CN106148267A - The induction of the stem cell in scape cambium layer source, Changchun and separation method - Google Patents
The induction of the stem cell in scape cambium layer source, Changchun and separation method Download PDFInfo
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- CN106148267A CN106148267A CN201510161811.4A CN201510161811A CN106148267A CN 106148267 A CN106148267 A CN 106148267A CN 201510161811 A CN201510161811 A CN 201510161811A CN 106148267 A CN106148267 A CN 106148267A
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Abstract
The invention discloses stem cell induction and the separation method in a kind of Apocynaceae (Apocynaceae) Vinca Herba Catharanthi Rosei (catharanthus roseus) stem cambium layer source, its step includes the Cambium tissue that will obtain from the stem of Apocynaceae Herba Catharanthi Rosei；The Cambium tissue gathered in the crops is cultivated in suitable culture medium；The cell of separated for above-mentioned Cambium tissue acquisition is cambium layer stem cell；By the stem cambium layer stem cell that obtained in 5L illumination bioreactor culture.The stem cell utilizing this law to produce scape cambium layer source, Changchun has fast-growth, the stable feature expressed, and has the biggest prospects for commercial application.
Description
Technical field
The present invention relates to Apocynaceae (Apocynaceae) Vinca Herba Catharanthi Rosei (Catharanthus roseus) stem cambium layer source stem cell and separation method.
Background technology
Plant as the most important ingredient of reproducibility living resources, is the basic source of the human being's production means of livelihood and living environment is rely the basic assurance maintained, be the basic source of necessary for human food, timber, fiber, oil plant, medicine and spiritual demand.Along with the continuous progress of science and technology, the bioactive medical value composition that has of plant origin is increasingly subject to the extensive concern of people.
But, the exploitation application based on plant amedica, produce specialization in various degree due to the cultivated since long of plant, often there is different kind matter features.Further, since people are for the unreasonable exploitation of natural vegetation and destruction and the strong pollution of local area, make the extinction in imminent danger of some floristics, and the loss planting matter has been the most recyclable.It addition, the secondary metabolite in plant, there is bioactive main component often, but its content is but extremely trace.And the speed of growth of plant is again slowly, this has just limited to the development and application of some materials with bioactive ingredients.
But, plant tissue culture has been widely used for life sciences every field as the important technology in cell engineering, is the important component part of biotech development.Its ledge mainly has the following aspects: first, and the production of metabolite is carried out under complete manual control condition, is not affected by geographical, season and weather.Second, reduce the farmland being largely used to plantation, reduce cost, with short production cycle, improve production efficiency.3rd, mutagenesis screening can be passed through, it is thus achieved that high yielding cell sarain, and the utility that the acquisition of specific bioconversion is new can be carried out.What is more important, current plant tissue culture has completed laboratory stage, and now progressively developing into alternative whole strain plant realizes continuous prodution in the factory.
In the expression system of plant, root, stem, leaf etc. belong to differentiated tissue, lose splitting ability, if wanting, making it be changed into the fissiparous cell line of tool must first experience Dedifferentiation.So-called dedifferentiation refers to that differentiated cell, under certain factor effect, recovers cell division capacity, loses the process of original differentiation state.But, Dedifferentiation easily causes chromosome disorder or gene mutation, thus causes the variation of cell line, therefore should avoid Dedifferentiation in plant tissue culture course as far as possible.
It is stably quickly to rise in value during long-term cultivation by the premise of the activated secondary metabolite of plant tissue culture industrialized production, and keeps stable hereditary character.But, most cell line is but due to long-term successive transfer culture, and there occurs variation.Therefore select a kind of effective position in plant to produce stable cell line, and then industrialized production, become as the task of top priority.
To this end, the present invention utilizes the Cambium tissue gathered from the stem of plant to induce stem cell.The class special cells group that plant stem cell is present in shoot apical meristem and Root apical meristem, there is self-renewal capacity, can produce again the daughter cell with lasting splitting ability, these special cells are the sources of the generation of the organs such as plant roots, stem, leaf and flower.The problem that instant invention overcomes plant cell dedifferentiation hereditary variation, improves hereditary stability, it is possible to quickly rise in value, and the most tentatively realizes Small Scale Industry metaplasia and produces.
The present inventor has had successfully been isolated the cambium layer in the scape of Changchun, and obtains cambium layer stem cell, the propagation that this cell line can be stable, confirms without variation, thus confirms the present invention.
Summary of the invention
It is an object of the invention to without Dedifferentiation obtains the method for the stem cell in cambium layer source in the scape of Changchun.
The feature of the stem cell in the cambium layer source of the Apocynaceae Changchun scape that the present invention utilizes is derived from Cambium tissue, and is the cell without dedifferentiation.
The separation method of the stem cell in the cambium layer source of the Apocynaceae Changchun scape that the present invention provides, comprises the steps:
The step of a Cambium tissue that () obtains from the stem of Apocynaceae Herba Catharanthi Rosei；
B step that the Cambium tissue gathered in the crops is cultivated in suitable culture medium by ()；
C the cell of the separated acquisition of () above-mentioned Cambium tissue is the step of cambium layer stem cell；
(d) by the stem cambium layer stem cell that obtained in the step of 5L illumination bioreactor culture.
Accompanying drawing explanation
Fig. 1 is the transverse section figure of plant Changchun scape, and the scale of lower end, right side is 500 μm.
Fig. 2 is the stem's stem cell induction according to the present invention and the outer implant lamination of Herba Catharanthi Rosei tender stem separated.
Fig. 3 is the solid culture figure of the stem cell of the Herba Catharanthi Rosei tender stem that the present invention is obtained.
Fig. 4 is the electron micrograph of the stem cell of the Herba Catharanthi Rosei tender stem that the present invention is obtained, and the scale of lower end, right side is 50 μm.
Fig. 5 is the stem cell concentration class situation under an optical microscope of the Herba Catharanthi Rosei tender stem that the present invention is obtained, and the scale of lower end, right side is 100 μm.
Fig. 6 is that the cambial stem cell of Changchun scape cultivation amount (dry weight) in 100 ml shaking flasks becomes.
Fig. 7 is cultivation amount (dry weight) change in 500 ml shaking flasks of the cambial stem cell of Changchun scape.
Fig. 8 is cultivation amount (dry weight) change in 5L bioreactor of the cambial stem cell of Changchun scape.
Detailed description of the invention
Hereinafter will be specifically described the present invention by embodiment.In this example, experimenter confirms the stem cell in scape cambium layer source, Changchun.
Embodiment 1: prepared by the stem cell in Changchun scape cambium layer source
The preparation of 1-1: vegetable material
Apocynaceae periwinkle plant Herba Catharanthi Rosei, by phloroglucinol stain it is observed that fiber between xylem and phloem, it may be determined that this dyeing part is cambium layer.In order to obtain the stem cell in Herba Catharanthi Rosei stem cambium layer source, first gather the tender stem of Herba Catharanthi Rosei early morning, gatherer process should be avoided select colored branch.The twig that newly gathers is removed blade and tender shoots, and cuts into the long section of 5-8 centimetre, be placed in flowing tap water and rinse 30 minutes to remove surface blot.Hereafter, the outer implant after cleaning uses filter paper adsorption surface moisture, with 75% ethanol sterilizing 30 seconds.Outer implant after ethanol sterilizing uses aseptic water washing 5 times, the most again with 0.05%(w/v) mercuric chloride solution sterilizing 6 minutes, with aseptic water washing 7 times.Outer implant after sterilizing is immersed in the citric acid solution of 150 mg/l.The instrument of high temperature sterilize is immersed in the ethanol solution of 75%.
1-2: obtain cambial preparation process from the stem tissue of outer implant
Outer implant after sterilizing being positioned in the culture dish equipped with sterilizing filter paper, after being cut to the segment of about 2 centimetres, rip cutting also removes marrow.The outer implant removing marrow is cut into further the fritter of about 0.5 square centimeter, cultivation will be affixed in culture medium containing cambial side.
The induction step of the stem cell in 1-3: Changchun scape cambium layer source
The cambium layer of the outer implant obtained in embodiment 1-2 is inoculated on the stem cell inducing culture of table 1.
Table 1: the inducing culture of the stem cell in Changchun scape cambium layer source
Cultivating and cultivate at 24 ± 1 DEG C of constant temperature culture framves, the photoperiod cultivates 12 hours.After about 7 ~ 12 days, the phenomenon of outer implant layering can be observed with the naked eye.As shown in Figure 2.The cell of layering is carefully peeled off outer implant, and transfers them to subculture medium is cultivated further.
The condition of successive transfer culture is the incubator of constant temperature 24 ± 1 DEG C, complete light culture.As shown in Figure 3.
Embodiment 2: the stem cells hyperplasia in Changchun scape cambium layer source is investigated with characteristic
The stem cell in the Changchun scape cambium layer source of described example 1 is put into liquid culture as described in Table 2 based on 25 DEG C, light culture on 100rpm shaking table.The cycle of Secondary Culture is 15 days, thus the cell of cultivation can be made to keep higher vigor at exponential phase all the time.
Table 2: the suspension medium of the stem cell in Changchun scape cambium layer source
According to the present invention it is observed that our cell of being obtained as shown in Figure IV, its cell size is little, and containing little and many vacuole.Such feature is present in the undifferentiated cell in plant, thus may determine that the cambial stem cell of Changchun scape of the present invention is in undifferentiated state.
One of the coherent condition of cell index determining that stem cell equally.As shown in Figure 5, being rolled into a ball and cell quantity by counting microscope observation of cell, after finding cell suspension, Herba Catharanthi Rosei stem cell is many to be existed with single celled state, and the unicellular rate that exists reaches 66.6%, and wherein maxicell group exists only 4.23%.
The amplification culture of the stem cell in 1-4: Changchun scape cambium layer source
The stem cell liquid culture in scape cambium layer source, Changchun is in the shaking flask of 100 ml, and it is 478% that the 15th day rate of growth reaches maximum；In the shaking flask of 500 ml, it is 502% that the 12nd day rate of growth reaches maximum；In 5 liters of bioreactors, it is 641% that the 9th day rate of growth reaches maximum.Result shows, the growth rate of stem cell is along with the increase of the scale of cultivation, and fermentation period constantly reduces, and rate of growth is also continuously increased.
Claims (11)
1. Apocynaceae periwinkle plant Herba Catharanthi Rosei (catharanthus roseus) stem cambium layer source stem cell, it is characterized in that being formed without dedifferentiation, and there is the special cells of cellular omnipotency or versatility.
2. the stem cell in the Changchun scape cambium layer source of claim 1 also should have following 1 feature:
(1), in morphology, stem cell has little and many vacuole；
(2) compared with Catharanthus roseus calli, cultivate in bioreactor and have insensitivity for shearing force；
(3) rapidly, there is the situation of planar growth to the character stable homogeneous of the stem cell in stem cambium layer source, and growth in regenerative cell；
(4), compared with callus, during stem cell suspension culture, in single cell culture state, aggregation tendency is greatly weakened；
(5) compared with other cells, the stem cell in Changchun scape cambium layer source is to radioactivity medicine, such as bleomycin, more sensitive.
3. the stem cell in the scape cambium layer source, Changchun of claim 2, its feature includes at least 2 features of feature of described (1)-(5).
4. the stem cell in the scape cambium layer source, Changchun of claim 2, its feature includes at least 3 features of feature of described (1)-(5).
5. the stem cell in the scape cambium layer source, Changchun of claim 2, its feature includes at least 4 features of feature of described (1)-(5).
6. the stem cell in the scape cambium layer source, Changchun of claim 2, its feature includes whole features of the feature of described (1)-(5).
7. the stem cell in the Changchun scape cambium layer source of claim 1 includes the step of following separation method:
(1) step of the Cambium tissue obtained from the stem of Apocynaceae Herba Catharanthi Rosei；
(2) step that the Cambium tissue gathered in the crops is cultivated in suitable culture medium；
(3) cell of separated for above-mentioned Cambium tissue acquisition is the step of cambium layer stem cell；
(4) by the stem cambium layer stem cell that obtained in the step of 5L illumination bioreactor culture.
8. the method for claim 7, is characterized in that comprising naphthalene acetic acid (NAA) in (2) described culture medium.
9. the method for claim 8, is characterized in that comprising concentration in (2) described culture medium is 1mg/L-2
Mg/L naphthalene acetic acid (NAA).
10. the method for claim 7, is characterized in that the stem cell that the stem cambium layer that step (3) is cultivated is originated is after the part propagation beyond cambium layer goes into unformed callus layer, the cell the most later obtained.
Any one of 11. claim 1-10 belong to Apocynaceae (Apocynaceae) periwinkle plant Herba Catharanthi Rosei (catharanthus roseus) stem cambium layer source stem cell.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109609433A (en) * | 2017-10-04 | 2019-04-12 | 超级拜客-环境保护有限公司 | The method for obtaining vegetable fibers from separation and culture growth cell |
| WO2024008874A1 (en) * | 2022-07-06 | 2024-01-11 | Green Bioactives Limited | Method for isolating plant stem cells from plant leaves and the associated cell lines obtained utilising the method |
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| CN109609433A (en) * | 2017-10-04 | 2019-04-12 | 超级拜客-环境保护有限公司 | The method for obtaining vegetable fibers from separation and culture growth cell |
| CN109609433B (en) * | 2017-10-04 | 2023-12-22 | 超级Bac生物技术解决方案公司 | Method for obtaining plant fibers from isolated and cultured growing cells |
| WO2024008874A1 (en) * | 2022-07-06 | 2024-01-11 | Green Bioactives Limited | Method for isolating plant stem cells from plant leaves and the associated cell lines obtained utilising the method |
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