CN106954552B - A kind of tissue culture propagation method of cinnamomum camphora stem section - Google Patents
A kind of tissue culture propagation method of cinnamomum camphora stem section Download PDFInfo
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- CN106954552B CN106954552B CN201710347398.XA CN201710347398A CN106954552B CN 106954552 B CN106954552 B CN 106954552B CN 201710347398 A CN201710347398 A CN 201710347398A CN 106954552 B CN106954552 B CN 106954552B
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The present invention relates to a kind of cinnamomum camphora tissue culture propagations, it is combined for the first time using improvement DCR culture medium and MS culture, by improving DCR culture medium constituent content, adjusting the measures such as hormone levels, increase active carbon, regulating and controlling temperature illumination, increase strong seedling culture, it has invented a kind of bud induction rate and has been up to 98% or more, growth coefficient average out to 4.8, melting brown rate is less than 3.2%, strong sprout rate (high 3cm or more) 81% or more, and transplanting survival rate reaches 87% or more cinnamomum camphora tissue culture system, meets market to the wilderness demand of cinnamomum camphora.
Description
Technical field
The present invention relates to a kind of cinnamomum camphora tissue culture propagations, belong to Tissue Culture of Trees raising technology field.
Background technique
Cinnamomum camphora (Cinnamomum camphora (L.) Presl) is the representative tree of east Asia Subtropical Evergreen Broad-leaf Forest
Kind, it is mainly distributed on number province on the south Taiwan Province of China and the Changjiang river, is the multipurpose native soil economic tree of southern region of China preciousness,
It is national second level Top-rated protected wild plants.Cinnamomum camphora limb is round and mellow, and hat is big shady dense, and leaf color is abundant, while beautifying the environment, also
There is the function of greening, sweetening treatment environment, have many ecological functions such as sound insulation, expelling parasite, purification air, is since ancient times exactly southern village
The best Feng Shui Woodland in the village, house, road, waterside and scenic forest tree species, and modern flower garden is ornamental, urban afforestation is indispensable
Tree species, market prospects are extensive, and promotional value is high.
Currently, cinnamomum camphora mainly uses three kinds of mode nursery.First is that nursery technique, this is the main of current cinnamomum camphora cultivating seedlings
Mode.But nursery technique is strong in the presence of seasonality, is difficult to the disadvantages of retaining merit.Second is that cuttage and seedling culture, but need more adopt
Fringe maternal plant, while cuttage is influenced by environmental conditions larger, rooting efficiency difficulty stabilization.Third is that tissue culture technology.Tissue culture technology is most suitable
The technology of fast-propagation is carried out when limited for female parent material.However, due to the own characteristic of cinnamomum camphora plant, in conventional tissue culture mistake
It is tired that stem section is easy browning, tissue-cultured seedling is thin and weak, transplanting survival rate is low in low, shoot proliferation culture that there are still bud induction rates in journey etc.
Difficulty, these are all the biggish major reasons of cinnamomum camphora industrialization promotion application difficulty.The present invention is stranded for present in the studies above
Difficulty is combined using through the improved DCR culture medium of inventor and MS culture for the first time, by improving DCR culture medium constituent content, adjusting
Hormone levels are saved, increases active carbon, regulating and controlling temperature illumination, increase the measures such as strong seedling culture, have invented a kind of bud induction rate
Up to 98% or more, growth coefficient average out to 4.8, melting brown rate less than 3.2%, strong sprout rate (high 3cm or more) 81% or more, and
Transplanting survival rate reaches 87% or more cinnamomum camphora tissue culture system, meets market to the wilderness demand of cinnamomum camphora.
Summary of the invention
The present invention provides a kind of tissue culture propagation method of cinnamomum camphora stem section, which comprises the following steps:
1) disinfection of explant material: current year raw spray tip 10-15cm disinfection is taken；
2) induction of bud: cutting section for the material disinfected, and every section has 1-2 axillary bud, is inoculated into initial medium MS,
It was cultivated by 10-20 days, axillary bud sprouts and grows sprouting；
3) differentiation of bud: the budlet grown is seeded on differential medium, is cultivated 20 days or so；
4) proliferation of bud: after sprout forms Multiple Buds, it is divided into single plant to be inoculated into subculture in proliferated culture medium sprouting
Culture, until the growth coefficient average out to 4.8 of bud；Melting brown rate is less than 3.2%；
5) strong seedling culture: obtained adventitious bud being placed in strong seedling culture base and is cultivated 10-20 days, and supreme 3cm's or more is strong
Seedling ratio is 81% or more；
6) rooting induction: the strong sprout is inoculated into root media, cultivate 10 days or so, rooting rate be up to 98% with
On, to root long 2cm, 3-5 pieces of blade progress hardening and transplanting；
7) hardening and transplanting: tissue-cultured seedling is placed in the greenhouse of 70% shade hardening 7-10 days, is transplanted into matrix；
Wherein differential medium described in the step 3) is to add plant hormone 6- based on improving DCR culture medium
BA 1.0mg/L,NAA 0.1mg/L；
The composition and its content for improving DCR culture medium are as follows: NH4NO3: 400mg/L, KNO3: 340mg/L, Ca
(NO3)2·4H2O:278mg/L, KH2PO4: 170mg/L, MgSO4·7H2O:370mg/L, CaCl2·2H2O:42.5mg/L,
H3BO4:6.2mg/L, MnSO4·4H2O:22.3mg/L, ZnSO4·7H2O:8.6mg/L, CuSO4·5H2O:0.25mg/L, KI:
0.83mg/L、CoCl2·6H2O:0.025mg/L, NiCl2: 0.025mg/L, Na2MoO4·2H2O:0.25mg/L, FeSO4·
7H2O:27.8mg/L, Na2- EDTA:37.3mg/L, VB1: 1.0mg/L, VB6: 0.5mg/L, VB3: 0.5mg/L, glycine:
2.0mg/L, inositol: 200mg/L, sucrose: 30g/L, agar: 6g/L.
It is in one embodiment, further comprising the steps of,
8) transplanting is placed on Miniature plastic film arched shed, and covers reality, is gradually opened after 7 days, fully open after two weeks, to
The management of normal water fertilizer is carried out after growing young leaves
In a preferred embodiment, in step 2), cultivation temperature is 24-26 DEG C, intensity of illumination 1800-
2000lx, illumination are daily 14-16h.
In a preferred embodiment, in step 3), cultivation temperature is 24-26 DEG C, intensity of illumination 1800-
2000lx, illumination are daily 12-14h.
In a preferred embodiment, in step 4), proliferated culture medium used is to add based on improving DCR culture medium
Add 6-BA 1.5-2.5mg/L, NAA 0.1-0.5mg/L, active carbon 0.5g/L.
In a preferred embodiment, in step 5), strong seedling culture base used is addition 6- based on MS culture medium
BA 1.5-2.5mg/L, IAA 0.1mg/L, active carbon 1g/L.
In a preferred embodiment, in step 6), root media is addition NAA based on 1/2MS culture medium
0.25-0.35mg/L, active carbon 2g/L；.
In a preferred embodiment, in step 6), cultivation temperature is 24-26 DEG C, intensity of illumination 2000-
2200lx, illumination are daily 10-12h.
In a preferred embodiment, in step 7), the matrix obtains thin river sand: peat soil by mixing ratio 1:1:3:
Yellow soil is constituted.
The composition and its content of MS culture medium used in the present invention are as follows: NH4NO3: 1650mg/L, KNO3: 1900mg/L,
CaCl2·2H2O:440mg/L, MgSO4·7H2O:370mg/L, KH2PO4: 170mg/L, KI:0.83mg/L, H3BO3: 6.2mg/
L、MnSO4·4H2O:22.3mg/L, ZnSO4·7H2O:8.6mg/L, Na2MoO4·2H2O:0.25mg/L, CuSO4·5H2O:
0.025mg/L、CoCl2·6H2O:0.025mg/L, FeSO4·7H2O:27.85mg/L, Na2-EDTA·2H2O:37.25mg/L,
Inositol: 100mg/L, niacin: 0.5mg/L, VB6: 0.5mg/L, VB1: 0.1mg/L, glycine: 2.0mg/L, sucrose: 30g/L, fine jade
Rouge: 7g/L.
Beneficial effect
This item tissue culture and rapid propagation method is to cultivate the cinnamomum camphora tissue culture technology combined using improvement DCR culture medium and MS for the first time,
The cinnamomum camphora tissue culture rapid propagation system that a kind of bud induction rate is high, growth coefficient is high, strong sprout rate is high, transplanting survival rate is high can be provided, effectively
The rapid scale nursery for solving the problems, such as cinnamomum camphora meets market to the wilderness demand of cinnamomum camphora.
Specific embodiment
Implemented according to following examples, the present invention can be better understood.However, as it will be easily appreciated by one skilled in the art that
Embodiment is merely to illustrate the present invention, without that should will not limit the present invention described in detail in claims.
Embodiment
Follow the steps below nursery:
1. the disinfection of explant material: taking cinnamomum camphora current year raw spray tip 10-15cm, remove blade after tap water cleaning, use
After detergent liquid impregnates 5-10min, rinsed well with tap water；By the material cleaned up with 70% wine on the sterile net platform of behaviour
Essence impregnates 30s, sterile washing 3 times；8-10min, aseptic water washing 3-5 times are sterilized with 0.1% mercuric chloride again.
2. the induction of bud: the material disinfected being cut section, every section has 1-2 axillary bud, is inoculated into initial medium MS；
By the culture of 10-20d, axillary bud sprouts and grows sprouting, and the inductivity of bud is up to 98% or more；Cultivation temperature is 24-26 DEG C,
Intensity of illumination is 1800-2000lx, and illumination is daily 14-16h.
3. the differentiation of bud: the budlet grown being seeded on differential medium, culture 20d or so buds differentiation rate reaches
95%；Differential medium adds plant hormone 6-BA 1.0mg/L, NAA 0.1mg/L based on improving DCR culture medium；Training
Supporting temperature is 24-26 DEG C, intensity of illumination 1800-2000lx, and illumination is daily 12-14h.
4. the proliferation of bud: after sprout forms Multiple Buds, being divided into single plant to be inoculated into subculture in proliferated culture medium sprouting
Culture, the growth coefficient average out to 4.8 of bud can reach 6 or more；Melting brown rate is less than 3.2%；Proliferated culture medium used is to change
Into DCR culture medium+6-BA 1.5-2.5mg/L, NAA 0.1-0.5mg/L, active carbon 0.5g/L.
5. strong seedling culture: obtained adventitious bud is placed in strong seedling culture base and cultivates 10-20d, gained strong sprout (high 3cm with
On) ratio be 81% or more；Used medium is MS+6-BA 1.5-2.5mg/L, IAA 0.1mg/L, active carbon 1g/L；Culture
Temperature is 24-26 DEG C, intensity of illumination 1800-2000lx, and illumination is daily 12-14h.
6. rooting induction: when sprout it is long to 3cm high when can be inoculated into root media, 10d or so starts to take root, raw
Root rate is up to 98% or more；Root media is 1/2MS+NAA 0.25-0.35mg/L, active carbon 2g/L；To root long 2cm, leaf
3-5 pieces of piece can acclimatization and transplants；Cultivation temperature is 24-26 DEG C, intensity of illumination 2000-2200lx, and illumination is daily 10-12h.
7. hardening and transplanting: tissue-cultured seedling is placed in hardening 7-10d in the greenhouse of 70% shade, cleans the culture medium of root,
In transplanting to the matrix through 0.1% disinfecting solution of potassium permanganate, the mixing ratio of matrix is thin river sand: peat soil: yellow soil=1:
1:3.Root system will unfold when transplanting, compacting, root and matrix close contact.
8. transplanting is placed on Miniature plastic film arched shed, and covers reality, it is gradually opened after 7d, it is fully open after two weeks, to
The management of normal water fertilizer is carried out after growing young leaves, transplanting survival rate reaches 87% or more.
Tissue culture propagation method of the invention and it is seen in formerly report (Ye Runyan, Tong Zaikang, Zhang Junhong, etc. camphor tree stem section group
Train fast numerous [J], Zhejiang A & F University's journal, 2016,33 (1): 177-182；Zhou Lihua, Cai Yanling, Zeng Linghai, etc. camphor tree is excellent
The tissue culture technical research [J] of respectable family system, tropical crops journal, 2013,34 (1): 67-73；Gong Zheng, Zhou Lihua, Zhang Weihua,
Deng, camphor tree tissue culture quick breeding Study on Seedling Cultivation Technique [J], Annual, 2007,23 (5): 35-39；Tang Guotao, Zhang Han
Forever, Zhu's former times is tender, etc. camphor tree Tissue culture assays [J], Fujian Forestry science and technology, 2013,40 (2): 70-72.) conventional tissue culture side
The technical indicator comparison of method is as follows:
1 present invention of table and conventional tissue culture technology Indexes Comparison
Claims (3)
1. a kind of tissue culture propagation method of cinnamomum camphora stem section, which comprises the following steps:
1) disinfection of explant material: current year raw spray tip 10-15cm disinfection is taken；
2) induction of bud: cutting section for the material disinfected, and every section has 1-2 axillary bud, is inoculated into initial medium MS, passes through
It cultivates within 10-20 days, axillary bud sprouts and grows sprouting；
3) differentiation of bud: the budlet grown is seeded on differential medium, is cultivated 20 days or so；
4) proliferation of bud: after sprout forms Multiple Buds, being divided into single plant to be inoculated into squamous subculture in proliferated culture medium sprouting,
To the growth coefficient average out to 4.8 of bud；Melting brown rate is less than 3.2%；
5) strong seedling culture: obtained adventitious bud being placed in strong seedling culture base and is cultivated 10-20 days, the strong sprout ratio of supreme 3cm or more
Rate is 81% or more；
6) rooting induction: the strong sprout is inoculated into root media, is cultivated 10 days or so, and rooting rate is up to 98% or more,
To root long 2cm, 3-5 pieces of blade progress hardening and transplanting；
7) hardening and transplanting: tissue-cultured seedling is placed in the greenhouse of 70% shade hardening 7-10 days, is transplanted into matrix；With
8) transplanting is placed on Miniature plastic film arched shed, and covers reality, is gradually opened after 7 days, fully open after two weeks, wait grow
The management of normal water fertilizer is carried out after young leaves；
Wherein in step 2), cultivation temperature is 24-26 DEG C, intensity of illumination 1800-2000lx, and illumination is daily 14-16h；
Wherein in step 3), cultivation temperature is 24-26 DEG C, intensity of illumination 1800-2000lx, and illumination is daily 12-14h；
Wherein differential medium described in the step 3) is to add plant hormone 6-BA based on improving DCR culture medium
1.0mg/L,NAA 0.1mg/L；
Wherein in step 4), based on proliferated culture medium used is improves DCR culture medium, addition 6-BA 1.5-2.5mg/L,
NAA 0.1-0.5mg/L, active carbon 0.5g/L；
Wherein in step 5), strong seedling culture base used is addition 6-BA 1.5-2.5mg/L, IAA based on MS culture medium
0.1mg/L, active carbon 1g/L；And
Wherein in step 6), cultivation temperature is 24-26 DEG C, intensity of illumination 2000-2200lx, and illumination is daily 10-12h；
The composition and its content for improving DCR culture medium are as follows: NH4NO3: 400mg/L, KNO3: 340mg/L, Ca (NO3)2·
4H2O:278mg/L, KH2PO4: 170mg/L, MgSO4·7H2O:370mg/L, CaCl2·2H2O:42.5mg/L, H3BO4:
6.2mg/L、MnSO4·4H2O:22.3mg/L, ZnSO4·7H2O:8.6mg/L, CuSO4·5H2O:0.25mg/L, KI:
0.83mg/L、CoCl2·6H2O:0.025mg/L, NiCl2: 0.025mg/L, Na2MoO4·2H2O:0.25mg/L, FeSO4·
7H2O:27.8mg/L, Na2- EDTA:37.3mg/L, VB1: 1.0mg/L, VB6: 0.5mg/L, VB3: 0.5mg/L, glycine:
2.0mg/L, inositol: 200mg/L, sucrose: 30g/L, agar: 6g/L.
2. according to the method described in claim 1, wherein in step 7), the matrix by mixing ratio 1:1:3 thin river sand:
Peat soil: yellow soil is constituted.
3. according to the method described in claim 1, the composition and its content of MS culture medium used in it are as follows: NH4NO3:
1650mg/L、KNO3: 1900mg/L, CaCl2·2H2O:440mg/L, MgSO4·7H2O:370mg/L, KH2PO4: 170mg/L,
KI:0.83mg/L, H3BO3: 6.2mg/L, MnSO4·4H2O:22.3mg/L, ZnSO4·7H2O:8.6mg/L, Na2MoO4·
2H2O:0.25mg/L, CuSO4·5H2O:0.025mg/L, CoCl2·6H2O:0.025mg/L, FeSO4·7H2O:27.85mg/
L、Na2-EDTA·2H2O:37.25mg/L, inositol: 100mg/L, niacin: 0.5mg/L, VB6: 0.5mg/L, VB1: 0.1mg/L,
Glycine: 2.0mg/L, sucrose: 30g/L, agar: 7g/L.
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| CN108142294B (en) * | 2018-01-09 | 2021-07-30 | 福建省永安林业(集团)股份有限公司 | Cinnamomum camphora tissue culture method and culture medium thereof |
| CN108668898A (en) * | 2018-05-23 | 2018-10-19 | 宿迁学院 | A kind of method for tissue culture of monkey camphor tree |
| CN108934700A (en) * | 2018-07-05 | 2018-12-07 | 紫云自治县紫香源农林科技有限责任公司 | A kind of mating system of cinnamomum camphora tree |
| CN111316919B (en) * | 2020-04-22 | 2021-12-10 | 安徽农业大学 | Method for improving regeneration efficiency in cinnamomum camphora tissue culture process |
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| CN102860258A (en) * | 2012-09-19 | 2013-01-09 | 广东省林业科学研究院 | Clonal tissue culture breeding method for camphor tree |
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| CN102860258A (en) * | 2012-09-19 | 2013-01-09 | 广东省林业科学研究院 | Clonal tissue culture breeding method for camphor tree |
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| 樟树组织培养快繁育苗技术研究;龚峥等;《广东林业科技》;20071231;第23卷(第5期);摘要，1 材料与方法，2 结果与分析 * |
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