soaking in 75% alcohol for 0.75min, sterilizing with 0.1% mercuric chloride solution for 18min, and sterilizing with 10% sodium hypochlorite aqueous solution for 14 min; and (3) induction culture of the bulb buds: inoculating the explant to an induction culture medium I, and culturing for 50d under the environmental conditions of the temperature of 20 +/-1 ℃, the illumination of 12h/d and the illumination intensity of 3000 Lx; culturing under the same environmental conditions for 70 d; after circulating heat treatment for 55 days, stripping off the explant, inoculating the explant to an induction culture medium II, culturing for 90 days, and then treating at 6 ℃ for 50 days; and (4) multiplication culture of the bulb buds: culturing at 20 + -1 deg.C in dark for 60 d; step (5), germplasm in vitro preservation and test tube bulb expansion synchronous culture: culturing at 20 + -2 deg.C in dark for 15 months; after the preservation period is prolonged and the germplasm is preserved for 4 times, the preservation period in vitro is satisfied by performing the steps again for 1 time after the field planting recovery; and (6) emergence and cold storage of the test tube bulbs: pretreating at 12 deg.C for 10 days, refrigerating at 3 deg.C for 60 days, and planting at selected time; the rest steps and the process are the same as the example 1.
Example 4: (method of measuring genetic stability of germplasm)
(1) Plant material: lily bulb and test tube bulb
(2) Lily DNA extraction and concentration determination: extracting total DNA of each lily sample by adopting a plant genome DNA extraction (TIANGEN) kit, and sterilizing all used reagent vessels, wherein the specific operation flow is as follows:
a) weighing about 0.1g of fresh lily sample, adding liquid nitrogen, and fully grinding;
b) rapidly transferring the ground powder into a centrifuge tube pre-filled with 700 mu L of 65 ℃ preheating buffer solution GP1 (adding mercaptoethanol into preheated GP1 before an experiment to enable the final concentration to be 0.1%), rapidly reversing and uniformly mixing, placing the centrifuge tube in a 65 ℃ water bath for 20min, and reversing the centrifuge tube in the water bath process to mix samples for a plurality of times;
c) adding phenol and chloroform (1: 1) in the same volume, mixing well, centrifuging for 5min at 13,400g, and transferring the supernatant to a new centrifuge tube;
d) adding 700 μ L chloroform, mixing, centrifuging at 13,400g for 5 min;
e) carefully transferring the upper aqueous phase obtained in the last step into a new centrifuge tube, adding 700 mu L of buffer solution GP2, and fully and uniformly mixing;
f) transferring the mixed liquid into an adsorption column CB3, centrifuging for 30s at 13,400g, and discarding the waste liquid (the volume of the adsorption column is about 700 mu L, and centrifuging can be added in times);
g) adding 500 mu L of buffer GD into an adsorption column CB3, centrifuging for 30s at 13 g to 400g, discarding waste liquid, and putting the adsorption column CB3 into a collection tube;
h) adding 700 μ L of rinsing liquid PW into adsorption column CB3, centrifuging for 30s at 13,400g, discarding waste liquid, and placing adsorption column CB3 into a collection tube;
i) adding 500 mu L of rinsing liquid PW into an adsorption column CB3, centrifuging for 30s at 13,400g, and discarding waste liquid;
j) putting the adsorption column CB3 back into the collecting pipe, centrifuging for 2min at 13,400g, discarding waste liquid, and standing the adsorption column CB3 at room temperature for several minutes to thoroughly dry the residual rinsing liquid in the adsorption material;
k) transferring the adsorption column CB3 into a clean centrifugal tube, suspending and dripping 80 mu L of elution buffer TE into the middle part of the adsorption film, standing at room temperature for 2-5min, centrifuging at 13,400g for 2min, and collecting the solution into the centrifugal tube; detecting DNA quality with 1% agarose gel, detecting DNA purity and concentration with U.S. BECKMAN DU2530 type nucleic acid/protein analyzer, diluting to 20 ng/. mu.L working solution, and storing at-20 deg.C;
(3) establishment and optimization of ISSR reaction system
The ISSR-PCR amplification reaction condition is determined as 20 mu L system by orthogonal optimization test, Taq enzyme and dNTP of Fermentas company are adopted, primers are synthesized by invitrogen company, and the dosage of each reaction component is respectively:
2.0. mu.L of 10 XPCR buffer, 25mmol/L MgCl21.2. mu.L, 2.5mmol/L dNTP 1.6. mu.L, 10. mu. mol/L primer 1. mu.L, 20 ng/. mu.L template DNA 1. mu.L, Taq enzyme (5U/. mu.L) 0.1. mu.L, ddH2O 13.1.1. mu.L;
the ISSR-PCR amplification procedure was: pre-denaturation at 94 ℃ for 4 min; denaturation at 94 deg.C for 45s, annealing at 52 deg.C for 45s, extension at 72 deg.C for 1.5min, circulating for 38 times, final extension at 72 deg.C for 7min, and storing at 4 deg.C;
(4) screening of ISSR primers
5 primers suitable for lily genetic stability detection are screened by using 50 public ISSR primers provided by ISSR primer sequences adopted by University of British Columbia (UBC) in Canada and Masumi Yamagishi et al in lily research through tests, (1)5 '-ACACACACACACACACYG-3'; (2) 5'-TCTCTCTCTCTCTCTCG-3', respectively; (3)5 '-CTCCTCCTCCTCRC-3'; (4) 5'-CTCTCTCTCTCTCTCTG-3', respectively; (5) 5'-CTCTCTCTCTCTCTGTG-3' are provided. According to the melting temperature (Tm) value of Yingjun biosynthesis primers, DNA of germplasm original materials and in-vitro preservation materials of lily varieties 'Siberia', '1038', '1068-2' and '1078-4' is used as a template for carrying out gradient PCR amplification, and screened primers obey the following principles: 1) the resulting DNA bands are clearly distinguishable; 2) higher polymorphisms were found between samples analyzed; 3) generating polymorphic DNA bands with repeatability; screening out primers with high polymorphism and the optimal annealing temperature thereof, and then carrying out PCR amplification on all materials;
(5) PCR amplification and electrophoresis
Carrying out 1.5% Agarose Gel electrophoresis separation on the PCR amplification product, detecting the Gel by a Gel electrophoresis imaging system, photographing and storing;
(6) data processing
Each band DNA fragment of the electrophoresis pattern is 1 molecular marker, represents the binding site of 1 primer, the PCR amplification fragment with the length within the range of 100-1500bp is used for statistical analysis, a binary data matrix is established by 0 or 1 statistics, and at the same migration position, an amplification band is marked as 1, and no band is marked as 0; analyzing genetic diversity indexes such as genetic distance, genetic similarity coefficient and the like by using POPGENE 1.32 software, carrying out UPGMA cluster analysis by using NTSYSpc 2.02c software, and analyzing the genetic distance between the in vitro preserved sample and the control sample so as to determine the genetic stability of the in vitro preserved material.
Claims (3)
1. The in vitro conservation method of oriental lily germplasm resources is characterized by comprising the following steps:
(1) preparation of a culture medium: comprises the components of a basic culture medium and a culture medium in each stage of in vitro preservation, and the weight of each component in each liter of culture medium is as follows:
a) basic culture medium: wherein,
the induction culture medium is an MS culture medium with 30-50 g/L of cane sugar, 4-5 g/L of agar powder and pH of 5.6-5.8;
the proliferation culture medium is 3/5MS culture medium with 50-60 g/L of cane sugar, 4-5 g/L of agar powder and pH of 5.6-5.8;
the synchronous culture medium for in vitro preservation and test tube bulb expansion is 1/2MS culture medium with sucrose of 60-75 g/L, agar powder of 4.0-5.0 g/L and pH of 5.6-6.0;
b) induction medium I: MS +6-BA 1.0-2.0 mg/L + IBA 0.1-0.5 mg/L + AC 1.0-3.0 mg/L;
c) induction medium II: MS +6-BA 0.5-1.0 mg/L + IBA 0.1-0.5 mg/L;
d) proliferation culture medium: 3/5MS +6-BA 0.2-1.0 mg/L + IBA 0.1-0.5 mg/L;
e) in-vitro preservation and test tube bulb expansion synchronous culture medium: 1/2MS + 0.1-0.5 mg/L NAA + 0.1-10 mg/L PP 3331 + 1.0-5.0 g/L mannitol + 1.0-5.0 mg/L AC;
(2) seed bulb treatment and sterilization: treating the seed bulbs at 5 ℃ for 3-4 months to break the dormancy; wrapping the bulbs with a breathable plastic film, placing the bulbs into moist peat with the water content of 60-70%, and placing the peat in a 50 ℃ incubator for heat treatment for 70-90 min; peeling off the outer layer scale, taking seed bulb buds with the length of 4-6 cm in the scale, soaking the seed bulb buds in 1% of detergent liquid for 10min, washing the seed bulb buds with clear water, and sterilizing the seed bulb buds for later use;
(3) and (3) induction culture of bulb buds: taking a stem tip 0.2-0.3 mm away from a sterilized seed scale stem bud as an explant, inoculating the explant to an induction culture medium I, and culturing for 50-60 days under the environmental conditions of the temperature of 20 +/-1 ℃, the illumination for 12h/d and the illumination intensity of 1000-3000 Lx until the scale stem bud is induced; cutting off top leaves of the bulb bud, longitudinally cutting into 2 pieces, transferring to an induction culture medium I, and culturing for 60-90 days under the same environmental condition until cluster bulb buds are induced to form; placing the stem in an illumination incubator, performing cyclic heat treatment for 50-60 days by changing the grade every 3 days according to the sequence of 30 ℃, 35 ℃ and 38 ℃, stripping 0.2-0.3 mm of stem tips in the illumination incubator as explants, inoculating the explants in an induction culture medium II for secondary induction culture, culturing for 60-90 days under the same environmental condition until the buds are induced to form bulb buds, and then performing low-temperature treatment for 45-60 days at 6 ℃ to recover the growth activity of the bulb buds;
(4) multiplication culture of bulb buds: cutting off leaves and part of base tissues of the bulb buds after low-temperature treatment, longitudinally cutting the bulb buds into 4-6 blocks, inoculating the 4-6 blocks on a proliferation culture medium, and culturing the 4-6 blocks at 20 +/-1 ℃ in the dark for 60-90 days to form robust bulb buds;
(5) and (3) germplasm in vitro preservation and test tube bulb expansion synchronous culture: cutting off the top and partial base tissues of the bulb buds in the step (4), longitudinally cutting into 4-6 pieces, inoculating the cut bulb buds into an in vitro preservation culture medium, and culturing at 20 +/-2 ℃ in the dark for 12-15 months until the bulb buds formed at the base of the cut pieces expand into test tube bulbs with the diameter of 0.8-1.2 cm; then seedling emergence is carried out or the root system of the test tube bulb is cut off and is inoculated in an in vitro preservation culture medium after being longitudinally cut into 4-6 blocks, and preservation and synchronous culture of test tube bulb expansion can be carried out for 1-4 times circularly under the same condition; after 4 times, the seeds need to be planted and recovered in the field and then preserved in vitro according to the steps until the requirement on the in vitro preservation period of the seeds is met;
(6) emergence and cold storage of the test tube bulb: washing off agar from the test tube bulb by using clean water, wrapping the test tube bulb in sterilized peat with the water content of 60-70%, pretreating for 10-20 days at 10-12 ℃, refrigerating for 50-60 days at 3-6 ℃ until the dormancy breaking period, and then planting or planting at a time selected within an extended storage period of 2-3 months at 1-3 ℃;
(7) planting: selecting a mountain area with an altitude of 1000-1900 m and a cool summer climate, planting the refrigerated bulbs obtained in the step (6) in a sterilized substrate in the late 4 th to middle 5 th months under the rain sheltering and insect prevention isolation conditions, and beginning to sprout, emerge and grow after 15-20 days;
(8) and (3) detecting the genetic stability of the germplasm: selecting plants which grow normally and vigorously and are detected to have no genetic variation through ISSR molecular marker, and culturing the plants into qualified oriental lily seedballs.
2. The method as claimed in claim 1, wherein the sterilization treatment of the seed-scale stem buds comprises soaking in 75% alcohol for 0.5-1.0 min, washing with sterile water for 6 times, sterilizing with 0.1% mercuric chloride solution for 15-20 min, washing with sterile water for 6 times, sterilizing with 10% sodium hypochlorite aqueous solution for 13-15 min, and washing with sterile water for 6 times.
3. The method of claim 1, wherein the planting substrate is peat perlite in a volume ratio of 7: 3.
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| CN110367125A (en) * | 2019-09-03 | 2019-10-25 | 南通大学 | The method and method for resuscitation of lily bulb preservation effect are improved with thermal stimulus |
| CN112251528A (en) * | 2020-10-19 | 2021-01-22 | 北京林业大学 | Microsatellite DNA molecular marker for identifying ploidy of lily and application thereof |
| CN112251528B (en) * | 2020-10-19 | 2021-07-06 | 北京林业大学 | Microsatellite DNA molecular marker for identifying ploidy of lily and application thereof |
| CN112450208A (en) * | 2020-12-28 | 2021-03-09 | 内蒙古蒙草生态环境（集团）股份有限公司 | Lily preservation method of lily |
| CN112450208B (en) * | 2020-12-28 | 2022-04-29 | 内蒙古蒙草生态环境（集团）股份有限公司 | Lily preservation method of lily |
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