CN104885948A - Method for directly regenerating plants by tea-oil tree cotyledonary nodes - Google Patents
Method for directly regenerating plants by tea-oil tree cotyledonary nodes Download PDFInfo
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Abstract
The invention discloses a method for directly regenerating plants by taking tea-oil tree cotyledonary nodes as explants through an organ generation manner, and belongs to the field of biotechnology. The method comprises the following steps: (1) culturing aseptic seedlings by taking seeds of common tea-oil trees as materials; (2) chopping off the cotyledonary nodes and inoculating the cotyledonary nodes into an adventitious bud induction culture medium, and culturing for 20-25 days to obtain adventitious buds; (3) transferring the adventitious buds into a subculture proliferation culture medium and culturing for 20-30 days, wherein the proliferation coefficient can reach 7.0; (4) transferring the proliferated adventitious buds into a strong seedling culture medium and culturing for 20-25 days until the seedling height is up to 3cm-4cm; and (5) transferring the obtained adventitious buds into a rooting culture medium to obtain complete test-tube plantlets. According to the method disclosed by the invention, a novel way is provided for rapidly proliferating high-quality tea-oil tree plants, and technical supports are provided for tea-oil tree genetic transformation and molecule improvement; and the method has important meanings on healthy production of tea-oil tree industries in China.
Description
Technical field
The invention belongs to biological technical field, is a kind of with the method for camellia oleosa seeds leaf segment directly regenerated plant.
Background technology
Oil tea (Camellia Oleifera) is Theaceae (Theaceae) Camellia (Camellia) evergreen shrubs or dungarunga, China distinctive woody edible oil material seeds, with olive, oil palm, coconut be called the large traditional oil tree in the world four.Tea oil unsaturated fatty acid content reaches more than 90%, and look good taste is fragrant, unique flavor, nutritious, storage tolerance, has the effect of hypotensive, blood fat and softening blood vessel, playing an important role to the prevention of angiocardiopathy, is one of 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 camellia oleifera lam 4531.2 ten thousand mu of China, produces tea oil more than 20 ten thousand tons per year, average yield per mu tea oil 5.8kg, and camellia oleiferaindustry ubiquity the low and deficiency in economic performance two large problems of yield per unit area.One of major reason of oil tea low yield poor efficiency is exactly that improved variety degree is low, and kind is chaotic, and most of camellia oleifera lam is in wild state, and output is lower, and the shortage of breeding nursery stock can not spread plantation.Meanwhile, Oil Tea Anthracnose Glomerella cingulata (Stonem.) Spauld et Schrenk is the Major Diseases of oil tea.The large area oil tea cultivation area of each province on the south the Yangtze river basin, and Henan, South Shaanxi occur general.After disease occurs, cause serious shedding, bud drop, branch withered, even whole strain decline, has had a strong impact on the sound development of camellia oleiferaindustry.Can Fast-propagation oil tea detoxic seedling by tissue cultures, transformed by genetic system on this basis and obtain disease-resistant plant, holding out broad prospects in traditional oil tree genetic improvement.
Tissue cultures is an important channel of Fast-propagation good plant kind, has wide Commercial Prospect.Research about oil tea tissue cultures is more, the people such as Zhang Zhijun utilize Camellia Oleifera Clones cotyledon to obtain regeneration plant by somatic embryo development ways, the people such as Li Ze utilize oil tea stem with bud to obtain regeneration plant, but more than research can only be significant in oil tea tissue culture quick breeding, oil tea genetic system can not be applied to and transform acquisition transfer-gen plant.It is that agrobacterium-mediated transformation infects the organs such as plant leaf blade, hypocotyl, petiole, cotyledonary node, radicle that plant genetic system transforms the most frequently used method, and the inductivity of oil tea blade evoking adventive bud is only 17.86%, can't be applied in oil tea genetic system conversion aspect at present, oil tea transgenosis does not also find suitable explant so far.Cotyledonary node has higher differentiation capability, and at present, researcher obtains regeneration plant by the direct evoking adventive bud of cotyledonary node in the plants such as watermelon, cucumber and soybean, and infects soybean cotyledon node by Agrobacterium and obtain transfer-gen plant.The research that camellia oleosa seeds leaf segment directly obtains regeneration plant have not been reported, the present invention is by utilizing the direct evoking adventive bud of camellia oleosa seeds leaf segment, be intended to set up efficient, easy, a stable breeding system, for oil tea Fast-propagation and factorial seedling growth lay the foundation, also for the transgenic research of oil tea provides a kind of technological means.
Summary of the invention
The object of the invention is to adopt leaf disk method to obtain plant regeneration technique for current oil tea also not set up, on this basis, provide and utilize camellia oleosa seeds leaf segment to obtain the method for regeneration plant by adventitious organogenesis, the method utilizes the direct evoking adventive bud of camellia oleosa seeds leaf segment, and its inductivity is high, and indefinite bud is healthy and strong, shoot proliferation coefficient is high, strong sprout is effective, and rooting rate is high, and key is also the possibility that can be realized oil tea genetic transformation by During Agrobacterium.
The object of the invention is to realize in the following manner.
A method for camellia oleosa seeds leaf segment directly regenerated plant, comprises the following steps:
1) evoking adventive bud: aseptically cut 0.5cm
2the cotyledonary node (cotyledonary node and hypocotyl connect the part of cotyledon) that expands of fritter oil tea, being inoculated into medium is 1/2MS+2.0-3.0mg/L 6-BA+0-0.5mg/L IAA+2.0mg/L GA
3evoking adventive bud;
2) squamous subculture: the indefinite 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 GA
3medium in carry out indefinite bud shoot proliferation cultivate;
3) strong seedling culture: the bud obtained by Multiplying culture is transferred to WPM+0.5mg/L NAA+3.0-5.0mg/L GA
3medium in carry 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 20-30d is cultivated under illumination condition.
Light culture 2d during strong seedling culture in said method, then cultivate 20-25d under forwarding illumination condition to.
The seedling in said method, strong seedling culture being obtained 3-4cm is received in the perlitic medium of 1/2MS+1.0-2.0mg/L IBA+1.0-2.0mg/L NAA+35g/L and is carried out culture of rootage;
Light culture 5d during culture of rootage in said method, then cultivate 20d under forwarding illumination to and namely take 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 equal additional saccharose 30g/L of the medium used in described method, agar 6g/L, pH is adjusted to 5.4-5.6.
The 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 GA
3
The medium that Multiplying culture uses is preferably: 1/2MS+2.0-3.0mg/L 6-BA+0.05mg/L IBA+1.0mg/L GA
3.
In said method, the detailed process of hardening, transplanting is as follows:
Oil tea test-tube plantlet after taking root first is carried out transplanting front hardening 3-5d in indoor, then take out from blake bottle, subsidiary perlite is directly transplanted in nutrition cup, matrix is peat soil: perlite: loess=2:1:1, plantlet in vitro keeps humidity more than 80% after transplanting, cover film is also sprayed water 1-2 time every day, namely progressively carries out normal management after two weeks.
In said method, the procurement process of oil tea aseptic seedling is as follows:
Choose ripe raw then Seed of Camellia oleifera, get except seed coat, benevolence tap water 3-5min will be planted, then 10-15h is soaked, water is changed 2-3 time in centre, in superclean bench, then first uses alcohol-pickled 20-30s aseptic water washing 3-5 time of 75%, then use the HgCl of 0.1%
2sterilization 3-6min, with aseptic water washing 5-6 time, cuts away the major part of endosperm, the embryo of subsidiary endosperm and cotyledon is seeded in WPM minimal medium, cultivates 20-25d and can obtain aseptic seedling after light culture 2-3d under forwarding illumination condition to; Cultivation temperature is 26 ± 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.
Main process of the present invention comprises: the acquisition of oil tea embryo aseptic seedling, the shoot proliferation cultivation of the direct evoking adventive bud of cotyledonary node, indefinite bud, strong seedling culture, culture of rootage, hardening and transplanting.The method is high to camellia oleosa seeds leaf segment adventitious bud induction frequency, and indefinite bud is healthy and strong, and shoot proliferation coefficient is high, and strong sprout is effective, and rooting rate is high; Be not only Fast-propagation oil tea plant and provide a new way, and by engineered method improvement oil tea resistance, cultivation disease-resistant plant after being, the foundation improving tea-oil tree yield and oil tea genetic system lays the first stone.This provides feasibility, for alleviating China's grain and oil secure context important in inhibiting later for oil tea strides forward from traditional breeding mode to molecular breeding direction.
In addition, the research of oil tea excised cotyledon is many, but major part obtains regeneration plant by somatic embryo development ways.Detailed advantage of the present invention is summarized as follows:
1, the present invention cuts endosperm after utilizing the kind benevolence of oil tea maturation to sterilize, embryo and cotyledon are inoculated on germination medium and grow, Deng cotyledon expand turn green after cut the medium that cotyledonary node is inoculated into adventitious bud inducing, by the direct evoking adventive bud of adventitious organogenesis, no matter from aspects such as the rugosity of growing way, sprout length, blade quantity, leaf color, bud and quality all better (Fig. 1-2) indefinite bud.In addition, the present invention is simple to operate, and the cycle is short, and indefinite bud growth is fast, and test-tube plantlet variation is less, is more conducive to oil tea genetic system and transforms.
2, the people such as Zhang Zhijun, Fan Xiaoming obtains regeneration plant with the cotyledon of excellent Camellia Oleifera Clones by somatic embryo development ways, although frequency of embryonic callus induction is up to more than 90%, but the cycle that the method is inoculated into indefinite bud generation needs more than 3 months from cotyledon just can induce indefinite bud, easily make a variation in incubation, the foundation of oil tea heritage system cannot be realized by agrobacterium-mediated transformation.The present invention passes through the adjustment repetition test of medium and hormone concentration, within 1 month, can induce indefinite bud, efficiently establish camellia oleosa seeds leaf segment Direct Regeneration system rapidly, lay the foundation for oil tea heredity has changed.
Accompanying drawing explanation
Fig. 1 is the photo of the indefinite bud of camellia oleosa seeds leaf segment of the present invention induction;
Fig. 2 is the photo of the indefinite bud of camellia oleosa seeds leaf segment of the present invention induction;
Fig. 3 is the photo of indefinite 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 indefinite bud strong seedling culture of the present invention;
Fig. 6 is the photo of indefinite 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 of transplanting after the present invention is taken root;
Figure 10 is the photo of the plantlet in vitro survived after the present invention transplants.
Embodiment
Be intended to further illustrate the present invention below in conjunction with embodiment, and unrestricted the present invention.
Embodiment 1
Carry out following operation successively:
1, choose ripe raw then Seed of Camellia oleifera, get except seed coat, benevolence tap water 3-5min will be planted, then soak 10-15h, water is changed 2-3 time in centre, in superclean bench, then first use the alcohol-pickled 20-30s of 75%, aseptic water washing 3-5 time, then the HgCl using 0.1%
2sterilization 3-5min, with aseptic water washing 5-6 time, cuts away the major part of endosperm, the embryo of subsidiary endosperm is seeded in WPM minimal medium, cultivate 20-25d and can obtain aseptic seedling after light culture 2-3d under forwarding illumination condition to; Cultivation temperature is 26 ± 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.
Table 1 difference disinfects mode to be affected the Disinfection Effect of oil tea embryo
2, cut the cotyledon that oil tea is expanded, aseptically cut and grow into 0.5cm
2fritter cotyledonary node, be inoculated into the A shown in table 2
1-A
12carry 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 GA
3, after light culture 2d, then cultivate 20-25d under illumination condition, the highest inductivity can reach 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)
Table 2 hormon proportioning is to the influential effect of camellia oleosa seeds leaf segment adventitious bud inducing
3, the indefinite bud of induction is cut the B be inoculated into shown in table 3
1-B
12the shoot proliferation carrying out indefinite bud in culture medium prescription is cultivated, preferred 1/2MS+2.0-3.0mg/L 6-BA+0.05-0.1mg/L IBA+0-1.0mg/L GA
3, light culture 3d, then 20-30d is cultivated under illumination condition, 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)
Table 3 hormon proportioning is on the impact of oil tea adventitious bud proliferation coefficient
4, the indefinite bud of propagation is carried out strong seedling culture, the bud of propagation is transferred to the C shown in table 4
1-C
12the strong seedling culture of indefinite bud is carried out, preferred WPM+0.5mg/L NAA+3.0-5.0mg/L GA in culture medium prescription
3, light culture 2d, then cultivate 20-25d under forwarding illumination condition 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, cultivation temperature is (26 ± 1) DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d; (see accompanying drawing 5-6)
Table 4 hormon proportioning is on the impact in oil tea indefinite bud strong sprout
5, when test-tube plantlet grows to 3-4cm, the D shown in table 5 is received
1-D
10the culture of rootage of indefinite bud is carried out in culture medium prescription, preferred root media 1/2MS+1.0-2.0mg/L IBA+1.0-2.0mg/L NAA+35g/L perlite, forward to after light culture 5d and add up rooting rate after 20d under illumination cultivation 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)
Table 5 hormon proportioning is on the impact of oil tea adventitious bud rooting
| Numbering | IBA(mg/L) | NAA(mg/L) | Perlite (g/L) | Rooting rate/% | To take root number/bar |
| D 1 | 0 | 1.0 | 35 | 0 | 0 |
| D 2 | 0.5 | 1.0 | 35 | 60.10 | 2.2 |
| D 3 | 1.0 | 1.0 | 35 | 87.26 | 4.0 |
| D 4 | 2.0 | 1.0 | 35 | 89.05 | 3.8 |
| D 5 | 0.5 | 2.0 | 35 | 72.14 | 4.1 |
| D 6 | 1.0 | 2.0 | 35 | 91.02 | 5.2 |
| D 7 | 2.0 | 2.0 | 35 | 85.33 | 3.3 |
| D 8 | 0.5 | 3.0 | 35 | 70.20 | 3.1 |
| D 9 | 1.0 | 3.0 | 35 | 76.29 | 3.0 |
| D 10 | 2.0 | 3.0 | 35 | 70.18 | 2.2 |
6, the test-tube plantlet after taking root first is carried out transplanting front hardening in indoor, about 3-5d, then take out from blake bottle, subsidiary perlite is directly transplanted in nutrition cup, matrix is peat soil: perlite: loess=2:1:1, and plantlet in vitro keeps humidity more than 80% after transplanting, and cover film is also sprayed water 1-2 time every day, progressively can carry out normal management after two weeks, transplanting survival rate can reach more than 86%.(see accompanying drawing 9-10)
Claims (10)
1. a method for camellia oleosa seeds leaf segment directly regenerated plant, is characterized in that, comprises the following steps:
1) evoking adventive bud: aseptically cut 0.5cm
2the cotyledonary node that expands of fritter oil tea, being inoculated into medium is 1/2MS+2.0-3.0mg/L 6-BA+0-0.5mg/L IAA+2.0mg/L GA
3evoking adventive bud;
2) squamous subculture: the indefinite bud of induction is cut and is inoculated into 1/2MS+2.0-3.0mg/L 6-BA+0.05-0.1mg/LIBA+0-1.0mg/L GA
3medium in carry out indefinite bud shoot proliferation cultivate;
3) strong seedling culture: the bud obtained by Multiplying culture is transferred to WPM+0.5mg/L NAA+3.0-5.0mg/L GA
3medium in carry out strong seedling culture;
4) culture of rootage;
5) hardening, transplanting.
2. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, is characterized 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, is characterized in that,
Light culture 3d when shoot proliferation is cultivated, then 20-30d is cultivated under illumination condition.
4. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, is characterized in that,
Light culture 2d during strong seedling culture, then cultivate 20-25d under forwarding illumination condition to.
5. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, is characterized in that,
Seedling strong seedling culture being obtained 3-4cm is received in the perlitic medium of 1/2MS+1.0-2.0mg/L IBA+1.0-2.0mg/L NAA+35g/L and is carried out culture of rootage.
6. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, is characterized in that,
Light culture 5d during culture of rootage, then cultivate 20d under forwarding illumination to and namely take root.
7. the method for the camellia oleosa seeds leaf segment directly regenerated plant according to any one of claim 1-6, it is characterized in that, cultivation temperature is 26 ± 1 DEG C, and during illumination cultivation, intensity of illumination is 2100-2200lx, light application time 12-14h/d; The equal additional saccharose 30g/L of the medium used in described method, agar 6g/L, pH is adjusted to 5.4-5.6.
8. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, is characterized in that,
The medium that the direct evoking adventive bud of cotyledonary node uses is: 1/2MS+2.0-3.0mg/L 6-BA+0.05-0.1mg/LIAA+2.0mg/L GA
3
The medium that Multiplying culture uses is: 1/2MS+2.0-3.0mg/L 6-BA+0.05mg/L IBA+1.0mg/L GA
3.
9. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, is characterized in that,
The detailed process of hardening, transplanting is as follows:
Oil tea test-tube plantlet after taking root first is carried out transplanting front hardening 3-5d in indoor, then take out from blake bottle, subsidiary perlite is directly transplanted in nutrition cup, matrix is peat soil: perlite: loess=2:1:1, plantlet in vitro keeps humidity more than 80% after transplanting, cover film is also sprayed water 1-2 time every day, namely progressively carries out normal management after two weeks.
10. the method for camellia oleosa seeds leaf segment directly regenerated plant according to claim 1, is characterized in that, the procurement process of described oil tea aseptic seedling is as follows:
Choose ripe raw then Seed of Camellia oleifera, get except seed coat, benevolence tap water 3-5min will be planted, then 10-15h is soaked, water is changed 2-3 time in centre, in superclean bench, then first uses alcohol-pickled 20-30s aseptic water washing 3-5 time of 75%, then use the HgCl of 0.1%
2sterilization 3-6min, with aseptic water washing 5-6 time, cuts away the major part of endosperm, the embryo of subsidiary endosperm is seeded in WPM minimal medium, cultivate 20-25d and can obtain aseptic seedling after light culture 2-3d under forwarding illumination condition to; Cultivation temperature is 26 ± 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.
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| CN105900845A (en) * | 2016-06-16 | 2016-08-31 | 南京晓庄学院 | Somatic embryo rapid propagation seedling raising method for camellia chekiangoleosa |
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| 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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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105230497A (en) * | 2015-11-23 | 2016-01-13 | 海南大学 | Method for producing camellia oleifera tissue culture seedlings in Hainan region |
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| CN105900845A (en) * | 2016-06-16 | 2016-08-31 | 南京晓庄学院 | Somatic embryo rapid propagation seedling raising method for camellia chekiangoleosa |
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| CN107743870A (en) * | 2017-11-22 | 2018-03-02 | 中南林业科技大学 | The method that a kind of 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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