CN102119657A - Tender stem callus plant regeneration induction culture medium for lonicera caerulea - Google Patents
Tender stem callus plant regeneration induction culture medium for lonicera caerulea Download PDFInfo
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Abstract
The invention relates to a tender stem callus plant regeneration induction culture medium for lonicera caerulea and solves the problems of low plant differentiation rate and high culture medium cost problem of the auxiliary bud or stem tip culture, which is the main tissue culture of lonicera caerulea. The culture medium consists of a large amount of macroelements, microelements and hormone, wherein the hormone consists of 6-benzyl aminopurine at a concentration of 1.0 to 2.0mg/L and indolebutyric acid or naphthylacetic acid at a concentration of 0.05 to 0.2mg. When the LD1 culture medium of the invention is used, the tender stems of lonicera caerulea can be easily induced to form calla, and the induction rates of the calla are all over 90 percent; and the probability of the induction of the calla into plants is high, the plant differentiation rate is up to 50 to 82 percent which is 66.6 to 173.3 percent higher than that of the conventional Murashige-Skoog (MS) culture medium, and the cost is saved by 45.1 percent compared with the reference MS culture medium.
Description
Technical field
The present invention relates to a kind of Lonicera edulis callus plant regeneration inducing culture.
Background technology
Lonicera edulis (Lonicera caerulea), another name indigo fruit, be commonly called as Brachybotrys paridiformis, goat milk, black bear fruit etc., be perennial machaka, be one of peculiar little oar fruit in Northeast Asian region, its distributed areas are mainly in forest zones such as Russia, NORTHERN KOREA, Japan and the Manchurian big Xiaoxinanlin Mountains, Changbai Mountain, Zhang Guangcai mountain ranges.
Lonicera edulis is another the emerging little berry after elements of black currant, raspberry, cowberry, mainly is grown in the woods wetland or border, swamp meadow, requires the soil of weak acid, the anti-low temperature more than-40 ℃ of top branch, and adaptability is stronger.
The Lonicera edulis fruit has higher healthy nutritive value, is rich in compositions such as carbohydrate, organic acid, mineral matter, vitamin.The total amount of contained 17 seed amino acids is higher than common fruit, and wherein essential amino acid accounts for total amount about 40%; Zinc in the mineral element, selenium, iron, calcium content are higher; Fruit can be eaten raw, also can be processed into beverage, jam, fruitcake and fruit wine etc.; In addition, cauline leaf and fruit can extract natural mauve pigment again.
In recent years along with the Lonicera edulis market demand constantly increases, its fresh fruit price also constantly increases, the cause per kilogram increased to 10～15 yuan of per kilograms for 4 yuan in nearly 4 years, the quick-frozen fruit market price increases to 9500 yuan/ton, the inspissated juice international market price is 6.0～8.0 ten thousand yuan/ton, the drink price is 5～10 yuan/bottle, and supply falls short of demand in market.
Along with to product developments such as wild indigo fruit beverage, fruit wine, pigments, the predation formula is gathered and is caused wild resource to fall sharply, and resource distribution is limited in addition, is difficult to satisfy the growing market demand, therefore carries out manually to foster and cultivate very necessary.And restrict tame subject matter is the sapling multiplication difficulty, and main employing cuttage and root division, seedling from seed breeding are planted in the production, and the former reproduction coefficient is low, and survival rate is not high, and destroys wild resource; Latter's seedling variability is bigger, influences the quality and the output of fruit, and because of seed grain is little, becomes the seedling difficulty, and the seedling growing process difficulty is big.Can address the above problem by tissue-culturing quick-propagation, fast numerous general the employings MS medium of domestic plant tissue culture, but because of cultivating floristics, the period of drawing materials, cultivating the position difference, its effect is difference also.The report of at present domestic relevant Lonicera edulis group training aspect is less, mainly take axillalry bud or Shoot Tip Culture, but effect is undesirable, still there is not directly to break up the report of bud clump, need by the callus of induce plant, plant differentiation rate less than 30%, and do not see on producing and use.
Summary of the invention
The objective of the invention is mainly to take axillalry bud or Shoot Tip Culture for the tissue culture that solves existing Lonicera edulis, there is the low and high problem of culture medium cost of plant differentiation rate, the invention provides a kind of Lonicera edulis children stem callus plant regeneration inducing culture.
Lonicera edulis children stem callus plant regeneration inducing culture of the present invention is made up of macroelement, trace element and hormone, and wherein said macroelement is the NH of 900～920mg/L by concentration
4NO
3, 90～100mg/L KH
2PO
4, 1000～1050mg/L KNO
3, 240～250mg/L CaCl
22H
2The MgSO of O, 200～210mg/L
47H
2The EDTA-Fe of O and 15～18mg/L forms, and described trace element is the KI of 0.83mg/L, the MnSO of 22.3mg/L by concentration
44H
2The H of O, 6.2mg/L
3BO
3, 8.6mg/L ZnSO
47H
2The Na of O, 0.025mg/L
2MoO
42H
2The CuSO of O, 0.25mg/L
45H
2The CoCl of O and 0.025mg/L
26H
2O, described hormone are that the 6-benzylaminopurine (6-BA) of 1.0～2.0mg/L or indolebutyric acid (IBA) or the methyl (NAA) of kinetin (KT) and 0.05～0.2mg/L are formed by concentration.
The present invention gathers its young stem in the growth of the beginning of June (on June one to No. six) Lonicera edulis peak period, and with the alcohol-pickled 10s of 70% (volume), aseptic water washing 3 times is used 0.1% (quality) mercuric chloride liquid disinfectant 8min again, and aseptic water washing is 3～5 times again; When being seeded to medium Lonicera edulis children stem two ends are cut off, be inoculated in LD of the present invention
1Media surface; Condition of culture is: 24～26 ℃ of cultivation temperature, and light application time 14～16h/d, intensity of illumination 1800～2000lx, 20～30 days subcultures are once.
Lonicera edulis children stem callus plant regeneration inducing culture of the present invention (is designated as LD
1Medium) mineral salt (macroelement and trace element) content is 2483.23～2586.23mg/L in, preferable inorganic salt content is 2534.73mg/L, with the mass concentration ratio of inorganic salt content (4604.005mg/L) in the existing MS medium be 1: 1.85～1.78, preferable ratio is 1: 1.82, and the promptly existing contained inorganic salt concentration of MS medium is LD of the present invention
11.85～1.78 times of the contained inorganic salt concentration of medium.LD of the present invention
1The concentration of mineral salt is lower than the existing contained inorganic salt concentration of MS medium in the medium, on the basis of MS medium the consumption of macroelement is adjusted, and has reduced NO
3 -, Mg
2+, ferro element concentration; The copper sulphate consumption is 10 times of MS medium in the trace element, contains the Mo of trace simultaneously, and it participates in the synthetic and metabolism of amino acid, and the effect that promotes that organ forms is arranged.
The present invention rationally reduces the content of medium mineral salt, helps differentiation culture, and regeneration, especially the phosphorus content of magnesium of callus plant are high more, big more its inhibitory action of ratio is obvious more and the high concentration mineral salt suppress; The increase of trace copper content can promote the generation of callus healthy growth and embryoid.Utilize LD of the present invention
1Medium carries out Lonicera edulis children stem to be cultivated, and Lonicera edulis children stem is easy to be induced the formation callus, and callus induction rate all can reach more than 90%; Again the light green callus that induces is forwarded to LD of the present invention
1Carry out subculture and differentiation culture on the medium, callus induction becomes the probability height of plant, regeneration plant inductivity (being the plant differentiation rate) can reach 50%～82%, plant differentiation rate (30%) than existing MS medium has improved 66.6%～173.3%, and comparison is saved input cost 45.1% according to the MS medium.
The invention solves Lonicera edulis and solved a series of groups of training difficult problems in proliferation, induce callus and regeneration plant by young stem cultivation, and kind property uniformity is strong, transplant plant strain growth and property determination as a result through tissue cultivating seedling, plant property purity more than 98%, and can reach the purpose of quick breeding good seed, and applied aborning, mu surplus popularizing area reaches 100 shows height uniformity and yielding ability.
LD of the present invention
1The medium compound method is identical with conventional medium compound method, but it is strict to fill a prescription, and element and hormone must be prepared in its concentration range in the various components.
Embodiment
Technical solution of the present invention is not limited to following cited embodiment, also comprises any combination between each embodiment.
Embodiment one: present embodiment is a Lonicera edulis children stem callus plant regeneration inducing culture, and medium is made up of macroelement, trace element and hormone, and wherein said macroelement is the NH of 900～920mg/L by concentration
4NO
3, 90～100mg/L KH
2PO
4, 1000～1050mg/L KNO
3, 240～250mg/L CaCl
22H
2The MgSO of O, 200～210mg/L
47H
2The EDTA-Fe of O and 15～18mg/L forms, and described trace element is the KI of 0.83mg/L, the MnSO of 22.3mg/L by concentration
44H
2The H of O, 6.2mg/L
3BO
3, 8.6mg/L ZnSO
47H
2The Na of O, 0.025mg/L
2MoO
42H
2The CuSO of O, 0.25mg/L
45H
2The CoCl of O and 0.025mg/L
26H
2O, described hormone are that the 6-benzylaminopurine (6-BA) of 1.0～2.0mg/L or indolebutyric acid (IBA) or the methyl (NAA) of kinetin (KT) and 0.05～0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment (is designated as LD
1Medium), rationally reduce the content of medium mineral salt, help differentiation culture, regeneration, especially the phosphorus content of magnesium of callus plant are high more, big more its inhibitory action of ratio is obvious more and the high concentration mineral salt suppress; The increase of trace copper content can promote the generation of callus healthy growth and embryoid.Utilize the LD of present embodiment
1Medium carries out Lonicera edulis children stem to be cultivated, and callus induction rate all can reach more than 90%, and regeneration plant inductivity (being the plant differentiation rate) can reach more than 50%, and comparison is saved input cost 45.1% according to the MS medium.Wherein, callus induction rate refer to can to induce behind the plants stems Duan Zupei callus ratio; Regeneration plant inductivity (being the plant differentiation rate) is meant that the callus that induces can differentiate the ratio of plant.
The LD of present embodiment
1Medium is cultivated by young stem and is induced callus and regeneration plant, it is strong to plant the property uniformity, transplant plant strain growth and property determination as a result through tissue cultivating seedling, plant property purity more than 98%, and can reach the purpose of quick breeding good seed, and applied aborning, mu surplus popularizing area reaches 100 shows height uniformity and yielding ability.
The LD of present embodiment
1The medium compound method is identical with conventional medium compound method, but it is strict to fill a prescription, and element and hormone must be prepared in its concentration range in the various components.
Embodiment two: what present embodiment and embodiment one were different is that described macroelement is the NH of 910mg/L by concentration
4NO
3, 95mg/L KH
2PO
4, 1025mg/L KNO
3, 245mg/L CaCl
22H
2The MgSO of O, 205mg/L
47H
2The EDTA-Fe of O and 16.5mg/L forms.Other parameter is identical with embodiment one.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment (is designated as LD
1Medium) mineral salt (macroelement and trace element) content is 2534.73mg/L in, with the mass concentration ratio of inorganic salt content (4604.005mg/L) in the existing MS medium be 1: 1.82.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment rationally reduces the content of medium mineral salt, help differentiation culture, regeneration, especially the phosphorus content of magnesium of callus plant are high more, big more its inhibitory action of ratio is obvious more and the high concentration mineral salt suppress; The increase of trace copper content can promote the generation of callus healthy growth and embryoid.Utilize the LD of present embodiment
1Medium carries out Lonicera edulis children stem to be cultivated, and callus induction rate all can reach more than 90%, and regeneration plant inductivity (being the plant differentiation rate) can reach more than 58%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment three: what present embodiment was different with embodiment one or two is that described hormone is that the 6-benzylaminopurine (6-BA) of 1.0～2.0mg/L and the indolebutyric acid (IBA) of 0.1～0.2mg/L are formed by concentration.Other parameter is identical with embodiment one or two.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach more than 65%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment four: what present embodiment was different with embodiment one or two is that described hormone is that the 6-benzylaminopurine (6-BA) of 2.0mg/L and the indolebutyric acid (IBA) of 0.2mg/L are formed by concentration.Other parameter is identical with embodiment one or two.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 82%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment five: what present embodiment was different with embodiment one or two is that described hormone is that the kinetin (KT) of 1.0～2.0mg/L and the methyl (NAA) of 0.05～0.1mg/L are formed by concentration.Other parameter is identical with embodiment one or two.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach more than 56%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment six: what present embodiment was different with embodiment one or two is that described hormone is that the kinetin (KT) of 2.0mg/L and the methyl (NAA) of 0.05mg/L are formed by concentration.Other parameter is identical with embodiment one or two.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 78%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment seven: present embodiment is different with one of embodiment one to six is that the condition of culture that Lonicera edulis children stem callus plant regeneration inducing culture is cultivated Lonicera edulis children stem is: 24～26 ℃ of cultivation temperature, light application time 14～16h/d, intensity of illumination 1800～2000lx, 20～30 days subcultures once.Other parameter is identical with one of embodiment one to six.
Embodiment eight: what present embodiment was different with one of embodiment one to seven is that Lonicera edulis children stem is seeded to before the Lonicera edulis children stem callus plant regeneration inducing culture, need carry out following processing to Lonicera edulis children stem: gather Lonicera edulis children stem in June 1 to June 6, with the alcohol-pickled 10s of 70% (volume), aseptic water washing 3 times, use 0.1% (quality) mercuric chloride liquid disinfectant 8min again, and then with aseptic water washing 3～5 times, when being seeded to medium, will cut off then, be inoculated in Lonicera edulis children stem callus plant regeneration inducing culture primary surface through the children of the Lonicera edulis after above-mentioned processing stem two ends.Other parameter is identical with one of embodiment one to seven.
Sterile water is the commercially available prod in the present embodiment.
Embodiment nine: present embodiment is a Lonicera edulis children stem callus plant regeneration inducing culture, and medium is made up of macroelement, trace element and hormone, and wherein said macroelement is the NH of 910mg/L by concentration
4NO
3, 95mg/L KH
2PO
4, 1025mg/L KNO
3, 245mg/L CaCl
22H
2The MgSO of O, 205mg/L
47H
2The EDTA-Fe of O and 16.5mg/L forms, and described trace element is the KI of 0.83mg/L, the MnSO of 22.3mg/L by concentration
44H
2The H of O, 6.2mg/L
3BO
3, 8.6mg/L ZnSO
47H
2The Na of O, 0.025mg/L
2MoO
42H
2The CuSO of O, 0.25mg/L
45H
2The CoCl of O and 0.025mg/L
26H
2O, described hormone are that the 6-benzylaminopurine (6-BA) of 1.0mg/L and the indolebutyric acid (IBA) of 0.05mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 58%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment ten: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 1.0mg/L and the indolebutyric acid (IBA) of 0.1mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 66%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 11: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 1.0mg/L and the indolebutyric acid (IBA) of 0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 68%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 12: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 1.0mg/L and the methyl (NAA) of 0.05mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 52%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 13: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 1.0mg/L and the methyl (NAA) of 0.1mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 64%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 14: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 1.0mg/L and the methyl (NAA) of 0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 56%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 15: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 2.0mg/L and the methyl (NAA) of 0.05mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 64%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 16: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 2.0mg/L and the methyl (NAA) of 0.1mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 72%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 17: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 2.0mg/L and the methyl (NAA) of 0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 70%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 18: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 2.0mg/L and the indolebutyric acid (IBA) of 0.05mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 68%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 19: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 2.0mg/L and the indolebutyric acid (IBA) of 0.1mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 80%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 20: what present embodiment and embodiment nine were different is that described hormone is that the 6-benzylaminopurine (6-BA) of 2.0mg/L and the indolebutyric acid (IBA) of 0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 82%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 21: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 1.0mg/L and the indolebutyric acid (IBA) of 0.05mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 56%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 22: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 1.0mg/L and the indolebutyric acid (IBA) of 0.1mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 60%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 23: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 1.0mg/L and the indolebutyric acid (IBA) of 0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 66%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 24: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 1.0mg/L and the methyl (NAA) of 0.05mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 58%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 25: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 1.0mg/L and the methyl (NAA) of 0.1mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 56%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 26: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 1.0mg/L and the methyl (NAA) of 0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 50%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 27: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 2.0mg/L and the indolebutyric acid (IBA) of 0.05mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 60%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 28: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 2.0mg/L and the indolebutyric acid (IBA) of 0.1mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 70%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 29: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 2.0mg/L and the indolebutyric acid (IBA) of 0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 74%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 30: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 2.0mg/L and the methyl (NAA) of 0.05mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 78%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 31: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 2.0mg/L and the methyl (NAA) of 0.1mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 76%, and comparison is saved input cost 45.1% according to the MS medium.
Embodiment 32: what present embodiment and embodiment nine were different is that described hormone is that the kinetin (KT) of 2.0mg/L and the methyl (NAA) of 0.2mg/L are formed by concentration.
The Lonicera edulis children stem callus plant regeneration inducing culture of present embodiment carries out Lonicera edulis children stem and cultivates, callus induction rate all can reach more than 90%, regeneration plant inductivity (being the plant differentiation rate) can reach 60%, and comparison is saved input cost 45.1% according to the MS medium.
Wherein, in the above-mentioned embodiment nine to 32, before Lonicera edulis children stem is inoculated in Lonicera edulis children stem callus plant regeneration inducing culture, need carry out following processing to Lonicera edulis children stem: gather Lonicera edulis children stem in June 1 to June 6, with the alcohol-pickled 10s of 70% (volume), aseptic water washing 3 times, use 0.1% (quality) mercuric chloride liquid disinfectant 8min again, and then with aseptic water washing 3～5 times, when being seeded to medium, will cut off then through the children of the Lonicera edulis after above-mentioned processing stem two ends, be inoculated in Lonicera edulis children stem callus plant regeneration inducing culture primary surface, the probability that this moment, young stem induced callus is callus induction rate; After inducing callus, again well-grown callus is forwarded to Lonicera edulis children stem callus plant regeneration inducing culture, induced growth plant again, the probability that can induce plant is regeneration plant inductivity (being the plant differentiation rate), and wherein Zhuan Jie callus number is 50.
In the embodiment nine to 32, condition of culture all is: 24～26 ℃ of cultivation temperature, and light application time 14～16h/d, intensity of illumination 1800～2000lx, 20～30 days subcultures are once.
To sum up embodiment nine to 32 as can be known, increase plant differentiation rate rising along with 6-BA concentration or KT concentration, when 6-BA concentration or KT concentration are 2.0mg/L, the best result rate can reach 82.0%, 78.0% respectively, wherein 6-BA is better than KT to the facilitation effect of callus differentiation, and average plant differentiation rate is higher.When 6-BA concentration one regularly, the plant differentiation rate presents the trend of rising with the increase of IBA concentration, is that 0.2mg/L is the best with IBA concentration; When one timing of 6-BA concentration, the plant differentiation rate presents the downward trend again that rises earlier with the increase of NAA concentration, and wherein the concentration with 0.1mg/L is the best.Different therewith, in the proportioning combination of KT and IBA, NAA, when one timing of KT concentration, the IBA change in concentration is identical during with 6-BA to the influence of plant differentiation rate, the concentration of IBA is the most suitable with 0.2mg/L, along with NAA concentration increase differentiation rate is then on a declining curve, NAA concentration is the most suitable with 0.05mg/L.
Claims (8)
1. Lonicera edulis children stem callus plant regeneration inducing culture, it is characterized in that Lonicera edulis children stem callus plant regeneration inducing culture is made up of macroelement, trace element and hormone, wherein said macroelement is the NH of 900～920mg/L by concentration
4NO
3, 90～100mg/L KH
2PO
4, 1000～1050mg/L KNO
3, 240～250mg/L CaCl
22H
2The MgSO of O, 200～210mg/L
47H
2The EDTA-Fe of O and 15～18mg/L forms, and described trace element is the KI of 0.83mg/L, the MnSO of 22.3mg/L by concentration
44H
2The H of O, 6.2mg/L
3BO
3, 8.6mg/L ZnSO
47H
2The Na of O, 0.025mg/L
2MoO
42H
2The CuSO of O, 0.25mg/L
45H
2The CoCl of O and 0.025mg/L
26H
2O, described hormone are that the 6-benzylaminopurine of 1.0～2.0mg/L or indolebutyric acid or the methyl of kinetin and 0.05～0.2mg/L are formed by concentration.
2. a kind of Lonicera edulis children stem callus plant regeneration inducing culture according to claim 1 is characterized in that described macroelement is the NH of 910mg/L by concentration
4NO
3, 95mg/L KH
2PO
4, 1025mg/L KNO
3, 245mg/L CaCl
22H
2The MgSO of O, 205mg/L
47H
2The EDTA-Fe of O and 16.5mg/L forms.
3. a kind of Lonicera edulis children stem callus plant regeneration inducing culture according to claim 1 and 2 is characterized in that described hormone is that the 6-benzylaminopurine of 1.0～2.0mg/L and the indolebutyric acid of 0.1～0.2mg/L are formed by concentration.
4. a kind of Lonicera edulis children stem callus plant regeneration inducing culture according to claim 1 and 2 is characterized in that described hormone is that the 6-benzylaminopurine of 2.0mg/L and the indolebutyric acid of 0.2mg/L are formed by concentration.
5. a kind of Lonicera edulis children stem callus plant regeneration inducing culture according to claim 1 and 2 is characterized in that described hormone is that the kinetin of 1.0～2.0mg/L and the methyl of 0.05～0.1mg/L are formed by concentration.
6. a kind of Lonicera edulis children stem callus plant regeneration inducing culture according to claim 1 and 2 is characterized in that described hormone is that the kinetin of 2.0mg/L and the methyl of 0.05mg/L are formed by concentration.
7. a kind of Lonicera edulis children stem callus plant regeneration inducing culture according to claim 1 and 2, the condition of culture that it is characterized in that Lonicera edulis children stem callus plant regeneration inducing culture is: 24～26 ℃ of cultivation temperature, light application time 14～16h/d, intensity of illumination 1800～2000lx, 20～30 days subcultures once.
8. a kind of Lonicera edulis children stem callus plant regeneration inducing culture according to claim 1 and 2, it is characterized in that Lonicera edulis children stem is seeded to before the Lonicera edulis children stem callus plant regeneration inducing culture, need carry out following processing to Lonicera edulis children stem: gather Lonicera edulis children stem in June 1 to June 6, with the alcohol-pickled 10s of 70% (volume), aseptic water washing 3 times, use 0.1% (quality) mercuric chloride liquid disinfectant 8min again, and then with aseptic water washing 3～5 times, when being seeded to medium, will cut off then, be inoculated in Lonicera edulis children stem callus plant regeneration inducing culture primary surface through the children of the Lonicera edulis after above-mentioned processing stem two ends.
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