CN101695280A - Tissue culture and rapid propagation method of raspberries - Google Patents
Tissue culture and rapid propagation method of raspberries Download PDFInfo
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- CN101695280A CN101695280A CN200910095104A CN200910095104A CN101695280A CN 101695280 A CN101695280 A CN 101695280A CN 200910095104 A CN200910095104 A CN 200910095104A CN 200910095104 A CN200910095104 A CN 200910095104A CN 101695280 A CN101695280 A CN 101695280A
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Abstract
The invention provides a tissue culture and rapid propagation method of raspberries. The method comprises the following steps: using the stem tips of raspberries or stem sections with buds as explants; directly inducing and culturing in an induction culture medium to generate bud bushes; then, alternately propagating and culturing a needed quantity of bud bushes in a propagation culture medium; and after rooting culture, directly putting in seedling bags for transplantation to obtain raspberry seedlings. The method is high-efficiency and rapid, is needless to collect a great quantity of explants to obtain sterile seedlings, achieves the aims of improving propagation rate and reducing variation by adjusting hormones at various culture stages and mixing and alternately using the hormones, eliminates a link for transplanting grouped culture seedling nutrition pans, improves the transplantation survival rate, facilitates the transmission of survival seedlings, directly plants after picking off the seedling bags in plantation and fully realizes the scaled and standardized production of fine-variety raspberry seedlings.
Description
Technical field
The present invention relates to tissue culture and the method for quickly breeding of tissue culture and propagation method, the especially raspberry of a plant species, belong to field of plant growing technology.
Background technology
Raspberry is the rose family, the rubus bush fruit, and the high 1～2m of plant, berry is bigger, and fruit is red or orange.Red raspberry fruits is nutritious, soft and succulency, fragrance uniqueness, lovely luster, the suitable high-grade products such as fruit juice, jam and natural colouring matter that are processed into.The propagation method of raspberry routine has root division method, offshoot, cuttage and the planting of layer etc.But above-mentioned propagation method reproduction speed is slow, and to maternal material requirements height, consumption is big, also is subjected to the restriction in season, is difficult to carry out the large tracts of land popularization of scale, standardized production and improved seeds.In addition, use the method cost of method for tissue culture propagation production seedling higher at present, cultivation cycle is longer.Therefore, need to seek the new vegetative propagation system that cost is low, the time is short, survival rate is high of a cover and enlarge the raspberry breeding amount, carry out the industrialization production of raspberry nursery stock, to meet the need of market.
Summary of the invention
At defective or the deficiency that the existing seedling growing process of raspberry exists, the object of the present invention is to provide tissue culture and the method for quickly breeding of a kind of raspberry, with the suitable for mass production high quality seedling.
The present invention is an explant with raspberry stem section, and carry out in medium provided by the invention that aseptic seedling is induced and enrichment culture, thereby set up aseptic fast traditional font system and aseptic seedling and transplant system, and in sterile system, improve the rate of increase and rooting rate, to shorten cultivation cycle, reduce cost, reach seedling breeding and production requirement fast.
The present invention finishes by following technical proposal: tissue culture of a kind of raspberry and method for quickly breeding is characterized in that comprising following process steps:
A, the raspberry explant that will handle through sterilization, i.e. stem apex of raspberry or stem with bud are inoculated in the following inducing culture:
The MS medium
Sugar 30～40g/L
Agar 5～7g/L
BA 0.5～1.5mg/L
NAA 0.1～0.3mg/L
pH 5.8～6.0
In intensity of illumination is 1500～2000lx, temperature is 20～27 ℃, photoperiod is under 8～10h/d condition of culture, be cultured to explant and sprout leaf differentiation and bud formation clump, downcut the bud clump, and explant continues in inducing culture, inducing culture downcuts to growing the sprouting clump, inducing culture was to transfer subculture the cycle once with 25～35 days so repeatedly;
B, bud clump that the A step is downcut samsara successively are forwarded among following proliferated culture medium I and the II:
Proliferated culture medium I:
The MS medium
Sugar 30～40g/L
Agar 5～7g/L
BA 0.5～1.0mg/L
NAA 0.2～0.3mg/L
pH 5.8～6.0
Proliferated culture medium II:
The MS medium
Sugar 30～40g/L
Agar 5～7g/L
BA 1.0～1.5mg/L
NAA 0.1～0.2mg/L
pH 5.8～6.0
And be 1500～2000lx in intensity of illumination, temperature is 20～27 ℃, photoperiod is under 8～10h/d condition of culture, carry out enrichment culture, and in proliferated culture medium I and II, per 25～30 days subcultures once respectively, must breed multiple and be 2.5～3 bud clump, so carry out the bud clump of enrichment culture repeatedly, and make the bud seedling on the bud clump grow to high 2～3cm, tool 2～3 joints, downcut to requirement;
C, the bud seedling that the B step is downcut are divided into individual plant, be inoculated in the following root media, and be 1500～2000lx in intensity of illumination, temperature is 20～27 ℃, and the photoperiod is under 8～10h/d condition of culture, and culture of rootage to bud seedling is grown up and is taken root:
The 1/2MS medium
Sugar 30～40g/L
Agar 5～7g/L
IAA 0.1～0.2mg/L
NAA 0.3～0.5mg/L
pH 5.8～6.0；
D, with the seedling of taking root of above-mentioned C step move to temper 6～10 days under the outdoor scattered light after, the taking-up seedling of taking root, with clear water medium on the seedling is cleaned, putting into mass concentration and be 0.1% carbendazim solution sterilized 1～2 minute, transplant to being equipped with in the Seedling bag of following quality than matrix: humus soil: laterite=1: 1, wherein contain mass ratio in this mixed-matrix and be 4～7% perlite, promptly get the raspberry seedling.
Described A step to the raspberry explant sterilization treatment that carries out disinfection be: in aseptic super-clean bench, 2～3cm the stem apex or the stem with bud that will clean with washing agent through conventional method, disinfecting 30～60 seconds in volumetric concentration is 70%～75% alcohol successively, is 0.1% HgCl again in mass concentration
2In sterilization treatment 6～10 minutes, in volumetric concentration is 1.5%～2% liquor natrii hypochloritis, disinfected 15～20 minutes at last, use rinsed with sterile water afterwards 2～3 times, get final product.
Described alcohol, HgCl
2, liquor natrii hypochloritis, sterile water, carbendazim be commercial conventional products.
The present invention is in the process of the group training of studying raspberry and quick-breeding method, by selecting raspberry stem with bud and stem apex is explant, directly induce and produce the bud clump, and utilize different proliferated culture mediums to replace cultivation, take to accelerate switching speed simultaneously and reach technical measures such as directly putting into the Seedling bag transplanting, the effect that has obtained the efficient breeding fast of raspberry seedling and produced.Its superiority is embodied in:
1, the complete set technology of the stripped tissue-culturing rapid propagation of raspberry is provided, has realized scale, the standardized production of raspberry seedling.
2, use stem with bud and stem apex as explant, and this explant is carried out repeated multiple times induce, obtain a large amount of bud clumps,, obtain a large amount of seedlings, need not to obtain aseptic seedling by gathering a large amount of explants again through enrichment culture, culture of rootage, acclimatization and transplants.
3, carry out the hormone regulation of different cultivation stages, take hormone to use and take turns usefulness with, reach the purpose that improves the rate of increase and reduce variation.
4, the sterile rootage seedling direct transplantation is in Seedling bag, saved the link that the tissue cultivating seedling nutritive cube is transplanted, and improves transplanting survival rate, survives seedling and is convenient to transportation, and during plantation, plucking behind the Seedling bag directly, plantation gets final product.
Embodiment
The present invention is described in further detail below in conjunction with embodiment.
Embodiment 1
1, explant sterilization: gathering raspberry children shoot, be cut into the long band bud segment of 2cm, after cleaning with washing powder water, in aseptic super-clean bench is 75% alcohol with volumetric concentration, to its surface sterilization 30 seconds, is 0.1% HgCl then with mass concentration
2Sterilizing 6 minutes, is sterilization 15 minutes among 2% the liquor natrii hypochloritis in volumetric concentration afterwards, uses rinsed with sterile water at last 3 times;
2, adventitious bud induction culture: will in aseptic working environment, be inoculated in the following inducing culture: MS medium+BA 1.0mg/L+NAA 0.1mg/L+ sugar 30g/L+ agar 5g/L through the above-mentioned explant of above-mentioned 1 step sterilization, pH 5.8, in intensity of illumination is 1500lx, cultivation temperature is 20 ℃, photoperiod is that the condition of culture of 8h/d was cultivated 18 days down, there is 35% explant pollution-free and grown the leaf bud clump, downcuts the bud clump; The explant that cuts the clump that newly sprouts continues in above-mentioned inducing culture, and cultivates 15 days under above-mentioned condition of culture, has 85% explant to grow the second generation leaf bud clump is arranged, and downcuts the bud clump; Cultivate explant so repeatedly, make it to induce to generate the sprouting clump, subculture switching in per 33 days once makes it to reach the required provenance quantity of production;
3, enrichment culture: the bud clump that above-mentioned 2 steps are downcut places following proliferated culture medium I: MS medium+BA 0.5mg/L+NAA 0.3mg/L+ sugar 30g/L+ agar 5g/L, pH 5.8, in intensity of illumination is 1500lx, cultivation temperature is 20 ℃, photoperiod is under the condition of culture of 8h/d, carried out enrichment culture 25 days, be forwarded among the following proliferated culture medium II: MS medium+BA 1.0mg/L+NAA0.1mg/L+ sugar 30g/L+ agar 5g/L, PH 5.8, at intensity of illumination 1500lx, and 20 ℃ of cultivation temperature, under the condition of photoperiod 8h/d, cultivated 25 days, and must breed multiple and be 2.5 bud clump, again gained bud clump is downcut separately and being transferred to successively among above-mentioned proliferated culture medium I and the II, at fate same as described above, under the same culture conditions, carry out enrichment culture, must breed multiple and be 2.5 bud clump, so repeatedly alternately in proliferated culture medium I and II, enrichment culture is required bud clump quantity extremely, and make the bud clump grow up to 2 joints, the bud seedling of high 2cm downcuts;
4, culture of rootage: the bud seedling that above-mentioned 3 steps are downcut is divided into individual plant and is inoculated in the following root media: 1/2MS medium+IAA 0.2mg/L+NAA 0.3mg/L+ sugar 30g/L+ agar 5g/L, pH 5.8, at intensity of illumination 1500lx, 20 ℃ of cultivation temperature, cultivated 30 days the seedling of taking root that must grow tall and become strong under the condition of photoperiod 8h/d;
5, hardening and transplanting: with the seedling of taking root of above-mentioned 4 steps move on to temper 6 days under the outdoor scattered light after, seedling is taken out in bottle, with clear water medium on the seedling is cleaned, put into mass concentration and be 0.1% carbendazim solution sterilization 1 minute, direct transplanting is to the Seedling bag that following matrix is housed: humus soil: in the matrix of laterite=1: 1 mass ratio, be mixed with mass ratio in this matrix and be 4% perlite, and carry out water conservation according to a conventional method, protect fertile, shelter from heat or light, management such as illumination, when treating that seedling leaf look dark green and producing a large amount of fibrous root, promptly get the raspberry seedling that to transplant plantation.
Embodiment 2
1, explant sterilization: gathering raspberry children shoot, be cut into the long band bud segment of 3cm, after cleaning with washing powder water, in aseptic super-clean bench is 70% alcohol with volumetric concentration, to its surface sterilization 60 seconds, is 0.1% HgCl then with mass concentration
2Sterilizing 10 minutes, is sterilization 20 minutes among 1.5% the liquor natrii hypochloritis in volumetric concentration afterwards, uses rinsed with sterile water at last 2 times;
2, adventitious bud induction culture: will in aseptic working environment, be inoculated in the following inducing culture: MS medium+BA 0.5mg/L+NAA 0.3mg/L+ sugar 40g/L+ agar 7g/L through the above-mentioned explant of above-mentioned 1 step sterilization, pH 6.0, in intensity of illumination is 2000lx, cultivation temperature is 27 ℃, photoperiod is that the condition of culture of 10h/d was cultivated 15 days down, there is 30% explant pollution-free and grown the leaf bud clump, downcuts the bud clump; The explant that cuts the clump that newly sprouts continues in above-mentioned inducing culture, and cultivates 20 days under above-mentioned condition of culture, has 87% explant to grow the second generation leaf bud clump is arranged, and downcuts the bud clump; Cultivate explant so repeatedly, make it to induce to generate the sprouting clump, subculture switching in per 35 days once makes it to reach the required provenance quantity of production;
3, enrichment culture: the bud clump that above-mentioned 2 steps are downcut places following proliferated culture medium I: MS medium+BA 1.0mg/L+NAA 0.2mg/L+ sugar 40g/L+ agar 7g/L, pH 6.0, in intensity of illumination is 2000lx, cultivation temperature is 27 ℃, photoperiod is under the condition of culture of 10h/d, carried out enrichment culture 30 days, be forwarded among the following proliferated culture medium II: MS medium+BA 1.5mg/L+NAA0.1mg/L+ sugar 40g/L+ agar 7g/L, PH 6.0, at intensity of illumination 2000lx, and 27 ℃ of cultivation temperature, under the condition of photoperiod 10h/d, cultivated 30 days, and must breed multiple and be 3 bud clump, again gained bud clump is downcut separately and being transferred to successively among above-mentioned proliferated culture medium I and the II, at fate same as described above, under the same culture conditions, carry out enrichment culture, must breed multiple and be 3 bud clump, so repeatedly alternately in proliferated culture medium I and II, enrichment culture is required bud clump quantity extremely, and make the bud clump grow up to 3 joints, the bud seedling of high 3cm downcuts;
4, culture of rootage: the bud seedling that above-mentioned 3 steps are downcut is divided into individual plant and is inoculated in the following root media: 1/2MS medium+IAA 0.1mg/L+NAA 0.5mg/L+ sugar 40g/L+ agar 7g/L, pH 6.0, at intensity of illumination 2000lx, 27 ℃ of cultivation temperature, cultivated 30 days the seedling of taking root that must grow tall and become strong under the condition of photoperiod 10h/d;
5, hardening and transplanting: with the seedling of taking root of above-mentioned 4 steps move on to temper 10 days under the outdoor scattered light after, seedling is taken out in bottle, with clear water medium on the seedling is cleaned, put into mass concentration and be 0.1% carbendazim solution sterilization 1 minute, direct transplanting is to the Seedling bag that following matrix is housed: humus soil: in the matrix of laterite=1: 1 mass ratio, be mixed with mass ratio in this matrix and be 7% perlite, and carry out water conservation according to a conventional method, protect fertile, shelter from heat or light, management such as illumination, when treating that seedling leaf look dark green and producing a large amount of fibrous root, promptly get the raspberry seedling that to transplant plantation.
Embodiment 3
1, explant sterilization: gathering raspberry children shoot, be cut into the long band bud segment of 2cm, after cleaning with washing powder water, in aseptic super-clean bench is 70% alcohol with volumetric concentration, to its surface sterilization 45 seconds, is 0.15% HgCl then with mass concentration
2Sterilizing 8 minutes, is sterilization 18 minutes among 2% the liquor natrii hypochloritis in volumetric concentration afterwards, uses rinsed with sterile water at last 3 times;
2, adventitious bud induction culture: will in aseptic working environment, be inoculated in the following inducing culture: MS medium+BA 1.5mg/L+NAA 0.2mg/L+ sugar 35g/L+ agar 6g/L through the above-mentioned explant of above-mentioned 1 step sterilization, pH 5.8, in intensity of illumination is 1800lx, cultivation temperature is 25 ℃, photoperiod is that the condition of culture of 9h/d was cultivated 10 days down, there is 25% explant pollution-free and grown the leaf bud clump, downcuts the bud clump; The explant that cuts the clump that newly sprouts continues in above-mentioned inducing culture, and cultivates 15 days under above-mentioned condition of culture, has 85% explant to grow the second generation leaf bud clump is arranged, and downcuts the bud clump; Cultivate explant so repeatedly, make it to induce to generate the sprouting clump, subculture switching in per 25 days once makes it to reach the required provenance quantity of production;
3, enrichment culture: the bud clump that above-mentioned 2 steps are downcut places following proliferated culture medium I: MS medium+BA 0.8mg/L+NAA 0.3mg/L+ sugar 35g/L+ agar 6g/L, pH 5.8, in intensity of illumination is 1800lx, cultivation temperature is 25 ℃, photoperiod is under the condition of culture of 9h/d, carried out enrichment culture 28 days, be forwarded among the following proliferated culture medium II: MS medium+BA 1.2mg/L+NAA0.2mg/L+ sugar 35g/L+ agar 6g/L, PH 5.8, at intensity of illumination 1800lx, and 25 ℃ of cultivation temperature, under the condition of photoperiod 9h/d, cultivated 28 days, and must breed multiple and be 2.8 bud clump, again gained bud clump is downcut separately and being transferred to successively among above-mentioned proliferated culture medium I and the II, at fate same as described above, under the same culture conditions, carry out enrichment culture, must breed multiple and be 2.8 bud clump, so repeatedly alternately in proliferated culture medium I and II, enrichment culture is required bud clump quantity extremely, and make the bud clump grow up to 2 joints, the bud seedling of high 2cm downcuts;
4, culture of rootage: the bud seedling that above-mentioned 3 steps are downcut is divided into individual plant and is inoculated in the following root media: 1/2MS medium+IAA 0.2mg/L+NAA 0.4mg/L+ sugar 35g/L+ agar 6g/L, pH 5.8, at intensity of illumination 1800lx, 25 ℃ of cultivation temperature, cultivated 30 days the seedling of taking root that must grow tall and become strong under the condition of photoperiod 9h/d;
5, hardening and transplanting: with the seedling of taking root of above-mentioned 4 steps move on to temper 8 days under the outdoor scattered light after, seedling is taken out in bottle, with clear water medium on the seedling is cleaned, put into mass concentration and be 0.1% carbendazim solution sterilization 2 minutes, direct transplanting is to the Seedling bag that following matrix is housed: humus soil: in the matrix of laterite=1: 1 mass ratio, be mixed with mass ratio in this matrix and be 5% perlite, and carry out water conservation according to a conventional method, protect fertile, shelter from heat or light, management such as illumination, when treating that seedling leaf look dark green and producing a large amount of fibrous root, promptly get the raspberry seedling that to transplant plantation.
Claims (2)
1. tissue culture of a raspberry and method for quickly breeding is characterized in that comprising following process steps:
A, the raspberry explant that will handle through sterilization---be stem apex or the stem with bud of raspberry, be inoculated in the following inducing culture:
The MS medium
Sugar 30～40g/L
Agar 5～7g/L
BA 0.5～1.5mg/L
NAA 0.1～0.3mg/L
pH 5.8～6.0
In intensity of illumination is 1500～2000lx, temperature is 20～27 ℃, photoperiod is under 8～10h/d condition of culture, be cultured to explant and sprout leaf differentiation and bud formation clump, downcut the bud clump, and explant continues in inducing culture, and inducing culture was to transfer subculture the cycle once to growing the sprouting clump with 25～35 days repeatedly;
B, the bud clump that the A step is downcut are forwarded among following proliferated culture medium I and the II successively:
Proliferated culture medium I:
The MS medium
Sugar 30～40g/L
Agar 5～7g/L
BA 0.5～1.0mg/L
NAA 0.2～0.3mg/L
pH 5.8～6.0
Proliferated culture medium II:
The MS medium
Sugar 30～40g/L
Agar 5～7g/L
BA 1.0～1.5mg/L
NAA 0.1～0.2mg/L
pH 5.8～6.0
And be 1500～2000lx in intensity of illumination, temperature is 20～27 ℃, photoperiod is under 8～10h/d condition of culture, carry out enrichment culture, and in proliferated culture medium I and II, per 25～35 days subcultures once respectively, must breed multiple and be 2.5～3 bud clump, so carry out the bud clump of enrichment culture repeatedly, and make the bud clump grow up to the bud seedling of 2～3 joints, high 2～3cm, downcut to requirement;
C, the bud seedling that the B step is downcut are divided into individual plant, be inoculated in the following root media, and be 1500～2000lx in intensity of illumination, temperature is 20～27 ℃, and the photoperiod is under 8～10h/d condition of culture, and culture of rootage to bud seedling is grown up and is taken root:
The 1/2MS medium
Sugar 30～40g/L
Agar 5～7g/L
IAA 0.1～0.2mg/L
NAA 0.3～0.5mg/L
pH 5.8～6.0；
D, with the seedling of taking root of above-mentioned C step move to temper 6～10 days under the outdoor scattered light after, the taking-up seedling of taking root, with clear water medium on the seedling is cleaned, putting into mass concentration and be 0.1% carbendazim solution sterilized 1～2 minute, transplant to the Seedling bag that following matrix is housed: humus soil: in the matrix of laterite=1: 1 mass ratio, be mixed with mass ratio in this matrix and be 4～7% perlite, promptly get the raspberry seedling.
2. tissue culture of raspberry as claimed in claim 1 and method for quickly breeding, what it is characterized in that described A step to the raspberry explant sterilization treatment that carries out disinfection is: in aseptic super-clean bench, 2～3cm the stem apex or the stem with bud that will clean through washing agent, in volumetric concentration is 70%～75% alcohol, disinfect successively 30～60 seconds, be 0.1%～0.15% HgCl in mass concentration
2In sterilization treatment 6～10 minutes, in volumetric concentration is 1.5%～2% liquor natrii hypochloritis, disinfected 15～20 minutes, use rinsed with sterile water afterwards 2～3 times, get final product.
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| CN2009100951044A CN101695280B (en) | 2009-10-27 | 2009-10-27 | Tissue culture and rapid propagation method of raspberries |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104145663A (en) * | 2014-07-25 | 2014-11-19 | 宁国市瑞龙生态农林开发有限公司 | Ecological imitated rubus idaeus planting method |
| CN105265310A (en) * | 2014-06-06 | 2016-01-27 | 王贵虹 | Method for breeding raspberry seedling through tissue culture |
| CN105494101A (en) * | 2016-01-09 | 2016-04-20 | 酒泉金硕元现代农业发展股份有限公司 | Industrialized seedling raising method of red raspberries in arid desertification regions |
| CN107094622A (en) * | 2017-04-18 | 2017-08-29 | 天津农学院 | A kind of method for inducing raspberry Callus formation globular embryo |
| CN107242136A (en) * | 2017-07-21 | 2017-10-13 | 广西桂平市蒙圩镇柱强种养专业合作社 | A kind of method for tissue culture of raspberry |
| CN110856463A (en) * | 2018-08-24 | 2020-03-03 | 安徽安盛农业科技发展有限公司 | Tissue culture and rapid propagation method of red raspberries |
-
2009
- 2009-10-27 CN CN2009100951044A patent/CN101695280B/en active Active
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105265310A (en) * | 2014-06-06 | 2016-01-27 | 王贵虹 | Method for breeding raspberry seedling through tissue culture |
| CN104145663A (en) * | 2014-07-25 | 2014-11-19 | 宁国市瑞龙生态农林开发有限公司 | Ecological imitated rubus idaeus planting method |
| CN105494101A (en) * | 2016-01-09 | 2016-04-20 | 酒泉金硕元现代农业发展股份有限公司 | Industrialized seedling raising method of red raspberries in arid desertification regions |
| CN107094622A (en) * | 2017-04-18 | 2017-08-29 | 天津农学院 | A kind of method for inducing raspberry Callus formation globular embryo |
| CN107242136A (en) * | 2017-07-21 | 2017-10-13 | 广西桂平市蒙圩镇柱强种养专业合作社 | A kind of method for tissue culture of raspberry |
| CN110856463A (en) * | 2018-08-24 | 2020-03-03 | 安徽安盛农业科技发展有限公司 | Tissue culture and rapid propagation method of red raspberries |
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|---|---|
| CN101695280B (en) | 2011-12-21 |
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