CN104885948B - A kind of method of camellia oleosa seeds leaf segment directly regenerated plant - Google Patents
A kind of method of camellia oleosa seeds leaf segment directly regenerated plant Download PDFInfo
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- CN104885948B CN104885948B CN201510308567.XA CN201510308567A CN104885948B CN 104885948 B CN104885948 B CN 104885948B CN 201510308567 A CN201510308567 A CN 201510308567A CN 104885948 B CN104885948 B CN 104885948B
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Abstract
The invention discloses a kind of camellia oleosa seeds leaf segment is explant, by the method for adventitious organogenesis directly regenerated plant, belongs to biological technical field.Comprise the following steps: (1) uses the seed of C. olelfera is Breeding aseptic seedling.(2) it cotyledonary node is cut is inoculated on adventitious bud induction culture base, through 20 25d, adventitious bud occurs.(3) being transferred to adventitious bud cultivate 20 30d in subculture multiplication medium, growth coefficient is up to 7.0.(4) adventitious bud of propagation being transferred to cultivate in strong seedling culture base 20 25d, height of seedling is up to 3 4cm.(5) adventitious bud obtaining is moved into root media, it is thus achieved that complete test tube seedling.It is not only Fast-propagation excellent oil tea plant by the present invention and provides a new way, and be oil tea genetic transformation and molecular improvement offer technical support, be that healthy production of China's camellia oleiferaindustry has great importance.
Description
Technical field
The invention belongs to biological technical field, be a kind of method with camellia oleosa seeds leaf segment directly regenerated plant.
Background technology
Oil tea (Camellia Oleifera) is Theaceae (Theaceae) Camellia (Camellia) evergreen shrubs or little
Arbor, is China distinctive woody edible oil material seeds, plants with olive, oil palm, coconut the referred to as big woody oleiferous plants in the world four
Thing.Tea oil unsaturated fatty acid content reaches more than 90%, and look good taste is fragrant, and unique flavor is nutritious, storage tolerance, have hypotensive,
Blood fat and the effect of softening blood vessel, the prevention to angiocardiopathy plays an important role, and is one of the edible oil of high-quality, is described as
" east olive oil ".Meanwhile, oil tea accessory substance still produces the quality raw materials of fertilizer, agricultural chemicals, tannin extract.At present, the total oil of China
Tea woods 4531.2 ten thousand mu, produces tea oil more than 20 ten thousand tons per year, and average yield per mu tea oil 5.8kg, camellia oleiferaindustry generally also exists unit are
Yield poorly and deficiency in economic performance two large problems.One of major reason of oil tea low yield poor efficiency is exactly that improved variety degree is low, and kind is mixed
Disorderly, major part camellia oleifera lam is in wild state, and yield is relatively low, and breeding nursery stock shortage can not spread plantation.Meanwhile, oil tea
Anthracnose Glomerella cingulata (Stonem.) Spauld et Schrenk is the Major Diseases of oil tea.At the Changjiang river stream
The large area oil tea cultivation area of each province on the south territory, and Henan, South Shaanxi generation are generally.After disease occurs, cause tight
Flumping fruit, bud drop, branch withered, even whole strain is become feeble and die, and has had a strong impact on the sound development of camellia oleiferaindustry.Can by tissue cultures
With Fast-propagation oil tea detoxic seedling, converted by genetic system on this basis and obtain disease-resistant plant, at traditional oil tree
Genetic improvement aspect holds out broad prospects.
Tissue cultures is an important channel of Fast-propagation good plant kind, has wide Commercial Prospect.Close
More in the research of oil tea tissue cultures, Zhang Zhijun et al. utilizes Camellia Oleifera Clones cotyledon to be obtained by somatic embryo development ways
Obtaining regeneration plant, Li Ze et al. utilizes oil tea stem with bud to obtain regeneration plant, but above research can only be trained at oil tea tissue
Support fast numerous aspect significant, it is impossible to be applied to the conversion of oil tea genetic system and obtain transfer-gen plant.Plant genetic system turns
Changing most common method is that agrobacterium-mediated transformation infects the organs such as plant leaf blade, hypocotyl, petiole, cotyledonary node, radicle, and oil tea
The inductivity of blade evoking adventive bud is only 17.86%, can't apply at present at oil tea genetic system conversion aspect, and oil tea turns
Gene does not also find suitable explant so far.Cotyledonary node has higher differentiation capability, and at present, researcher is in west
The plants such as melon, cucumber and soybean obtain regeneration plant by the direct evoking adventive bud of cotyledonary node, and is invaded by Agrobacterium
Dye soybean cotyledon node obtains transfer-gen plant.The research that camellia oleosa seeds leaf segment directly obtains regeneration plant have not been reported, this
Bright by utilizing the direct evoking adventive bud of camellia oleosa seeds leaf segment, it is intended to set up efficient, easy, a stable breeding system, for oil
Tea Fast-propagation and industrial seedling rearing lay the foundation, and also the transgenic research for oil tea provides a kind of technological means.
Content of the invention
It is an object of the invention to use leaf disk method to obtain plant regeneration technique for current oil tea also not set up, basis at this
On, providing and utilize camellia oleosa seeds leaf segment to obtain the method for regeneration plant by adventitious organogenesis, the method utilizes oil tea cotyledon
Saving direct evoking adventive bud, its inductivity is high, and adventitious bud is healthy and strong, and shoot proliferation coefficient is high, and strong sprout, effect was good, and rooting rate is high, closes
Key also resides in the possibility that can be realized oil tea genetic transformation by During Agrobacterium.
It is an object of the invention to be accomplished by.
A kind of method of camellia oleosa seeds leaf segment directly regenerated plant, comprises the following steps:
1) evoking adventive bud: aseptically cut 0.5cm2Fritter oil tea expand cotyledonary node (cotyledonary node i.e. under
Plumular axis connects the part of cotyledon), being inoculated into culture medium is 1/2MS+2.0-3.0mg/L 6-BA+0-0.5mg/L IAA+2.0mg/
L GA3Evoking adventive bud；
2) squamous subculture: the adventitious bud of induction is cut and is inoculated into 1/2MS+2.0-3.0mg/L 6-BA+0.05-0.1mg/
L IBA+0-1.0mg/L GA3Culture medium in carry out adventitious bud shoot proliferation cultivate；
3) strong seedling culture: the bud obtaining Multiplying culture is transferred to WPM+0.5mg/L NAA+3.0-5.0mg/L GA3's
Culture medium carries out strong seedling culture；
4) culture of rootage；
5) hardening, transplanting.
In said method during evoking adventive bud after light culture 2d, then cultivate 20-25d under illumination condition.
Light culture 3d during subculture Multiplying culture in said method, then cultivate 20-30d under illumination condition.
Light culture 2d during strong seedling culture in said method, returns again to cultivate under illumination condition 20-25d.
The seedling in said method, strong seedling culture obtaining 3-4cm receives 1/2MS+1.0-2.0mg/L IBA+1.0-2.0mg/
The perlitic culture medium of L NAA+35g/L carries out culture of rootage；
Light culture 5d during culture of rootage in said method, returns again to cultivate 20d under illumination and i.e. takes root.
In said method, cultivation temperature is 26 ± 1 DEG C, and during illumination cultivation, intensity of illumination is 2100-2200lx, light application time
12-14h/d；The culture medium equal additional saccharose 30g/L using in described method, agar 6g/L, pH are adjusted to 5.4-5.6.
The culture medium that in said method, the direct evoking adventive bud of cotyledonary node uses is preferably: 1/2MS+2.0-3.0mg/L 6-
BA+0.05-0.1mg/L IAA+2.0mg/L GA3
The culture medium that Multiplying culture uses is preferably: 1/2MS+2.0-3.0mg/L 6-BA+0.05mg/L IBA+1.0mg/
L GA3。
Hardening in said method, transplanting detailed process as follows:
Oil tea test tube seedling after taking root first carries out transplanting front hardening 3-5d in indoor, then takes out from blake bottle, attached
Band perlite is directly transplanted in nutrition cup, and matrix is peat soil: perlite: loess=2:1:1, and plantlet in vitro keeps wet after transplanting
Degree more than 80%, cover film every day spray water 1-2 time, i.e. progressively carry out normal management after two weeks.
In said method, the acquisition process of oil tea aseptic seedling is as follows:
That chooses maturation gives birth to Seed of Camellia oleifera then, removes seed coat, kind of a benevolence running water is rinsed 3-5min, then soaks
10-15h, centre changes water 2-3 time, then in superclean bench first with 75% alcohol-pickled 20-30s aseptic water washing 3-5
Secondary, then with 0.1% HgCl2Sterilization 3-6min, with aseptic water washing 5-6 time, cuts away the major part of endosperm, incidentally endosperm with
The embryo of cotyledon is seeded in WPM minimal medium, and forwarding cultivate 20-25d under illumination condition after light culture 2-3d to can obtain
Aseptic seedling；Cultivation temperature is 26 ± 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.
The main processes of the present invention includes: the acquisition of oil tea embryo aseptic seedling, the direct evoking adventive bud of cotyledonary node, adventitious bud
Shoot proliferation cultivation, strong seedling culture, culture of rootage, hardening and transplanting.The method is high to camellia oleosa seeds leaf segment adventitious bud induction frequency, no
Normal bud is healthy and strong, and shoot proliferation coefficient is high, and strong sprout, effect was good, and rooting rate is high；It is not only Fast-propagation oil tea plant and provide one
New way, and for later by engineered method improvement oil tea resistance, cultivation disease-resistant plant, improving tea-oil tree yield and oil
The foundation of tea genetic system lays the first stone.This for oil tea stride forward from traditional breeding mode to molecular breeding direction provide feasible
Property, alleviate China's grain and oil secure context important in inhibiting for later.
Additionally, the research of oil tea excised cotyledon is relatively more, but major part is to be obtained by somatic embryo development ways
Obtain regeneration plant.The detailed advantage of the present invention is summarized as follows:
1st, the present invention cuts endosperm after being sterilized by the kind benevolence utilizing oil tea ripe, and embryo and cotyledon are inoculated into sprouting training
Support growth on base, wait cotyledon to expand and turn the culture medium that cotyledonary node under green rear cutout is inoculated into adventitious bud inducing, by organ, way occurs
The direct evoking adventive bud in footpath, no matter adventitious bud is from growing way, sprout length, blade quantity, leaf color, the rugosity of bud and quality etc.
Aspect is all preferable (Fig. 1-2).Additionally, the present invention is simple to operate, the cycle is short, and adventitious bud growth is fast, and the variation of test tube seedling is less, more has
It is beneficial to the conversion of oil tea genetic system.
2nd, Zhang Zhijun, Fan Xiaoming et al. are obtained by somatic embryo development ways with the cotyledon of excellent Camellia Oleifera Clones again
Raw plant, although frequency of embryonic callus induction is up to more than 90%, but the method is inoculated into adventitious bud generation needs 3 from cotyledon
The cycle of more than individual month just can induce adventitious bud, easily makes a variation in incubation, cannot be realized by agrobacterium-mediated transformation
The foundation of oil tea heritage system.The present invention passes through the regulation repetition test of culture medium and hormone concentration, within 1 month, can induce not
Normal bud, efficiently and rapidly establishes camellia oleosa seeds leaf segment Direct Regeneration system, has changed for oil tea heredity and laid the foundation.
Brief description
Fig. 1 is the photo of the adventitious bud of camellia oleosa seeds leaf segment of the present invention induction；
Fig. 2 is the photo of the adventitious bud of camellia oleosa seeds leaf segment of the present invention induction；
Fig. 3 is the photo of adventitious bud squamous subculture of the present invention；
Fig. 4 is the photo that adventitious bud proliferation of the present invention is cultivated；
Fig. 5 is the photo of adventitious bud strong seedling culture of the present invention；
Fig. 6 is the photo of adventitious bud strong seedling culture of the present invention；
Fig. 7 is the photo that adventitious bud rooting of the present invention is cultivated；
Fig. 8 is the photo that adventitious bud rooting of the present invention is cultivated；
Fig. 9 is the photo of the plantlet in vitro transplanted after the present invention is taken root；
Figure 10 is the photo of the plantlet in vitro surviving after the present invention transplants.
Detailed description of the invention
It is intended to further illustrate the present invention below in conjunction with embodiment, and the unrestricted present invention.
Embodiment 1
Carry out following operation successively:
That the 1st, chooses maturation gives birth to Seed of Camellia oleifera then, removes seed coat, kind of a benevolence running water is rinsed 3-5min, then soaks
10-15h, centre changes water 2-3 time, then in superclean bench first with 75% alcohol-pickled 20-30s, aseptic water washing 3-5
Secondary, then with 0.1% HgCl2Sterilization 3-5min, with aseptic water washing 5-6 time, cuts away the major part of endosperm, subsidiary endosperm
Embryo is seeded in WPM minimal medium, forwards cultivate 20-25d under illumination condition to and can obtain aseptic seedling after light culture 2-3d；
Cultivation temperature is 26 ± 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.
Table 1 difference disinfects the Disinfection Effect on oil tea embryo for the mode to be affected
2nd, cut the cotyledon that oil tea is expanded, be aseptically cut into a length of 0.5cm2Fritter cotyledonary node, be inoculated into table 2
Shown A1-A12Carrying out evoking adventive bud in culture medium prescription, preferred culture medium is 1/2MS+2.0-3.0mg/L 6-BA+0-
0.5mg/L IAA+2.0mg/L GA3, after light culture 2d, then to cultivating 20-25d under illumination condition, the highest inductivity up to
87.28%.Additional saccharose 30g/L, agar 6g/L, pH 5.4.Cultivation temperature is 26 ± 1 DEG C, and intensity of illumination is 2100-
2200lx, light application time 12-14h/d；(see accompanying drawing 1-2)
The influential effect to camellia oleosa seeds leaf segment adventitious bud inducing for the table 2 hormon proportioning
3rd, it the adventitious bud of induction is cut is inoculated into the B shown in table 31-B12Culture medium prescription carries out the subculture of adventitious bud
Multiplying culture, preferably 1/2MS+2.0-3.0mg/L 6-BA+0.05-0.1mg/L IBA+0-1.0mg/L GA3, light culture 3d,
Cultivating 20-30d under illumination condition again, growth coefficient reaches as high as 7.0；Additional saccharose 30g/L, agar 6g/L, pH 5.4-
5.8；Cultivation temperature is 26 ± 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d；(see accompanying drawing 3-4)
The impact on oil tea adventitious bud proliferation coefficient for the table 3 hormon proportioning
4th, the adventitious bud of propagation is carried out strong seedling culture, the bud of propagation is transferred to the C shown in table 41-C12Culture medium prescription
In carry out the strong seedling culture of adventitious bud, preferably WPM+0.5mg/L NAA+3.0-5.0mg/L GA3, light culture 2d, return again to light
Cultivating 20-25d under the conditions of according to, height of seedling is 3-4cm, and the number of blade is 3-6.Additional saccharose 30g/L, agar 6g/L, pH 5.5, training
Supporting temperature is (26 ± 1) DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d；(see accompanying drawing 5-6)
The impact on oil tea adventitious bud strong sprout for the table 4 hormon proportioning
5th, when test tube seedling grows to 3-4cm, the D shown in table 5 is received1-D10Culture medium prescription carries out the training of taking root of adventitious bud
Supporting, preferably root media 1/2MS+1.0-2.0mg/L IBA+1.0-2.0mg/L NAA+35g/L perlite, after light culture 5d
Forward under illumination cultivation, after 20d, add up rooting rate to and reach 91.02%, additional saccharose 20g/L, agar (sigma) 2.5g/L, pH
5.5.Cultivation temperature is 26 ± 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.(see accompanying drawing 7-8)
The impact on oil tea adventitious bud rooting for the table 5 hormon proportioning
| Numbering | IBA(mg/L) | NAA(mg/L) | Perlite (g/L) | Rooting rate/% | Take root number/bar |
| D1 | 0 | 1.0 | 35 | 0 | 0 |
| D2 | 0.5 | 1.0 | 35 | 60.10 | 2.2 |
| D3 | 1.0 | 1.0 | 35 | 87.26 | 4.0 |
| D4 | 2.0 | 1.0 | 35 | 89.05 | 3.8 |
| D5 | 0.5 | 2.0 | 35 | 72.14 | 4.1 |
| D6 | 1.0 | 2.0 | 35 | 91.02 | 5.2 |
| D7 | 2.0 | 2.0 | 35 | 85.33 | 3.3 |
| D8 | 0.5 | 3.0 | 35 | 70.20 | 3.1 |
| D9 | 1.0 | 3.0 | 35 | 76.29 | 3.0 |
| D10 | 2.0 | 3.0 | 35 | 70.18 | 2.2 |
6th, the test tube seedling after taking root first carries out transplanting front hardening, about 3-5d in indoor, then takes out from blake bottle, attached
Band perlite is directly transplanted in nutrition cup, and matrix is peat soil: perlite: loess=2:1:1, and plantlet in vitro keeps wet after transplanting
Degree more than 80%, cover film every day spray water 1-2 time, can progressively carry out normal management after two weeks, transplanting survival rate up to
More than 86%.(see accompanying drawing 9-10).
Claims (6)
1. the method for a camellia oleosa seeds leaf segment directly regenerated plant, it is characterised in that comprise the following steps:
1) evoking adventive bud: aseptically cut 0.5cm2The cotyledonary node that expands of fritter oil tea, being inoculated into culture medium is 1/
2MS+2.0-3.0mg/L 6-BA+0-0.5mg/L IAA+2.0mg/L GA3Evoking adventive bud；
2) squamous subculture: the adventitious bud of induction is cut and is inoculated into 1/2MS+2.0-3.0mg/L 6-BA+0.05-0.1mg/L
IBA+0-1.0mg/L GA3Culture medium in carry out adventitious bud shoot proliferation cultivate；
3) strong seedling culture: the bud obtaining Multiplying culture is transferred to WPM+0.5mg/L NAA+3.0-5.0mg/LGA3Culture medium
In carry out strong seedling culture；
4) culture of rootage: the seedling that strong seedling culture obtains 3-4cm is received 1/2MS+1.0-2.0mg/LIBA+1.0-2.0mg/L
The perlitic culture medium of NAA+35g/L carries out culture of rootage；
5) hardening, transplanting: the oil tea test tube seedling after taking root first carries out transplanting front hardening 3-5d, then from blake bottle in indoor
Taking out, subsidiary perlite is directly transplanted in nutrition cup, and matrix is peat soil: perlite: loess=2:1:1, and plantlet in vitro is transplanted
Rear holding humidity more than 80%, sprays water 1-2 time cover film every day, i.e. progressively carries out normal management after two weeks；
Cultivation temperature is 26 ± 1 DEG C, and during illumination cultivation, intensity of illumination is 2100-2200lx, light application time 12-14h/d；Described side
The culture medium equal additional saccharose 30g/L using in method, agar 6g/L, pH are adjusted to 5.4-5.6.
2. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, it is characterised in that
During evoking adventive bud after light culture 2d, then cultivate 20-25d under illumination condition.
3. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, it is characterised in that
Light culture 3d when shoot proliferation is cultivated, then cultivate 20-30d under illumination condition.
4. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, it is characterised in that
Light culture 2d during strong seedling culture, returns again to cultivate under illumination condition 20-25d.
5. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, it is characterised in that
Light culture 5d during culture of rootage, returns again to cultivate 20d under illumination and i.e. takes root.
6. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, it is characterised in that
The culture medium that the direct evoking adventive bud of cotyledonary node uses is: 1/2MS+2.0-3.0mg/L6-BA+0.05-0.1mg/L IAA
+2.0mg/L GA3,
The culture medium that Multiplying culture uses is: 1/2MS+2.0-3.0mg/L 6-BA+0.05mg/L IBA+1.0mg/L GA3。
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| CN105230497B (en) * | 2015-11-23 | 2018-03-02 | 海南大学 | A kind of production method of Hainan Region white flower oil tea tissue-cultured seedling |
| CN105900845B (en) * | 2016-06-16 | 2018-03-13 | 南京晓庄学院 | Somatic embryo rapid propagation seedling raising method for camellia chekiangoleosa |
| CN107410025A (en) * | 2017-06-22 | 2017-12-01 | 兰溪市奥而特农业科技有限公司 | A kind of method for cultivating oil tea sterile bud |
| CN107743870B (en) * | 2017-11-22 | 2019-09-24 | 中南林业科技大学 | A kind of method that oil tea half is dehydrated the sterile regeneration plant of embryo |
| CN109892156A (en) * | 2019-03-01 | 2019-06-18 | 广西生态工程职业技术学院 | A kind of control method of tea oil tree sooty mould |
| CN113966716A (en) * | 2021-06-17 | 2022-01-25 | 陕西理工大学 | Camellia oleifera callus in-vitro culture and karyotype analysis method |
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| US6599743B2 (en) * | 2001-03-22 | 2003-07-29 | Council Of Scientific & Industrial Research | Method for microproduction of tea plants from leaf explants |
| CN103461143B (en) * | 2013-09-30 | 2014-10-29 | 中南林业科技大学 | Method for tissue culture and rapid propagation of camellia oleifera |
| CN104094747B (en) * | 2014-06-24 | 2016-02-10 | 江西省林业科学院 | A kind of oil tea plantlet in vitro outside sprout-cultivating-bottle method |
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