CN105941150B - Cinnamomum camphora gushes golden adventitious buds proliferation method - Google Patents
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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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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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
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Abstract
The invention discloses a kind of cinnamomum camphoras to gush golden adventitious buds proliferation method, includes the following steps：The cinnamomum camphora that axillary bud is not yet sprouted is taken to gush golden young sprout, is inoculated in inducing culture after aseptic process and forms Multiple Buds；It the cinnamomum camphora for forming Multiple Buds is gushed into golden young sprout is transferred in proliferated culture medium culture and cultivate 25d, be then transferred to the second medium culture 25d, finally transfer to proliferated culture medium culture 25d, complete adventitious buds proliferation.The method of the present invention effectively improves the problem of gushing weak golden squamous subculture room Multiple Buds growing way, yellow leaf, browning, improves proliferation efficiency by the investigation to factors such as minimal medium, hormone, added substance, condition of culture.
Description
Technical field
The invention belongs to biological self reproducing technical field, specifically, being related to a kind of cinnamomum camphora gushes golden adventitious buds proliferation method.
Background technology
Camphor tree (Cinnamomum caphora) also known as cinnamomum camphora are the subtropical evergreen broad-leaved arbor of Lauraceae Cinnamomum.It is given birth to
Long rapid, flourishing, the four seasons are evergreen, leaf beautiful and distribute giving off a strong fragrance, are both precious fragrant oils seeds and city
Important landscape and green tree species.Cinnamomum camphora ' gushing gold ' (Cinnamomum camphora ' Yongjin ') is cinnamomum camphora seed variation product
Kind, limb cerise, young leaves, flower are in golden yellow, and pericarp is in yellow, and limb, leaf, pericarp color are made with Seasonal dynamics change
For tall and big color leaf arbor, there is very high ornamental plantation and promote and apply potentiality and value.But ' gushing gold ' seed heredity object
Matter is unstable, and nursery offspring's degradation ratio is up to more than 99%, and kind preserves and breeding difficulty is larger.It is excellent asexual to obtain at present
System has carried out Grafting experiments to obtain the filial generation for keeping maternal merit, however, as elite stand branch limited amount, i.e.,
Cutting propagation is made also to be difficult to meet the needs of real.
Plant Tissue Breeding is the important channel of quick breeding elite germplasm, and tissue culture technique can keep the excellent of female parent
Good hereditary capacity, and the breeding nursery stock of neat and consistent can be provided in a short time, and can also be that seed improvement utilizes and heredity turns
Change lays the first stone, and is applied in many botanical seedling culturings at present.
At present obtain cinnamomum camphora ' gushing gold ' although tissue culture regeneration technology at home and abroad possess some special knowledge, plant with
The increase of subculture number, Multiple Buds breeding coefficient reduce, and phenomena such as weak growth potential, yellow leaf, browning occur, proliferation efficiency is significantly
Reduce, can not stability and high efficiency acquisition Multiple Buds, and then be difficult to realize the purpose quickly bred.To realize cinnamomum camphora ' gushing gold ' test tube
The factorial praluction of seedling, the proliferation of system research Multiple Buds are very necessary.
Invention content
In view of this, the present invention to be solved existing cinnamomum camphora and gushes golden tissue culture regeneration plant with the increase of subculture number,
The problem of proliferation efficiency substantially reduces, provides a kind of cinnamomum camphora and gushes golden adventitious buds proliferation method.
In order to solve the above-mentioned technical problem, the invention discloses a kind of cinnamomum camphoras to gush golden adventitious buds proliferation method, including following
Step：The cinnamomum camphora that axillary bud is not yet sprouted is taken to gush golden young sprout, is inoculated in inducing culture after aseptic process and forms Multiple Buds；By shape
Cinnamomum camphora into Multiple Buds is gushed golden young sprout and is transferred in proliferated culture medium and cultivates 25d, is then transferred to the second medium culture 25d,
Proliferated culture medium culture 25d is finally transferred to, completes adventitious buds proliferation.
Further, the proliferated culture medium includes MS culture mediums, potassium dihydrogen phosphate (KH2PO4), 6- benzyl aminoadenines
(6-BA) and heteroauxin (IAA).
Further, KH in the proliferated culture medium2PO4A concentration of 50mg/L, 6-BA a concentration of 3mg/L, IAA are a concentration of
0.02mg/L。
Further, the proliferated culture medium further includes sucrose, and the sucrose concentration is 20-25g/L.
Further, the proliferated culture medium pH value is 5.8-6.0.
Further, second culture medium includes MS culture mediums, KH2PO4, 6-BA and IAA.
Further, KH in second culture medium2PO4A concentration of 50mg/L, 6-BA a concentration of 1mg/L, IAA are a concentration of
0.02mg/L。
Further, the inducing culture is to include MS culture mediums, 6-BA and indolebutyric acid (IBA).
Further, a concentration of 0.02mg/L of 6-BA a concentration of 1.0mg/L, IBA in the inducing culture.
Compared with prior art, the present invention can be obtained including following technique effect：
1) method of the invention is by the way that minimal medium, the investigation of the factors such as hormone, added substance, condition of culture has
Effect improves the problem of cinnamomum camphora gushes weak golden squamous subculture room Multiple Buds growing way, yellow leaf, browning, improves proliferation efficiency.
Certainly, implementing any of the products of the present invention must be not necessarily required to reach all the above technique effect simultaneously.
Description of the drawings
Attached drawing described herein is used for providing further understanding of the present application, forms the part of the application, this Shen
Illustrative embodiments and their description please do not form the improper restriction to the application for explaining the application.In the accompanying drawings：
Fig. 1 is the adventitious buds proliferation figure of culture medium A 1 in the embodiment of the present application；
Fig. 2 is the adventitious buds proliferation figure of culture medium A 2 in the embodiment of the present application；
Fig. 3 is the adventitious buds proliferation figure of culture medium A 3 in the embodiment of the present application；
Fig. 4 is the adventitious buds proliferation figure of culture medium A 4 in the embodiment of the present application；
Fig. 5 is the adventitious buds proliferation figure of culture medium A 5 in the embodiment of the present application；
Fig. 6 is the adventitious buds proliferation figure of culture medium A 6 in the embodiment of the present application；
Fig. 7 is the adventitious buds proliferation figure of culture medium A 7 in the embodiment of the present application；
Fig. 8 is the adventitious buds proliferation figure of culture medium A 8 in the embodiment of the present application；
Fig. 9 is the adventitious buds proliferation figure of culture medium A 9 in the embodiment of the present application；
Figure 10 is the adventitious buds proliferation figure of culture medium B1 in the embodiment of the present application；
Figure 11 is the adventitious buds proliferation figure of culture medium B2 in the embodiment of the present application；
Figure 12 is the adventitious buds proliferation figure of culture medium B3 in the embodiment of the present application；
Figure 13 is the adventitious buds proliferation figure of culture medium B4 in the embodiment of the present application；
Figure 14 is the adventitious buds proliferation figure of culture medium B5 in the embodiment of the present application；
Figure 15 is the adventitious buds proliferation figure of culture medium B6 in the embodiment of the present application.
Specific embodiment
Carry out the embodiment that the present invention will be described in detail below in conjunction with accompanying drawings and embodiments, thereby how the present invention is applied
Technological means can fully understand and implement according to this to solve technical problem and reach the realization process of technical effect.
Embodiment
First, material
Cinnamomum camphora ' gushing gold ' stem with bud is collected in 14 age period cinnamomum camphora of Ningbo Forest Bureau's seedling center Qiu Ai bases and ' gushes
On gold ' tree, the young sprout that clip axillary bud is not yet sprouted takes back laboratory, and inducing culture (MS+6- is seeded to after aseptic process
BA 1.0mg/L+IBA 0.02mg/L) in formed Multiple Buds.
2nd, data processing
The total bud numbers of proliferation times=25d/inoculation bud number (being effective bud highly for more than 1cm)；Data using average value ±
Standard error (SE) represents, data are counted using SPSS17.0 statistical softwares, and mean value compares using the one-factor analysis of variance,
The Different treatments significance difference opposite sex uses Duncan ' s multiple range tests method (SSR, P ＜ 0.05).
3rd, the proliferation of cinnamomum camphora ' gushing gold ' Multiple Buds
Influence of the 3.1 different minimal mediums, plant growth regulator type and concentration to adventitious buds proliferation
The culture medium being constantly proliferated to filter out suitable cinnamomum camphora ' gushing gold ' Multiple Buds is studied and selectes three kinds of different basic trainings
Support base：The KH of MS, MS and 50mg/L2PO4、1/2MS；6-BA concentration is designed as：0.5、1、3mg/L；Auxin is IAA：0、
0.02、0.04mg/L；GA3(gibberellin)：0th, 0.05,0.1mg/L, experiment is optimized using orthogonal scheme, is trained
It supports base design and is shown in Table 1.
The design of 1 cinnamomum camphora of table ' gushing gold ' adventitious buds proliferation culture medium
| Number | Minimal medium | 6-BA mg/L | IAA mg/L | GA3 mg/L |
| A1 | MS | 0.5 | 0 | 0 |
| A2 | MS | 1 | 0.02 | 0.05 |
| A3 | MS | 3 | 0.04 | 0.1 |
| A4 | The KH of MS+50mg/L2PO4 | 0.5 | 0.02 | 0.1 |
| A5 | The KH of MS+50mg/L2PO4 | 1 | 0.04 | 0 |
| A6 | The KH of MS+50mg/L2PO4 | 3 | 0 | 0.05 |
| A7 | 1/2MS | 0.5 | 0.04 | 0.05 |
| A8 | 1/2MS | 1 | 0 | 0.1 |
| A9 | 1/2MS | 3 | 0.02 | 0 |
Cinnamomum camphora ' gushing gold ' tissue that Multiple Buds are formed in inducing culture is taken, puts the different proliferated culture mediums of above-mentioned A1-A9
Middle Multiplying culture, cultivation results are shown in Fig. 1-Fig. 9 and table 2.
Influence of 2 different culture media of table to cinnamomum camphora ' gushing gold ' adventitious buds proliferation
Note：Different lowercase letter significant differences under 0.05 level with after column data.
As shown in figs 1-9, by cinnamomum camphora ' gushing gold ' adventitious buds proliferation the study found that Multiple Buds in the only culture of 6-BA
There is a small amount of bud point to be formed in base (A1, A6, A8), but grow it is difficult, and in A1 culture mediums Multiple Buds yellow leaf, base portion is brown
Change seriously, and with the extension leaf abscission of incubation time, plant death (such as Fig. 1).And clump can be made by adding suitable auxin
Sprouting, growth is vigorous, and leaf color is light green, and proliferative capacity enhances with the raising of auxin.If in culture medium the basic element of cell division with
The ratio of auxin is appropriate, then the proliferation times of cinnamomum camphora ' gushing gold ' Multiple Buds increase rapidly (A3, A4, A5).
The IAA that suitable concentration is added in proliferated culture medium is good to the cultivation effect of cinnamomum camphora ' gushing gold ' Multiple Buds, and seedling growth is prosperous
It contains, leaf color is light green.As a concentration of 0.04mg/L of IAA, inhibiting effect is generated to proliferation, seedling base portion forms larger callus group
It knits, generates a circle brown inhibiting substances and base portion leaf is caused to redden, then send out withered, influence the growth (A3, A5, A7) of whole strain.
GA3To the growth and cultivation effect of cinnamomum camphora ' gushing gold ' Multiple Buds without apparent facilitation.GA3Concentration is followed successively by 0,
0.05th, 0.1mg/L, there is no because of GA for the upgrowth situation of Multiple Buds3The raising of concentration and change, and with GA3Leaf burnt hair, shape
Into bud point cannot develop, almost without proliferation.
Experimental result also shows that different minimal mediums has a major impact the growth of cinnamomum camphora ' gushing gold ' Multiple Buds：By training
Base junction fruit is supported as it can be seen that being added to 50mg/L KH2PO4MS minimal mediums and 1/2MS in, browning during adventitious buds proliferation
It has clear improvement (A4, A5, A6).
Influence of 3.2 different sucroses to cinnamomum camphora ' gushing gold ' adventitious buds proliferation effect
On the basis of above-mentioned experiment, influence of the research different sucrose to cinnamomum camphora ' gushing gold ' adventitious buds proliferation effect.
Sucrose concentration level is respectively：10th, 20,30,40,50,60g/L.Culture medium is the KH of MS, 50mg/L2PO4, 2mg/L 6-BA
With the IAA of 0.02mg/L, agar concentration 7g/L, pH value 5.8.Experimental result is shown in Figure 10-Figure 15 and table 3.
Influence of 3 sucrose concentration of table to cinnamomum camphora ' gushing gold ' adventitious buds proliferation
Note：Different lowercase letter significant differences under 0.05 level with after column data.
As shown in fig. 10-15, in Plant Tissue Breeding, since the photosynthetic capacity of culture is weaker, so needing
Some carbohydrates are added in the medium, on the one hand as carbon source needed for growth and development and the energy, while can also be maintained certain
Osmotic pressure.In cinnamomum camphora ' gushing gold ' adventitious buds proliferation culture, the addition of different sucrose amounts generates proliferation and growth conditions
Certain influence.Multiple Buds show as that leaf pale green, bud are thin and delicate on the sucrose culture medium B1 of low concentration, and close to the leaf of base portion
Piece also occurs withered and yellow；On B3 culture mediums, seedling growth is more vigorous, and leaf color is light green, occurs substantially without withered seedling；And in higher sugarcane
On the B4 of sugared concentration, B5 medium, seedling is slow-growing, and differentiation is few, and leaf is yellowish green, thicker and aging, and base portion aggregation brown material has
Withered seedling is entrained among bud clump.As can be seen from Table 3, with the increasing of sucrose concentration, growth coefficient substantially first increase and
After reduce.Simultaneously by table 3 it is recognised that sucrose concentration is 20,30g/L in B2 and B3 culture mediums, proliferation times at this moment are most
Greatly, reaching 2.80 and 3.38, but because B3 growth coefficients are higher, bud is partially thin and delicate, general production considers more effective seedlings, so training
Supporting sucrose concentration in base selects 20~25g/L to be advisable.
Influence of the 3.3 different pH value to cinnamomum camphora ' gushing gold ' adventitious buds proliferation
PH value influences the Multiplying culture of Multiple Buds, therefore the following pH value of this experimental design, to select best training
The condition of supporting, culture medium include the KH of MS, 50mg/L2PO4, 2mg/L 6-BA and 0.02mg/L IAA, agar concentration 7g/L.
Experimental result is shown in Table 4.
Influence of the different pH value of table 4 to cinnamomum camphora ' gushing gold ' adventitious buds proliferation
Note：Different lowercase letter significant differences under 0.05 level with after column data.
PH in culture medium value is also to influence a key factor of Plant Tissue Breeding.The variation of pH value influence some from
Son solubility and influence plant to the absorption of each element and nutritional deficiency symptom occur.Too high or too low pH value influences whether simultaneously
The solidification of culture medium, pH value is bigger than normal, culture medium can be made to harden, on the contrary then culture medium is too soft.PH value is cultivated at 5.5 after sterilizing
Base is softer, and bud-leaf of growing thickly is grayish green, and the Multiple Buds of differentiation have vitrifying trend and bud is slow-growing；Culture of the pH value 5.8~6.5
On base, cinnamomum camphora ' gushing gold ' Multiple Buds can more normally be grown, and proliferation times are high, and effective bud is more；When pH value is higher than 7.0, clump
Leaf of sprouting shows yellowish green situation, and bud is slow-growing, and proliferation times reduce.As shown in Table 4, cinnamomum camphora ' gushing gold ' Multiple Buds are in pH
Be worth be 5.8~6.0 between when, sprout can vigorous growth, and higher proliferation times can be maintained.
Influence of the 3.4 different subculture modes to cinnamomum camphora ' gushing gold ' adventitious buds proliferation
Following 5 kinds of culture mediums are subjected to alternate culture, cultivation results are shown in Table 5.
1. the KH of MS, 50mg/L2PO4, 3mg/L 6-BA and 0.02mg/L IAA；
2. the KH of MS, 50mg/L2PO4, 2mg/L 6-BA and 0.02mg/L IAA；
3. the KH of MS, 50mg/L2PO4, 1mg/L 6-BA and 0.02mg/L IAA；
4. the KH of MS, 50mg/L2PO4, 0.5mg/L 6-BA and 0.02mg/L IAA；
5. the KH of MS, 50mg/L2PO4With the IAA of 0.02mg/L.
Not influence of the subculture mode to cinnamomum camphora ' gushing gold ' adventitious buds proliferation of table 5
Note：Different lowercase letter significant differences under 0.05 level with after column data.
Tissue culture seedling proliferation be typically the continuous subculture of explant in containing 6-BA proliferated culture mediums, this mode frequently results in
The vitrifying of tissue-cultured seedling or growth deformity, therefore different subculture modes are compared to cinnamomum camphora ' gushing gold ' adventitious buds proliferation and growth
It influences.Explant is inoculated in the KH of the proliferated culture medium MS+50mg/L of filtered out Multiple Buds first2PO4The 6- of+3mg/L
In the IAA (1.) of BA+0.02mg/L, (2., 3., 4.) or remove 6-BA cultures that concentration continuously decreases are transferred to after 25d respectively
In base (5.), in being transferred to after 25d in proliferated culture medium 1..As a result (table 5) is shown, in five kinds of subculture modes, D3 modes are most
Be conducive to the proliferation of cinnamomum camphora ' gushing gold ' Multiple Buds, average coefficient of proliferation highest, and bud fast growing；Continuously in culture medium 1. middle training
It is foster growth coefficient to be caused to be substantially reduced, and easily there is vitrifying in Multiple Buds.
3.5 different subculture cycles grow thickly to cinnamomum camphora ' gushing gold ' influence of proliferation
Find that subculture cycle is affected to cinnamomum camphora ' gushing gold ' adventitious buds proliferation in experiment, so the following experiment side of design
Case, to filter out suitable Subculture Time, the KH of culture medium MS+50mg/L2PO4The IAA of the 6-BA+0.02mg/L of+2mg/L,
Agar concentration is 7g/L, and pH value 5.8, experimental result is shown in Table 6.
Table 6 is with continuation for influence of the period to cinnamomum camphora ' gushing gold ' adventitious buds proliferation
Note：Different lowercase letter significant differences under 0.05 level with after column data.
Material does not shift in incubation, can lead to culture materials brown stain, and final material is all dead.It timely updates
Culture medium is one of effective important measures for reducing browning.For the proliferative properties of cinnamomum camphora ' gushing gold ' Multiple Buds, difference is compared
Influence of the subculture cycle to growth.The result shows that culture 10d is shifted, proliferation times are minimum, and proliferation times are with subculture week
The extension of phase occurs first increasing the trend reduced afterwards.Subculture cycle 25d~30d proliferation times compared with other processing in notable
Difference (p<0.05), and subculture cycle is average reachable in 25d its proliferation times.Although the proliferation times of 30d subculture Multiple Buds
It is higher, but Multiple Buds growing state starts to deteriorate, and the phenomenon that yellow leaf and dead seedling occurs, is unfavorable for continuing to cultivate.Therefore selection 25
~30d carries out subculture and is preferred, and is conducive to development and the proliferation of Multiple Buds.
4th, cinnamomum camphora ' gushing gold ' adventitious buds proliferation method finally determined
Cinnamomum camphora ' gushing gold ' young sprout that axillary bud is not yet sprouted is taken, inducing culture (MS+6-BA is inoculated in after aseptic process
1.0mg/L+IBA 0.02mg/L) form Multiple Buds；Cinnamomum camphora ' gushing gold ' young sprout for forming Multiple Buds is transferred to proliferated culture medium
(MS+KH2PO450mg/L+6-BA 3.0mg/L+IAA 0.02mg/L+ sucrose 20-25g/L, pH=5.8-6.0) culture 25d,
It is then transferred to the second culture medium (MS+KH2PO450mg/L+6-BA 1.0mg/L+IAA 0.02mg/L) culture 25d, finally again
It is transferred to proliferated culture medium (MS+KH2PO450mg/L+6-BA 3.0mg/L+IAA 0.02mg/L+ sucrose 20-25g/L, pH=
25d 5.8-6.0) is cultivated, completes adventitious buds proliferation.
The method of the present invention is by minimal medium, the investigation of the factors such as hormone, added substance, condition of culture, effectively
The problem of improving weak cinnamomum camphora ' gushing gold ' squamous subculture room Multiple Buds growing way, yellow leaf, browning, improve proliferation efficiency.
Some vocabulary has such as been used to censure special component or method in specification and claim.Art technology
Personnel are, it is to be appreciated that different regions may call same ingredient with different nouns.This specification and claims are not
In a manner that the difference of title is used as and distinguishes ingredient.As the "comprising" of the specification in the whole text and claim mentioned in is
One open language, therefore should be construed to " including but not limited to "." substantially " refer in receivable error range, this field
Technical staff can solve the technical problem within a certain error range, basically reach the technique effect.Specification is follow-up
It is described as implementing the better embodiment of the present invention, so description is for the purpose of illustrating the rule of the present invention, not
To limit the scope of the present invention.Protection scope of the present invention is when subject to appended claims institute defender.
It should also be noted that, term " comprising ", "comprising" or its any other variant are intended to nonexcludability
Comprising so that commodity or system including a series of elements not only include those elements, but also including without clear and definite
It the other element listed or further includes as this commodity or the intrinsic element of system.In the feelings not limited more
Under condition, the element that is limited by sentence "including a ...", it is not excluded that in the commodity including the element or system also
There are other identical elements.
Several preferred embodiments of the present invention have shown and described in above description, but as previously described, it should be understood that the present invention
Be not limited to form disclosed herein, be not to be taken as the exclusion to other embodiment, and available for various other combinations,
Modification and environment, and the above teachings or related fields of technology or knowledge can be passed through in the scope of the invention is set forth herein
It is modified.And changes and modifications made by those skilled in the art do not depart from the spirit and scope of the present invention, then it all should be in this hair
In the protection domain of bright appended claims.
Claims (1)
1. cinnamomum camphora gushes golden adventitious buds proliferation method, which is characterized in that includes the following steps：The cinnamomum camphora that axillary bud is not yet sprouted is taken to gush gold
Young sprout is inoculated in inducing culture after aseptic process and forms Multiple Buds；The cinnamomum camphora for forming Multiple Buds is gushed golden young sprout to be transferred to
25d is cultivated in proliferated culture medium, the second medium culture 25d is then transferred to, finally transfers to proliferated culture medium culture
25d completes adventitious buds proliferation；
The proliferated culture medium is by MS culture mediums, KH2PO4, 6-BA, IAA and sucrose composition；
Second culture medium is by MS culture mediums, KH2PO4, 6-BA and IAA composition；
The inducing culture is made of MS culture mediums, 6-BA and IBA；
KH in the proliferated culture medium2PO4It is a concentration of 0.02mg/L of a concentration of 50mg/L, 6-BA a concentration of 3mg/L, IAA, described
Sucrose concentration is 20-25g/L, and the proliferated culture medium pH value is 5.8-6.0；
KH in second culture medium2PO4A concentration of 0.02mg/L of a concentration of 50mg/L, 6-BA a concentration of 1mg/L, IAA；
A concentration of 0.02mg/L of 6-BA a concentration of 1.0mg/L, IBA in the inducing culture.
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