CN102511397A - Method for inducing populus calli and induction culture medium - Google Patents
Method for inducing populus calli and induction culture medium Download PDFInfo
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Abstract
The invention discloses a method for inducing populus calli and an induction culture medium, and belongs to the field of the tissue culture of populus. The sieved and optimized induction culture medium for the populus calli is an H culture medium which, based on one liter, contains 0.5 to 1.5 mg of naphthylacetic acid, 0.5 to 1.5 mg of 6-aminopurine, 20 to 40 g of cane sugar and 4.5 to 6.5 g of agar. According to the method, populus anthers in the single nuclear aside period are used as explants, and are inoculated to the sieved induction culture medium for the calli to induce the calli to obtain the populus calli with high growth speed and high differentiation rate of organs. The induction culture medium for the calli can improve the inductivity of the calli; and the obtained calli are of high quality, and a large number of high-quality populus test tube seedlings can be grown in a short period. The method can propagate populus quickly and also can be applied to improvement of populus plants, germplasm storage and the like.
Description
Technical field
The present invention relates to the tissue culture medium (TCM) of a plant species, the particularly inducing culture of willow callus and the application in cultivating the willow anther callus thereof belongs to the field of tissue culture of willow.
Background technology
Beijing poplar (Populus beijingensis) is the filial generation of Cathay poplar and Lombardy poplar; It is the new varieties of poplar that forestry circle is bred with artificial cross breeding method for the first time after the founding of New; It has characteristics such as cold-resistant, happiness liquid manure, tree attitude U.S.A; Be good timber forest, shelter forest, street tree and " four is other " green tree species, liked by the masses.Mainly be distributed in areas such as Chinese North China and northwest.
Because poplar is a dioecism, self-pollination is impossible realize basically.The strain system of isozygotying through the crossbreeding acquisition of routine needs very long breeding cycle.For the willow strain system that obtains to isozygoty, the researcher adopted self-pollination or the method for backcrossing in the past, but the breeding time limit that this breeding process needs is very long.Then can obtain the monoploid of target strain system earlier through anther culture, after it is doubled to become double haploid (doubled haploids DHs), so just can significantly shorten breeding cycle.In addition; Along with modern biomolecule technology rapid development; Plant haploid and product thereof are in many-sided successful Application such as the excavation of ploidy breeding, gene order-checking, map construction and functional gene and evaluations and obtained very big achievement, and haplophyte material and product thereof are favored by more and more researchers.
At present, the ways of regeneration of plant haploid comprises flower pesticide and pollen cultivation, parthenogenesis, chromosome elimination etc., and wherein anther culture is a kind of comparatively otherwise effective technique that obtains plant haploid.Callus culture is a kind of modal training method, and except that the shoot apical meristem cultivation with a part of organ culture, other several plant tissue culture form finally all will experience callus could produce plant.Callus also usually is the material source of suspension cultured cells and protoplast in addition.Callus is divided into embryo callus and non-embryonic callus tissue, and embryo callus is easy to regeneration bud, can develop into the tender plant of children.
Under isolated condition, cultivate flower pesticide, the development pathway of artificial change microspore with the method that exsomatizes; Its gametophyte development pathway is stopped, turn to sporophyte to grow, through the approach of organ differentiation; Obtain complete haplobiont, produce monoploid in a large number for manual work means effectively are provided.Haplobiont through this approach regeneration doubles through spontaneous doubling or artificial induction, obtains pure and mild liploid plant, thereby is the material that further breeding and genetic research provide usefulness.Utilize flower pesticide pollen induction callus; It is the technical major reform of plant breeding that callus differentiation culture acquisition monoploid carries out breeding; It can combine with methods such as the present crossbreeding of adopting, induced mutations breeding, distant hybridization; In order to overcome the deficiency in these methods, also can directly create new type with the method.But there is the low problem of callus induction rate all the time in the anther culture process, makes plant haploid can not satisfy researchers' demand far away.
Summary of the invention
Technical problem to be solved by this invention is to the low problem of existing callus induction rate in the existing willow flower pesticide incubation; A kind of abductive approach of new willow callus is provided; The inducing culture and the condition of culture thereof of optimum willow anther callus groped and screened to the inventive method; This method can form a large amount of good willow callus within a short period of time; Callus induction rate is high, and the organ differentiation rate of the callus of acquisition is high, can carry out scale, batch production production.
In order to solve the problems of the technologies described above, the technical scheme that the present invention adopted is following:
One aspect of the present invention provides a kind of willow flower pesticide callus inducing medium of optimization, and said willow flower pesticide callus inducing medium is the H medium that has added 0.5-1.5mg/L methyl (NAA), 0.5-1.5mg/L 6-chaff aminopurine (KT), 20-40g/L sucrose, 4.5-6.5g/L agar.
Wherein, the concentration of said NAA is preferably 0.5-1.0mg/L, further is preferably 1.0mg/L; The concentration of said KT is preferably 0.5-1.0mg/L, further is preferably 1.0mg/L; Said concentration of sucrose is preferably 30g/L; The concentration of said agar is preferably 5.5g/L.
Preferably, willow according to the invention is Beijing poplar (Populus beijingensis).
The present invention provides a kind of abductive approach of willow callus on the other hand, may further comprise the steps:
1) from disinfect back willow inflorescence, takes out flower pesticide, obtain aseptic flower pesticide explant;
2) aseptic flower pesticide is seeded in carries out callus induction on the described willow flower pesticide callus inducing medium and cultivate, obtain callus.
Willow described in the present invention is preferably Beijing poplar (Populus beijingensis).
In order to reach better cultivation effect, when the microspore development of willow inflorescence was in monokaryon keeps to the side the phase in period, took out the inducing culture that carry out callus with flower pesticide wherein this moment; Can effectively improve the inducing culture rate of callus; Induce the callus growth speed that obtains fast, be yellow or brown, rough surface; Quality is loose frangible, has higher vigor.Wherein, the willow microspore development period can be through form of observing bud and the developmental stage that diameter is judged microspore roughly.Comparatively effectively detection method is the developmental stage that microspore is observed in dyeing, and the present invention observes the developmental stage of confirming microspore through aceto-camine dyeing.
Because poplar flower is a catkin, the scale on bud surface is little by the felt hair, disinfects described in the step 1) and adopts following processing mode to have better aseptic result:
1-A) use the aseptic water washing bud;
1-B) use alcohol-pickled bud;
1-C) bud is carried out surface sterilizing, use aseptic water washing with the liquor natrii hypochloritis;
1-D) take out flower pesticide after blotting the bud surface moisture, promptly get.
Preferably, the aseptic water washing number of times is 4-5 time step 1-A); The concentration of volume percent of alcohol step 1-B) is 70.0%; Soak time is 30s; Step 1-C) mass percent concentration of clorox is 7.0% in; The surface sterilizing time is 5min; The aseptic water washing number of times is 3-5 time.
The composition of the inducing culture of callus is directly connected to the inductivity of callus and the quality height of callus; In order to improve the inductivity of willow anther callus; The present invention optimizes the inducing culture of willow anther callus; Finally confirm through a large amount of screening tests; The callus induction that adopts following callus inducing medium to carry out flower pesticide is cultivated, callus induce effect best: callus inducing medium is preferably: H minimal medium+naa (NAA) 0.5-1.5mg/L+6-chaff aminopurine (KT) 0.5-1.5mg/L+ sucrose 20-40g/L+ agar 4.5-6.5g/L; Further be preferably: H minimal medium+naa 0.5-1.0mg/L+6-chaff aminopurine 0.5-1.0mg/L+ sucrose 20-40g/L+ agar 4.5-6.5g/L; Most preferably be: H minimal medium+naa 1.0mg/L+6-chaff aminopurine 1.0mg/L+ sucrose 30g/L+ agar 5.5g/L.
Preferably, callus induction step 2) is cultivated and carried out under the following condition: under the dark condition, cultivation temperature is 25 ± 1 ℃.
The present invention carries out cultured in vitro to Beijing poplar bloassom medicine, and the microspore induced development is to have the high callus of vigor in the flower pesticide, and willow flower pesticide callus induction rate is high, reaches 30.0%.The callus growth speed that obtains is fast, and callus organ differentiation culture obtains indefinite bud, and the differentiation rate of indefinite bud is up to 100.0%, for willow quality-improving and breeding of new variety provide strong means.The callus that utilizes the inventive method to induce to obtain is cultivated the willow haplobiont, and plant strain growth is healthy and strong, reproduction coefficient is high, obtains test tube seedling transplanting survival rate up to 92.0%, is the simple, fast technical system of batch production large-scale production willow plant.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment with form or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall in protection scope of the present invention the details of technical scheme of the present invention.
One, test material
1, supplies the examination material: Beijing poplar (Populus beijingensis);
2, plant growth regulator
Employed plant growth regulating substance adopts homemade methyl (NAA), 6-benzyl aminoadenine (BAP), 6-chaff aminopurine (KT), gibberellin (GA among the present invention
3), the 2,4 dichloro benzene ethoxyacetic acid (2,4-D), indolebutyric acid (IBA).
3, culture medium preparation:
(1) composition or the compound method of " MS minimal medium ":
Table 1 MS medium (Murashige and Skoog, 1962)
More than the consumption of various nutrient components, except mother liquor I was 20 times of concentrates, remaining was 200 times of concentrates.After above-mentioned MS medium mother liquor prepared, it was for use to be stored in 4 ℃ of refrigerators.According to the total amount of preparing culture medium, agar that weighing is required and sucrose are poured into and desire to join in the distilled water of culture volume 3/4, and the limit heating edge stirs with glass bar, up to the liquid shape that is translucent.From various mother liquors, take out required consumption respectively with graduated cylinder or pipette then, add hormone then according to the kind and the concentration of hormone in the culture medium prescription, it is put into enamel graduate with various mother liquors like need.Every adding is a kind of all fully stirs, and last adding distil water is settled to cumulative volume, stirs.With pH meter test media acid-base value, the NaOH solution of drawing 1.0mol/L with dropper dropwise adds in the medium of thawing, stirs while dripping, and is adjusted to 5.8 up to the pH of medium value.
(2) composition and the compound method of " H medium ":
Table 2H medium
More than the consumption of various nutrient components, except mother liquor I was 20 times of concentrates, remaining was 200 times of concentrates.After above-mentioned H medium mother liquor prepared, it was for use to be stored in 4 ℃ of refrigerators.According to the total amount of preparing culture medium, agar that weighing is required and sucrose are poured into and desire to join in the distilled water of culture volume 3/4, and the limit heating edge stirs with glass bar, up to the liquid shape that is translucent.From various mother liquors, take out required consumption respectively with graduated cylinder or pipette then, add hormone then according to the kind and the concentration of hormone in the culture medium prescription, it is put into enamel graduate with various mother liquors like need.Every adding is a kind of all fully stirs, and last adding distil water is settled to cumulative volume, stirs.With pH meter test media acid-base value, the NaOH solution of drawing 1.0mol/L with dropper dropwise adds in the medium of thawing, stirs while dripping, and is adjusted to 5.5 up to the pH of medium value.
(3) N
6The composition of minimal medium and compound method:
Table 3 N
6Medium (Beijing plant institute, Heilungkiang Academy of Agricultural Sciences, 1974)
More than the consumption of various nutrient components, except mother liquor I was 20 times of concentrates, remaining was 200 times of concentrates.Above-mentioned N
6After the medium mother liquor prepared, it was for use to be stored in 4 ℃ of refrigerators.According to the total amount of preparing culture medium, agar that weighing is required and sucrose are poured into and desire to join in the distilled water of culture volume 3/4, and the limit heating edge stirs with glass bar, up to the liquid shape that is translucent.From various mother liquors, take out required consumption respectively with graduated cylinder or pipette then, add hormone then according to the kind and the concentration of hormone in the culture medium prescription, it is put into enamel graduate with various mother liquors like need.Every adding is a kind of all fully stirs, and last adding distil water is settled to cumulative volume, stirs.With pH meter test media acid-base value, the NaOH solution of drawing 1.0mol/L with dropper dropwise adds in the medium of thawing, stirs while dripping, and is adjusted to 5.8 up to the pH of medium value.
(4) differentiation adventitious buds culture medium prescription: MS minimal medium+BAP 0.6-1.0mg/L+NAA 0.2-0.5mg/L+GA
30.02-02mg/L+ sucrose 20g/L+ agar 5.5g/L; Regulating the pH value is 5.8, and medium was 121 ℃ of following constant temperature sterilizations 20 minutes.
(5) the adventitious bud rooting culture medium prescription is: 1/2MS minimal medium+IBA 0.2-0.5mg/L sucrose 20g/L+ agar 5.5g/L; Regulating the pH value is 5.8, and medium was 121 ℃ of following constant temperature sterilizations 20 minutes.
The screening and the optimization of Test Example 1 Beijing poplar bloassom medicine callus inducing medium
1, the suitable developmental stage of the flower pesticide of cultured in vitro and microspore
Clip has Beijing poplar bloassom branch of full bud in 25 ℃ of following water planting a few days of room temperature.The willow bud is placed on the slide, strips out flower pesticide, adopt aceto-camine dyeing then, sediments microscope inspection is observed the developmental stage of microspore, to confirm pollen development period.This test is chosen microspore and is in the keep to the side flower pesticide of phase of monokaryon and inoculates, and result of study shows anther cultural just best period this moment.
2, explant sterilization
To be in the keep to the side bud of phase of monokaryon and take off from catkin, earlier with aseptic water washing 5 times, the filter paper suck dry moisture.Explant is placed on the superclean bench; The use concentration of volume percent is 70.0% alcohol-pickled bud 30s; Then using mass percent concentration is to carry out surface sterilization 5 minutes among 7.0% the liquor natrii hypochloritis, uses aseptic water washing 3-5 time then, takes out bud.
Under anatomical lens, the scale of bud is peelled off, got the flower pesticide at inflorescence middle part, be inoculated in the culture dish, seal, secretly cultivate with preservative film, 30 flower pesticide of each culture dish inoculation, each handles repetition 5 times.Condition of culture: dark condition, 25 ± 1 ℃ of cultivation temperature.
3, the screening of callus inducing medium
The keep to the side flower pesticide of phase of monokaryon be will be in and MS, H and N will be inoculated in respectively
6In three kinds of minimal mediums, add 1.0mg/L 2,4-D and 1.0mg/L BAP, sucrose 30g/L, agar 5.5g/L, pH5.8 in every kind.30 flower pesticide of each culture dish inoculation, each handles repetition 5 times.Condition of culture: dark condition, 25 ± 1 ℃ of cultivation temperature are cultivated the callus number that begins to add up each processing formation after 35 days, cultivate and calculate callus induction rate after 120 days, statistics and result of calculation such as table 4.
The different medium of table 4 are to the effect of inducing of callus
| Minimal medium | Inoculation flower pesticide number (individual) | Produce the flower pesticide number (individual) of callus | Callus of induce rate (%) |
| MS | 150 | 3 | 2.0 |
| H | 150 | 5 | 3.3 |
| N 6 | 150 | 0 | 0.0 |
The result of the test of table 4 shows:
1) adopt the H medium, the callus induction rate of willow flower pesticide is high.
2) the flower pesticide inoculation is about 35 days, begins to have observed callus and forms.In MS, H medium, all observe the formation of callus, this callus quality consolidation, faint yellow opaque, the surface is graininess, poor growth.At N
6Do not observe the formation of callus in the medium.
4, callus inducing medium hormone kind screening
According to the H minimal medium of screening, screening different hormone kind.
Be inoculated in respectively in the H minimal medium that adds different hormone kinds and concentration being in the keep to the side flower pesticide of phase of monokaryon, the H minimal medium that adds different hormone kinds and concentration is as shown in table 5.
Table 5 contains the H medium of different hormones
Add sucrose 30g/L, agar 5.5g/L, pH5.5 in every kind of medium.30 flower pesticide of each culture dish inoculation, each handles repetition 3 times.Condition of culture: dark condition, 25 ± 1 ℃ of cultivation temperature are cultivated the callus number that begins to add up each processing formation after 35 days, cultivate and calculate callus induction rate after 120 days, statistics and result of calculation such as table 6.
The different hormone kinds of table 6 are to the influence of callus induction
Result of the test shows: hormone kind NAA and KT combination are the highest to the anther callus inductivity, and average inductivity reaches 9.3%, secondly is 2, and 4-D and BAP combination are used.Testing result shows that the hormone combinations of NAA and KT induces effect more satisfactory to callus.
5, callus inducing medium hormone concentration screening
According to the minimal medium and the hormone kind of screening, the proportioning of screening hormone.
Be inoculated in respectively in the H medium of the NAA that adds variable concentrations, KT being in the keep to the side flower pesticide of phase of monokaryon, and add sucrose 30g/L, agar 5.5g/L, pH5.5 in every kind of medium.30 flower pesticide of each culture dish inoculation, each handles repetition 3 times.Condition of culture: dark condition, 25 ± 1 ℃ of cultivation temperature are cultivated the callus number that begins to add up each processing formation after 35 days, cultivate and calculate callus induction rate after 120 days, and statistics and result of calculation are as shown in table 7.
Table 7 different hormone combinations is to the influence of callus induction
Visible according to table 7 result of the test; NAA that in the H medium, is added and the concentration of KT are respectively within the 0.5-1.0mg/L scope; Help improving the inductivity of willow anther callus; Wherein, the inductivity of callus is the highest in the H medium of interpolation 1.0mg/LNAA and 1.0mg/L KT, and the inductivity of callus has reached 30.0%; Next is to add 1.0mg/L NAA and 0.5mg/L KT in the H medium, and the inductivity of callus has reached 17.8%;
Be inoculated in the inducing culture of callus: the willow flower pesticide among H medium+NAA1.0mg/L+KT1.0mg/L was being cultivated about 10 days; Can be observed flower pesticide expands gradually; Cultivating can observe in about 35 days has callus to form in the medium, this callus quality consolidation is faint yellow opaque; The surface is graininess, poor growth.After this in inducing culture successive observed to the formation that callus is arranged, be cultured to 120 days after callus seldom form again.
Test Example 2
1, prepares aseptic flower pesticide
Get the keep to the side willow bud of phase of monokaryon; With aseptic water washing bud 3-5 time; The use concentration of volume percent is 70.0% alcohol-pickled bud 30s behind the use filter paper suck dry moisture; Then using mass percent concentration is 7.0% the liquor natrii hypochloritis 5min that sterilizes, and uses aseptic water washing bud 3-5 time then.On superclean bench, peel off the scale of bud, take out the flower pesticide at inflorescence middle part, promptly get.
2, callus induction is cultivated
Flower pesticide is inoculated in the callus inducing medium secretly cultivates; Condition of culture: under the dark condition, cultivation temperature is 25 ± 1 ℃, cultivates 120 days; Statistics anther callus inductivity reaches 30.0%; Wherein, callus inducing medium is: H+NAA 1.0mg/L+KT 1.0mg/L+ sucrose 30g/L+ agar 5.5g/L, pH5.5.
3, differentiation adventitious buds is cultivated
The diameter that cultivation is obtained is that the willow anther callus about 3mm is inoculated into and carries out differentiation adventitious buds in the differentiation adventitious buds medium and cultivate, and condition of culture: temperature is 25 ± 1 ℃, illumination 1500～2000lx, light application time 16 hours/day.In the differentiation culture process; The willow anther callus transfers yellow green to by yellow and becomes green then, can see the bud point of green projection about a week on the callus surface, and then vane extension is come out; Indefinite bud is put forth then, and no offspring growing height can reach 1.5～2cm about 20 days.The differentiation rate of cultivating statistics indefinite bud after 20 days is 100.0%, and wherein, the differentiation adventitious buds culture medium prescription is: MS+BAP 0.6-1.0mg/L+NAA 0.2-0.5mg/L+GA
30.02-0.2mg/L+ sucrose 20g/L+ agar 5.5g/L, pH5.8.
Callus organ differential medium is the disclosed any willow callus organ differential medium of inducing poplar callus differentiation phase; These willow callus organ differential mediums all can be applicable to the present invention; Its constituent is open in various documents or textbook, except adopting above-mentioned medium: MS+BAP 0.6-1.0mg/L+NAA0.2-0.5mg/L+GA
30.02-0.2mg/L+ outside the sucrose 20g/L+ agar 5.5g/L, other willow callus differentiation adventitious buds medium also are applicable to the present invention, for example: MS+BAP 3.0mg/L+IAA 0.2mg/L and MS+KT 2.0mg/L+IAA 0.5mg/L etc.
4, culture of rootage
Be inoculated into and carry out culture of rootage in the root media, condition of culture being cultured to the indefinite bud with 3-5 sheet young leaflet tablet: cultivation temperature is 25 ± 1 ℃, illumination 1500～2000lx; Light application time 16 hours/day, the situation of taking root of cultivating statistics indefinite bud after 20 days, rooting rate reaches 100.0%; Obtain willow test tube seedling, wherein, the adventitious bud rooting culture medium prescription is: 1/2MS+IBA 0.2-0.5g/L+ sucrose 20g/L+ agar 5.5g/L; PH5.8, the main root of test tube seedling is sturdy, and fibrous root is numerous.
Disclosed any willow adventitious bud rooting medium when the adventitious bud rooting medium is the cultivation of inducing poplar adventitious bud rooting; These willow adventitious bud rooting medium all can be applicable to the present invention; Its constituent is open in various documents or textbook; Except adopting above-mentioned medium: the 1/2MS+IBA0.2-0.5g/L+ sucrose 20g/L+ agar 5.5g/L; Other willow adventitious bud rooting medium also are applicable to the present invention, for example: H+IAA 1.0-1.5g/L+ sucrose 20g/L+ agar 5.5g/L, 1/2MS+IAA 1.0-1.5g/L+ sucrose 20g/L+ agar 5.5g/L etc.
5, refining seedling, transplanting
Choose the test tube seedling preferably of taking root; Be placed on and refine seedling cultivation 2-3 days in the shade in the greenhouse; Take out seedling then and clean the root medium; Be transplanted in the willow soilless culture substrate (mixture of perlite and vermiculite), soilless culture substrate is a perlite and the ratio of the volume of vermiculite is 1: 1 a mixture.Water permeablely after the transplanting, add up transplanting survival rate after 15 days, survival rate is 92.0%.
Practical application shows: adopt callus of the present invention to organize the willow anther callus of inducing culture institute inducing culture repeatedly obtaining the willow haplobiont, good reliability in the different repeated tests; Haplobiont is doubled or artificial handles growing point with colchicin, the dliploid that obtains isozygotying through natural.
Claims (10)
1. a willow flower pesticide callus inducing medium is characterized in that its composition is: H medium+methyl 0.5-1.5mg/L+6-chaff aminopurine 0.5-1.5mg/L+ sucrose 20-40g/L+ agar 4.5-6.5g/L.
2. willow flower pesticide callus inducing medium as claimed in claim 1 is characterized in that its composition is: H medium+methyl 0.5-1.0mg/L+6-chaff aminopurine 0.5-1.0mg/L+ sucrose 20-40g/L+ agar 4.5-6.5g/L.
3. willow flower pesticide callus inducing medium as claimed in claim 2 is characterized in that its composition is: H medium+methyl 1.0mg/L+6-chaff aminopurine 1.0mg/L+ sucrose 30g/L+ agar 5.5g/L.
4. the abductive approach of a willow callus comprises the steps:
1) from disinfect back willow inflorescence, takes out flower pesticide, obtain aseptic flower pesticide explant;
2) aseptic flower pesticide is seeded in carries out callus induction on any one described willow flower pesticide callus inducing medium of claim 1-3 and cultivate, obtain the willow callus.
5. abductive approach as claimed in claim 4 is characterized in that step 2) described in callus induction cultivate and to carry out under the following condition: under the dark condition, cultivation temperature is 25 ± 1 ℃.
6. abductive approach as claimed in claim 4 is characterized in that flower pesticide described in the step 1) is that developmental stage is that microspore is in the keep to the side willow flower pesticide of phase of monokaryon.
7. abductive approach as claimed in claim 4 is characterized in that described willow is Beijing poplar (Populus beijingensis).
8. abductive approach as claimed in claim 4 is characterized in that disinfecting described in the step 1) and comprises the steps:
1-A) use the aseptic water washing bud after gently brushing bud scale surface with writing brush;
1-B) use alcohol-pickled bud;
1-C) bud is carried out surface sterilizing, use aseptic water washing with the liquor natrii hypochloritis;
1-D) blot behind the bud surface moisture and take out flower pesticide, promptly get from inflorescence.
9. abductive approach as claimed in claim 8 is characterized in that step 1-B) described in the concentration of volume percent of alcohol be 70.0%.
10. abductive approach as claimed in claim 8 is characterized in that step 1-C) described in liquor natrii hypochloritis's mass percent concentration be 7.0%.
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| CN112352675B (en) * | 2020-04-06 | 2021-09-10 | 东北林业大学 | Populus deltoides hormone autotrophic cell line capable of realizing plant regeneration |
| CN113403254A (en) * | 2021-07-08 | 2021-09-17 | 华中农业大学 | Preparation method of chimonanthus nitens protoplast |
| CN113403254B (en) * | 2021-07-08 | 2022-10-11 | 华中农业大学 | Preparation method of chimonanthus nitens protoplast |
| CN114467754A (en) * | 2022-02-24 | 2022-05-13 | 吉林农业大学 | Method for obtaining populus deltoids aneuploid plant |
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