CN101558742A - Method for regenerating plant from camellia callus - Google Patents
Method for regenerating plant from camellia callus Download PDFInfo
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- CN101558742A CN101558742A CNA2009100983986A CN200910098398A CN101558742A CN 101558742 A CN101558742 A CN 101558742A CN A2009100983986 A CNA2009100983986 A CN A2009100983986A CN 200910098398 A CN200910098398 A CN 200910098398A CN 101558742 A CN101558742 A CN 101558742A
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Abstract
The invention mainly relates to a method for regenerating a plant from camellia callus. The method has the following steps: stripping off the inner and outer seed coats of a camellia fruit seed, and inoculating the seed to a 1/2MS culture medium; when a sterile seedling grows above 3 cm, inoculating the soft leaf of the young plant to a callus induction culture medium which is MS + 0.5mg.L <-1>6-BA+1.0mg.L <-1>2,4-D; when the callus grows to get a diameter about 1 cm, shifting the callus to a callus differentiation culture medium which is MS+mg.L <-1>6-BA20+0.1mg.L <-1>I BA+ mg.L <-1> KT0.1; when an indefinite bud grows to 0.5 cm, carrying out the separation and inoculating to a strong bud culture medium which is MS + 0.2mg.L <-1>6-BA+0.05mg.L <-1>NAA; and when a bud stick grows to 4 to 5 cm, cutting off the basal of the bud stick, immersing the basal of the bud stick in 1,000 g.L <-1> I BA, and then inoculating to a MW + 0.2mg.L <-1>I BA+0.2mg.L <-1>NAA culture medium. The method has the advantages that: the method has a simple culture medium recipe, a simple and convenient operating process, short culture time, high regeneration frequency and a high propagation expansion coefficient, and facilitates the large-scale production of rare camellia plants and the realization of the genetic transformation of exogenous genes.
Description
Technical field
The present invention relates to the camellia method for tissue culture, belong to biological technical field, is a kind of method of regenerating plant from camellia callus.
Background technology
Camellia is the characteristic rare flower of China, also is the world-famous woody plant of viewing and admiring.Camellia is the important materials of afforestation, has pattern U.S., and the florescence is long, and blade is bright green, and tree crown is colourful, and the habit that can well grow under tall and big tree crown, is widely used in park green land, natural scenic spot and the scenic spots and historical sites.Camellia also is potted plant good merchantable brand, and the shrub type kind of some short life often is used as potted plant application.Except ornamental value, camellia also has very high economic worth, and its flower, root all can be used as medicine, and fruit can extract oil, and the leaf of Camellia nitidissima can be for tea-drinking, supplemental treatment hypertension, but flower treating hematochezia, and red, the yellow leaching liquor of flower can be made food coloring.
Be to keep the merit of camellia breed parent, the breeding of camellia does not adopt the seed that grows directly from seeds to broadcast the seedling mode usually, also the rose polyphyll, fully camellia kind such as polyphyll type degenerate fully because of female, stamen can not be solid.Adopt cuttage and grafting method, for some rare camellia kinds, its reproduction coefficient is lower, is difficult to carry out large-scale production, realizes the industrialization purpose.
Utilizing callus approach regenerated adventitious bud can obtain a large amount of asexual plant division, is the necessary means of quick breeding of camellia and genetic transformation.The explant that evoked callus is commonly used in camellia is leaf and stem section, minority is also arranged with petal and microspore.Pedroso etc. (1993) with 50 age Red Hill tea bearing tree blade be explant, find easily to form callus at petiole and main lobe arteries and veins place.Shults etc. (1996) are explant with the petal of Red Hill tea, and callus has growth preferably on the medium that contains BA and NAA, but does not obtain its regeneration plant.The grand hero (1981) that shakes as explant, can obtain the pollen of oil tea higher callus induction frequency, and obtain green bud point and root on proper culture medium, but fails to obtain complete regeneration plant because of pollution.
Yet there are no the report that from the camellia callus, obtains indefinite bud, also do not see patent application.Therefore the present invention provides a valid approach for the market demand that satisfies rare camellia kind, also lays the foundation for the camellia molecular breeding simultaneously.
Summary of the invention
The objective of the invention is provides a kind of method of regenerating plant from camellia callus at camellia callus induction and the very difficult technical bottleneck of regeneration plant.
The object of the invention is achieved through the following technical solutions: the method for this regenerating plant from camellia callus, and this method is carried out according to the following steps:
(1) the camellia fruit is rinsed well with running water, used detergent immersion again 10 minutes, the running water flushing; Transfer to then on the aseptic super-clean bench remove pericarp after, be 75% ethanol surface sterilization 30 seconds with volume ratio, aseptic water washing 2 times is 0.1% HgCl with weight ratio
2Middle immersion 15-20 minute, aseptic water washing 5-6 time;
(2) peel off the interior exosper of seed, be seeded on the 1/2MS medium;
When (3) aseptic seedling is grown to more than 3 centimetres, plantlet young leaflet tablet and stem section are seeded on the callus inducing medium, callus inducing medium is MS+0.5mgL
-1-BA+1.0mgL
-12,4-D;
When (4) callus grows to diameter and is 1 centimetre big or small, be transferred on the callus differential medium, the callus differential medium is MS+mgL
-16-BA20+0.1mgL
-1IBA+mgL
-1KT0.1;
Separate when (5) indefinite bud grows to 0.5cm, strong bud medium is MS+0.2mgL
-16-BA+0.05mgL
-1NAA;
(6) when budling grows to 4-5cm, cut away the budling base portion, with 1000gL
-1IBA soak the base portion of budling, be seeded in MW+0.2mgL then
-1IBA+0.2mgL
-1On the medium of NAA.
As preferably, described camellia fruit is the ripening fruits of gathering September.
As preferably, described condition of culture is that cultivation temperature is 25 ± 2 ℃, and the illumination light intensity is 2500～3000Lx, and light application time is 12h/d.
The invention has the beneficial effects as follows: at present also not from the report of camellia callus induction indefinite bud and plant regeneration.Culture medium prescription of the present invention is simple, and operating procedure is easy, and incubation time is short, the regeneration frequency height, and it is big to expand numerous coefficient, helps the genetic transformation of rare camellia plant of large-scale production and realization foreign gene.
Embodiment
The present invention is described in further detail by following examples, but content of the present invention is not limited thereto.
The method of this regenerating plant from camellia callus of the present invention, material are Zhejiang Red Hill tea ripening fruits in September.The fruit of gathering is rinsed well with running water, used detergent immersion again 10 minutes, the running water flushing.Transfer to then on the aseptic super-clean bench remove pericarp after, be 75% ethanol surface sterilization 30 seconds with volume ratio, aseptic water washing 2 times, weight ratio is 0.1% HgCl
2Middle immersion 15---20 minutes, aseptic water washing 5---6 times.Be seeded on the 1/2MS medium behind the exosper in peelling off, cultivation temperature is 25 ± 2 ℃, and the illumination light intensity is 2500～3000Lx, and light application time is 12h/d, and following condition of culture is all identical.
Seedling grows to 3 centimetres high after cultivating for 2 weeks, and plantlet young leaflet tablet and stem section are seeded on the callus inducing medium, and callus inducing medium is MS+0.5mgL
-16-BA+1.0mgL
-12,4-D.After cultivating a week, the leaf stripping and slicing begins to purse up, and the first quarter moon back edge has a small amount of callus to grow, and the callus diameter reaches about 1 centimetre after 1 month.
Callus forms the graininess kick after differential medium is cultivated about first quarter moon, promptly growing point is divided into indefinite bud again after cultivating, can form a large amount of indefinite buds on the callus, behind the bigger bud of excision, also have indefinite bud constantly to break up to come out, best medium is MS+20mgL
-16-BA+0.1mgL
-1IBA+0.1mgL
-1KT.
After cultivating first quarter moon, it is promptly separable that indefinite bud grows to 0.5cm, at strong bud medium MS+0.2mgL
-16-BA+0.05mgL
-1The last cultivation of NAA after 1 month budling can grow to 4---5cm, cut away the budling base portion, with 1000gL
-1IBA soak the base portion of budling, be seeded in MW+0.2mgL then
-1IBA+0.2mgL
-1On the medium of NAA, cultivate prosperity of root system after 1 month, rooting rate reaches 100%.
In addition to the implementation, the present invention can also have other embodiments.All employings are equal to the technical scheme of replacement or equivalent transformation formation, all drop on the protection domain of requirement of the present invention.
Claims (3)
1, a kind of method of regenerating plant from camellia callus is characterized in that: this method is carried out according to the following steps:
(1) the camellia fruit is rinsed well with running water, used detergent immersion again 10 minutes, the running water flushing; Transfer to then on the aseptic super-clean bench remove pericarp after, be 75% ethanol surface sterilization 30 seconds with volume ratio, aseptic water washing 2 times is 0.1% HgCl with weight ratio
2Middle immersion 15-20 minute, aseptic water washing 5-6 time;
(2) peel off the interior exosper of seed, be seeded on the 1/2MS medium;
When (3) aseptic seedling is grown to more than 3 centimetres, plantlet young leaflet tablet and stem section are seeded on the callus inducing medium, callus inducing medium is MS+0.5mgL
-16-BA+1.0mgL
-12,4-D;
When (4) callus grows to diameter and is 1 centimetre big or small, be transferred on the callus differential medium, the callus differential medium is MS+mgL
-16-BA20+0.1mgL
-1IBA+mgL
-1KT0.1;
Separate when (5) indefinite bud grows to 0.5cm, strong bud medium is MS+0.2mgL
-16-BA+0.05mgL
-1NAA;
(6) when budling grows to 4-5cm, cut away the budling base portion, with 1000gL
-1IBA soak the base portion of budling, be seeded in MW+0.2mgL then
-1IBA+0.2mgL
-1On the medium of NAA.
2, the method for regenerating plant from camellia callus according to claim 1 is characterized in that: described camellia fruit is the ripening fruits of gathering September.
3, the method for regenerating plant from camellia callus according to claim 1 is characterized in that: described condition of culture is that cultivation temperature is 25 ± 2 ℃, and the illumination light intensity is 2500～3000Lx, and light application time is 12h/d.
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|---|---|---|---|
| CN2009100983986A CN101558742B (en) | 2009-05-12 | 2009-05-12 | Method for regenerating plant from camellia callus |
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|---|---|---|---|
| CN2009100983986A CN101558742B (en) | 2009-05-12 | 2009-05-12 | Method for regenerating plant from camellia callus |
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| CN101558742A true CN101558742A (en) | 2009-10-21 |
| CN101558742B CN101558742B (en) | 2012-11-21 |
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Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102613089A (en) * | 2012-04-25 | 2012-08-01 | 江苏省林业科学研究院 | High-efficient in-vitro propagation method of 20-year-old schima superba big tree |
| CN102696482A (en) * | 2012-06-18 | 2012-10-03 | 湖南省林业科学院 | Method for detoxifying clonal tissue culture explant of Camellia oleifera Abel |
| CN103081807A (en) * | 2013-02-01 | 2013-05-08 | 中国林业科学研究院亚热带林业研究所 | Method for regenerating plant by use of callus of camellia japonica |
| CN103329799A (en) * | 2013-06-08 | 2013-10-02 | 上海辰山植物园 | Rapid propagation and seedling growing method for Shuhua camellia tissue culture |
| CN103548686A (en) * | 2013-11-01 | 2014-02-05 | 重庆文理学院 | Rooting culture medium for tissue culture seedling of camellia japonica |
| CN104285813A (en) * | 2014-10-27 | 2015-01-21 | 南宁市金花茶公园 | Camellia chrysantha tissue culture propagation method |
| CN104488713A (en) * | 2014-12-11 | 2015-04-08 | 柳州博泽科技有限公司 | Camellia nitidissima tissue culture and rapid propagation method |
| CN105941159A (en) * | 2016-06-17 | 2016-09-21 | 句容市下蜀窑业茶场 | Seedling strengthening and rooting culture medium for tea tree tissue culture seedlings |
| CN106234226A (en) * | 2016-08-23 | 2016-12-21 | 中央民族大学 | A kind of in-vitro culture method of Ligularia rumicifolia (Drumm.) S.W. Liu. |
| 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 |
| CN107646701A (en) * | 2017-11-15 | 2018-02-02 | 范瑶飞 | A kind of method for obtaining polyphyll camellia plant |
| CN110604060A (en) * | 2019-10-17 | 2019-12-24 | 中国林业科学研究院亚热带林业研究所 | Method for obtaining regeneration plant by in vitro culture of camellia japonica anther |
| CN111705033A (en) * | 2020-07-08 | 2020-09-25 | 中南林业科技大学 | Method for callus suspension culture and protoplast separation of camellia oleifera |
| CN112175989A (en) * | 2020-09-23 | 2021-01-05 | 中国林业科学研究院亚热带林业研究所 | Genetic transformation method for winter-tolerant camellia |
| CN115413578A (en) * | 2021-06-18 | 2022-12-02 | 重庆市风景园林科学研究院 | Method for cultivating new camellia variety seedlings by using immature hybrid seeds |
-
2009
- 2009-05-12 CN CN2009100983986A patent/CN101558742B/en not_active Expired - Fee Related
Cited By (18)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102613089A (en) * | 2012-04-25 | 2012-08-01 | 江苏省林业科学研究院 | High-efficient in-vitro propagation method of 20-year-old schima superba big tree |
| CN102696482A (en) * | 2012-06-18 | 2012-10-03 | 湖南省林业科学院 | Method for detoxifying clonal tissue culture explant of Camellia oleifera Abel |
| CN103081807A (en) * | 2013-02-01 | 2013-05-08 | 中国林业科学研究院亚热带林业研究所 | Method for regenerating plant by use of callus of camellia japonica |
| CN103081807B (en) * | 2013-02-01 | 2014-08-20 | 中国林业科学研究院亚热带林业研究所 | Method for regenerating plant by use of callus of camellia japonica |
| CN103329799A (en) * | 2013-06-08 | 2013-10-02 | 上海辰山植物园 | Rapid propagation and seedling growing method for Shuhua camellia tissue culture |
| CN103329799B (en) * | 2013-06-08 | 2015-01-07 | 上海植物园 | Rapid propagation and seedling growing method for Shuhua camellia tissue culture |
| CN103548686A (en) * | 2013-11-01 | 2014-02-05 | 重庆文理学院 | Rooting culture medium for tissue culture seedling of camellia japonica |
| CN104285813A (en) * | 2014-10-27 | 2015-01-21 | 南宁市金花茶公园 | Camellia chrysantha tissue culture propagation method |
| CN104488713A (en) * | 2014-12-11 | 2015-04-08 | 柳州博泽科技有限公司 | Camellia nitidissima tissue culture and rapid propagation method |
| CN105941159A (en) * | 2016-06-17 | 2016-09-21 | 句容市下蜀窑业茶场 | Seedling strengthening and rooting culture medium for tea tree tissue culture seedlings |
| CN106234226A (en) * | 2016-08-23 | 2016-12-21 | 中央民族大学 | A kind of in-vitro culture method of Ligularia rumicifolia (Drumm.) S.W. Liu. |
| 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 |
| CN107646701A (en) * | 2017-11-15 | 2018-02-02 | 范瑶飞 | A kind of method for obtaining polyphyll camellia plant |
| CN110604060A (en) * | 2019-10-17 | 2019-12-24 | 中国林业科学研究院亚热带林业研究所 | Method for obtaining regeneration plant by in vitro culture of camellia japonica anther |
| CN111705033A (en) * | 2020-07-08 | 2020-09-25 | 中南林业科技大学 | Method for callus suspension culture and protoplast separation of camellia oleifera |
| CN111705033B (en) * | 2020-07-08 | 2022-04-01 | 中南林业科技大学 | Method for callus suspension culture and protoplast separation of camellia oleifera |
| CN112175989A (en) * | 2020-09-23 | 2021-01-05 | 中国林业科学研究院亚热带林业研究所 | Genetic transformation method for winter-tolerant camellia |
| CN115413578A (en) * | 2021-06-18 | 2022-12-02 | 重庆市风景园林科学研究院 | Method for cultivating new camellia variety seedlings by using immature hybrid seeds |
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|---|---|
| CN101558742B (en) | 2012-11-21 |
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