CN104823861A - Method for acquiring regeneration plants by adventitious bud inducement of camellia oleifera radicles - Google Patents
Method for acquiring regeneration plants by adventitious bud inducement of camellia oleifera radicles Download PDFInfo
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- CN104823861A CN104823861A CN201510310417.2A CN201510310417A CN104823861A CN 104823861 A CN104823861 A CN 104823861A CN 201510310417 A CN201510310417 A CN 201510310417A CN 104823861 A CN104823861 A CN 104823861A
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Abstract
The invention belongs to the field of plant cell engineering and discloses a method for acquiring regeneration plants by adventitious bud inducement of camellia oleifera radicles. The method mainly includes steps of direct inducement of adventitious buds through camellia oleifera radicles , subculture multiplication, best seedling cultivation, rooting cultivation, acclimatization, transplantation and the like. Not only is an approach for rapid propagation of excellent camellia oleifera varieties provided but also a technical support is provided for shortening camellia oleifera breeding process and improving resistibility of camellia oleifera by means of gene engineering.
Description
Technical field
The invention belongs to oil tea cell engineering field, be specifically related to a kind of with oil tea radicle for explant induction indefinite bud, then obtain the method for complete test-tube plantlet 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.Oil tea is mainly distributed in Yangtze river basin areas to the south of China, and Hunan and Jiangxi are the main growing areas of oil tea.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, 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 forwarding aspect at present, oil tea transgenosis does not also find suitable explant so far.The present invention is by utilizing oil tea radicle evoking adventive bud, again by the shoot proliferation of indefinite bud, take root and a series of process such as acclimatization and transplants, be intended to the oil tea radicle rapid propagation in vitro system setting up an efficient stable, for oil tea Fast-propagation and factorial seedling growth lay the foundation.Key is also the possibility that can be realized oil tea genetic transformation by During Agrobacterium, for oil tea Fast-propagation and genetic transformation provide wider approach and technical support.
Summary of the invention
The object of the invention is for adopting oil tea blade evoking adventive bud also not obtain regeneration plant at present, oil tea transgenosis is also in the exploratory stage at initial stage, main cause is the group training system not being applicable to the conversion of oil tea genetic system, on this basis, the invention provides a kind of method that oil tea radicle evoking adventive bud obtains regeneration plant, the method utilizes oil tea radicle to induce indefinite bud, its inductivity is higher, indefinite bud sprouts several more, shoot proliferation coefficient is high, strong sprout is effective, rooting rate is high, the crucial possibility that can also be realized oil tea genetic transformation by During Agrobacterium.
The object of the invention is to realize in the following manner.
Oil tea radicle adventitious bud inducing obtains the method for regeneration plant, comprises the following steps:
1) evoking adventive bud: aseptically cut the long oil tea aseptic seedling radicle root segment for 1.0-1.5cm, be inoculated into 1/2MS+2.0mg/L 6-BA+0.05-0.1mg/L IAA+0.05mg/L IBA+2.0mg/L GA
3medium in evoking adventive bud;
2) squamous subculture: the indefinite bud that radicle is induced 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 the squamous subculture of indefinite bud;
3) strong seedling culture: the bud obtained by Multiplying culture is transferred to WPM+0.1-1.0mg/L NAA+3.0-5.0mg/L GA
3medium in carry out strong seedling culture;
4) culture of rootage;
5) hardening, transplanting.
Light culture after 3 days during evoking adventive bud in said method, then 30-35 days is cultivated under illumination condition.
Light culture 3 days during subculture Multiplying culture in said method, then 20-30 days is cultivated under illumination condition.
Light culture 2 days during strong seedling culture in said method, then cultivate 20-25 days 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/LNAA+35g/L and is carried out culture of rootage.
Light culture 5 days during culture of rootage in said method, then cultivate under forwarding illumination to and can take root for 20 days.
In said method, cultivation temperature is 26 scholar 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, radicle evoking adventive bud uses is preferably: 1/2MS+2.0mg/L 6-BA+0.05mg/L IAA+0.05mg/L IBA+2.0mg/L GA
3;
The medium that Multiplying culture uses is preferably: 1/2MS+2.0mg/L 6-BA+0.05mg/L IBA+1.0mg/L GA
3;
The medium that strong seedling culture uses is preferably: WPM+0.5mg/L NAA+3.0-5.0mg/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 3-5 days 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.
The procurement process of the oil tea aseptic seedling described in said method is as follows:
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 3-6min, 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 and namely obtain aseptic seedling in 20-25 days after light culture 2-3 days under forwarding illumination condition to; Cultivation temperature is 26 scholar 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.
Main process of the present invention comprises: oil tea radicle adventitious bud inducing, shoot proliferation cultivation, strong seedling culture, culture of rootage, hardening and transplanting.The method is higher to oil tea radicle adventitious bud induction frequency, and sprout several more, 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 technological approaches, and after being by engineered method improvement oil tea resistance, cultivate disease-resistant plant, 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.
Accompanying drawing explanation
Fig. 1 is the photo that oil tea radicle of the present invention induces the indefinite bud initial stage;
Fig. 2 is the oil tea radicle evoking adventive bud of the present invention photo of 30 days;
Fig. 3 is the photo that oil tea adventitious bud proliferation of the present invention is cultivated;
Fig. 4 is the photo of oil tea indefinite bud strong seedling culture of the present invention;
Fig. 5 is the photo that oil tea adventitious bud rooting of the present invention is cultivated;
Fig. 6 is the photo that oil tea adventitious bud rooting of the present invention is cultivated;
Fig. 7 is the photo of the plantlet in vitro of transplanting after oil tea of the present invention takes root;
Fig. 8 is the photo of the plantlet in vitro survived after oil tea of the present invention is transplanted.
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 and can obtain aseptic seedling in 20-25 days after light culture 2-3 days under forwarding illumination condition to; Cultivation temperature is 26 scholar 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
| 75% alcohol | 0.1% mercuric chloride | Pollution rate | Melting brown rate |
| 0 | 2 | 83.23 | 0 |
| 0 | 3 | 49.80 | 0 |
| 0 | 5 | 22.03 | 0 |
| 0 | 8 | 20.37 | 5.27 |
| 30 | 2 | 36.46 | 0 |
| 30 | 3 | 2.60 | 0 |
| 30 | 5 | 0 | 0 |
| 30 | 8 | 0 | 59.36 |
2, choose oil tea aseptic seedling radicle, aseptically cut the root segment of growing into 1.0-1.5cm, be inoculated into the B shown in table 2
1-B
18carry out evoking adventive bud in culture medium prescription, preferred culture medium is 1/2MS+2.0mg/L 6-BA+0.05-0.1mg/L IAA+0.05mg/L IBA+2.0mg/L GA
3, light culture after 3 days, then cultivates 30-35 days under illumination condition, and the highest inductivity can reach 68.31%.Additional saccharose 30g/L, agar 6g/L, pH 5.4.Cultivation temperature is (26 scholar 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 oil tea radicle 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 3 days, then under illumination condition, cultivate 20-30 days, additional saccharose 30g/L, agar 6g/L, pH 5.4-5.8; Cultivation temperature is 26 scholar 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d; (see accompanying drawing 3)
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+0.1-1.0mg/L NAA+3.0-5.0mg/L GA in culture medium prescription
3, light culture 2 days, then cultivate 20-25 days under forwarding illumination condition to, height of seedling is 3-5cm, and the number of blade is 3-6.Additional saccharose 30g/L, agar 6g/L, pH 5.5, cultivation temperature is (26 scholar 1) DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d; (see accompanying drawing 4)
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 the highest rooting rate after 20d under illumination cultivation and reach 89.11%, additional saccharose 20g/L, agar (sigma) 2.5g/L, pH 5.5.Cultivation temperature is (26 scholar 1) DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.(see accompanying drawing 5-6)
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 | 65.23 | 2.2 |
| D 3 | 1.0 | 1.0 | 35 | 87.26 | 4.9 |
| D 4 | 2.0 | 1.0 | 35 | 86.04 | 4.1 |
| D 5 | 0.5 | 2.0 | 35 | 72.21 | 4.3 |
| D 6 | 1.0 | 2.0 | 35 | 89.11 | 5.5 |
| D 7 | 2.0 | 2.0 | 35 | 85.30 | 3.7 |
| D 8 | 0.5 | 3.0 | 35 | 73.23 | 3.6 |
| D 9 | 1.0 | 3.0 | 35 | 79.24 | 3.1 |
| D 10 | 2.0 | 3.0 | 35 | 71.10 | 2.8 |
6, the test-tube plantlet after taking root first is carried out transplanting front hardening in indoor, about 3-5 days, 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 7-8).
Claims (10)
1. oil tea radicle adventitious bud inducing obtains the method for regeneration plant, it is characterized in that, comprises the following steps:
1) evoking adventive bud: aseptically cut the long oil tea aseptic seedling radicle root segment for 1.0-1.5cm, be inoculated into 1/2MS+2.0mg/L 6-BA+0.05-0.1mg/L IAA+0.05mg/L IBA+2.0mg/L GA
3medium in evoking adventive bud;
2) squamous subculture: the indefinite bud that radicle is induced 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 the squamous subculture of indefinite bud;
3) strong seedling culture: the bud obtained by Multiplying culture is transferred to WPM+0.1-1.0mg/L NAA+3.0-5.0mg/L GA
3medium in carry out strong seedling culture;
4) culture of rootage;
5) hardening, transplanting.
2. oil tea radicle adventitious bud inducing according to claim 1 obtains the method for regeneration plant, and it is characterized in that, during evoking adventive bud, light culture is after 3 days, then cultivates 30-35 days under illumination condition.
3. oil tea radicle adventitious bud inducing according to claim 1 obtains the method for regeneration plant, it is characterized in that, light culture 3 days when shoot proliferation is cultivated, then cultivates 20-30 days under illumination condition.
4. oil tea radicle adventitious bud inducing according to claim 1 obtains the method for regeneration plant, it is characterized in that, light culture 2 days during strong seedling culture, then cultivates 20-25 days under forwarding illumination condition to.
5. oil tea radicle adventitious bud inducing according to claim 1 obtains the method for regeneration plant, it 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. oil tea radicle adventitious bud inducing according to claim 1 obtains the method for regeneration plant, it is characterized in that, light culture 5 days during culture of rootage, then cultivates under forwarding illumination to and can take root for 20 days.
7. the oil tea radicle adventitious bud inducing according to any one of claim 1-6 obtains the method for regeneration plant, and it is characterized in that, cultivation temperature is 26 scholar 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. oil tea radicle adventitious bud inducing according to claim 1 obtains the method for regeneration plant, it is characterized in that,
The medium that radicle evoking adventive bud uses is: 1/2MS+2.0mg/L 6-BA+0.05mg/L IAA+0.05mg/LIBA+2.0mg/L GA
3;
The medium that Multiplying culture uses is: 1/2MS+2.0mg/L 6-BA+0.05mg/L IBA+1.0mg/L GA
3;
The medium that strong seedling culture uses is: WPM+0.5mg/L NAA+3.0-5.0mg/L GA
3.
9. oil tea radicle adventitious bud inducing according to claim 1 obtains the method for regeneration plant, it 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 3-5 days 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. oil tea radicle adventitious bud inducing according to claim 1 obtains the method for regeneration plant, and it 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 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 3-6min, 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 and namely obtain aseptic seedling in 20-25 days after light culture 2-3 days under forwarding illumination condition to; Cultivation temperature is 26 scholar 1 DEG C, and intensity of illumination is 2100-2200lx, light application time 12-14h/d.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN113293176A (en) * | 2021-05-31 | 2021-08-24 | 福建农林大学 | Preparation method of pineapple agrobacterium transformation receptor and application of pineapple agrobacterium transformation receptor in pineapple transformation |
-
2015
- 2015-06-08 CN CN201510310417.2A patent/CN104823861B/en active Active
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| 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 |
| 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 |
| CN113293176A (en) * | 2021-05-31 | 2021-08-24 | 福建农林大学 | Preparation method of pineapple agrobacterium transformation receptor and application of pineapple agrobacterium transformation receptor in pineapple transformation |
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