CN1826878A - Tissue-culturing rapid propagation method of Actinidia macrosperma - Google Patents
Tissue-culturing rapid propagation method of Actinidia macrosperma Download PDFInfo
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- CN1826878A CN1826878A CN 200610050251 CN200610050251A CN1826878A CN 1826878 A CN1826878 A CN 1826878A CN 200610050251 CN200610050251 CN 200610050251 CN 200610050251 A CN200610050251 A CN 200610050251A CN 1826878 A CN1826878 A CN 1826878A
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- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Cultivation Of Plants (AREA)
Abstract
The invention provides a method of rapid breeding of kiwi fruit with large seed by tissue culture. The invention comprises steps of selecting and treating explant, inducing culture medium, root culturing and replanting, etc. The invention takes MS culture medium as basis and adds BA, NAA, GA, ZT and other external plant growth regulator to induce the growth of axillary bud of the said kiwi fruit and strengthen sprout and root. The root growth rate is as high as 100%, the average bud height is 6.28 cm and the average replanting survival rate is 91% after 35 days culture. The invention solves the technique problem of rapid breeding of said kiwi fruit and provides a method of short period, high breeding rate and low cost for the industrial production of kiwi fruit with large seed.
Description
Technical field
The present invention relates to biological technical field, be specifically related to a kind of tissue culture quick propagation culturing method of big seed kiwi fruit.
Background technology
Plant is the natural treasure-house of medicine, and people utilize the historical of long standing and well established of medicinal plant, the whole world have approximately 75% population with plant as treatment prophylactic medicament sources (Xing Jianmin, 2001).The mankind have found many medicines with height physiologically active from plant, the medicine from plant origin accounts for more than 25% of medicine total amount (Zheng Guangzhi, 1987) at present.The history in existing thousands of years of traditional Chinese herbal medicine of China is still widely used in China and many countries and regions so far.But because traditional Chinese herbal medicine acquisition methods is to be cost to gather and to consume a large amount of wild plant resources,, will inevitably cause this species reduction when gathering and consumption when surpassing the regeneration capacity of natural resources, in imminent danger even become extinct.Along with the destruction day by day of natural ecological environment, also further cause the scarcity of resources of medicinal plant simultaneously.Biotechnology flourish for the production that fundamentally changes traditional medicinal material provides a brand-new method, plant tissue and cell culture technology then are one of them important means.The medicinal plant Study on tissue culture of China can be traced back to the 1950's.1964, professor Luo Shiwei has at first reported since the achievement in research of ginseng tissue cultivation achieving success, the scientific worker of China is obtaining huge achievement aspect the medicinal plant tissue culture, the tissue culture technique level is also in continuous progress: develop into the liquid continuous culture from solid, liquid, suspension culture, deep layer bulk fermentation as cultural method; Culture materials also goes so far as cell from tissues such as the root of medicinal plant, stem, leaf, flower, embryo, fruit, seed or organ.China obtains the existing roxburgh anoectochilus terminal bud (Anoectochilus formosanus) of medicinal plant, the bletilla striata (Bletilla striata), Crocus sativus (Crocus sativus), dendrobium candidum (Dendrobiumofficinale), the gynostemma pentaphylla (Gynostemma pentaphyllum) of test tube plantlet over nearly 40 years through cultured in vitro, tens of kinds of Folium llicis Latifoliae (Ilex latifolia) etc., wherein great majority are precious medicinal plant (Xue Huai, 2002).
Big seed kiwi fruit (Actinidia macrosperma C.F.Liang) has another name called the valvate actinidia root, it is the local tradition medicinal plant in Zhejiang Province, main product is in Fuyang, Zhejiang, Linan, Hangzhou, rivers and mountains and other places, in Hubei, also there is distribution (Zhejiang flora, 1993) in province such as Jiangxi and Anhui.Its root skin is rufous, is commonly called as " pearls and jewels ".The effect of have removing toxicity for detumescence, dispelling rheumatism.Be used for the treatment of deep abscess clinically, osteomyelitis, arthralgia due to wind-dampness, sore swollen toxin, cirrhosis jaundice ascites etc. among the peoplely are usually used in treating all kinds of tumor in digestive tract (Yao Gan etc., 1989).
Increasingly extensive along with valvate actinidia root's clinical practice, the also corresponding increase of its raw medicinal herbs consumption, only the year usage amount in Zhejiang Province can reach 100 tons, wherein more than halfly be used for oncotherapy, become the Chinese gooseberry of continuing (Chinese gooseberry) root later another cancer therapy drug from Actinidiaceae (Xia Jinpei, 1997; Jin Ruizhi etc., 1998; Next ordinary etc., 2002).But increasing along with valvate actinidia root's clinical practice, its medicinal material consumption also increases sharply year by year.Only usage amount promptly reaches 70 tons Fuyang year, nearly 100 tons of the whole province's year usage amount, therefore as far back as the eighties in 20th century Fuyang big seed kiwi fruit almost run out, the Anhui that main dependence closes on, the supply in Jiangxi, according to our field investigation and local medicinal herb grower reflection, the wild big seed kiwi fruit resource in Zhejiang Province remains little at present, has almost reached the stage of extinction, and closes on the also atrophy promptly even the exhaustion of wild resource in province.If untimely again adopting an effective measure contained the excessive exploitation of resource, improve the effective rate of utilization of big seed kiwi fruit, then may cause the extinction of this species wild resource.Simultaneously, had a strong impact on the quality and the curative effect of this medicine, made orthodox school's " pearls and jewels " almost run out (come all flat, 2002) because the wretched insufficiency of historical reasons and crude drug supply causes the increase of valvate actinidia root's substitute and adulterant.
The report of relevant kiwi fruit group training research is quite a lot of, is the research about edible kiwi fruit breed improvement and germ plasm resource preservation mostly.At present, the successful kiwi fruit kind of group training has Chinese gooseberry (A.chinensis), delicious kiwi fruit (A.deliciosa), actinidia eriantha (A.eriantha), tara vine (A.arguta), Actinidia kolomicta (A.kolomikta) and spends more kiwi fruit (Zhang Yuanji, 1996 such as (A.latifolia); Margarida OM, 1991; Kovae J, 1993; Jia Shuzhong, 1991), do not see the research report that big seed kiwi fruit group is trained as yet.
List of references
Jin Ruizhi, Wang Guoqiang. valvate actinidia root's soup treatment bulging example is released. and the Zhejiang Journal of Traditional Chinese Medicine, 1998,33 (1): 35 merchant's book loyalties are opened merit. spend more the kiwi fruit tissue culture. biology magazine, 1991, (2): 25-26
Come ordinary, Zhang Hongyan. the Zhejiang area Chinese medicine valvate actinidia root progress of commonly seeing. Zhejiang Chinese medicine institute journal, 2002,26 (1): 77-78
Xing Jianmin, Zhao Xiude etc. saussurea medusa bio-reactor suspension culture. Botany Gazette, 2000,42 (1): 98-101.
Xue Huai, Liu Min etc. Chinese medicinal plant Study on tissue culture progress. the plant magazine, 2002 (1): 6-7 thanks to Qi Kun. the medicinal plant tissue culture. Shanghai, 1986, Shanghai science tech publishing house.
Xia Jinpei. valvate actinidia root's soup associating elemene treatment tumor in digestive tract clinical experience in late period. Shenzhen combination of Chinese tradiational and Western medicine magazine, 1997,7 (4): 29-30
Yao Gan, Wang Tie monk. the arrangement of East China Actinidia medicinal plant. traditional Chinese medicine, 1989,12 (2): 15-16 Zheng Guang plants. and the new development of Secondary Metabolism of Plant cell engineering research: the 6th international plant tissue and cell culture meeting are commented. Plant Physiology Communications, 1987 (2): 75-79.
Zhang Yuanji, Qian Yingqian. actinidia eriantha callus induction and plant regeneration. Guangxi science .1994,1 (4): 1-5Margarida OM, Pais M S S.Plant regeneration from protoplasts of long-termcallus cultures of Actinidia deliciosa var.Hayward (kiwifruit) .Plant CellRep, 1991,9 (11): 643-646
Kovae?J.Micropropagation?of?Actinidia?Koaomikta.Plant?Cell?Tissue?OrgCult，1993，35(3)：301-303
The meaning of mentioned abbreviation letter is seen following breviary vocabulary in the literary composition:
MS Murashige and Skoog medium MS medium
NAA α-naphthal acetic acid α-Nai Yisuan
BA 6-benzylaminopurine 6-benzyl aminopurine
GA gibberellic acid gibberellin
ZT zeatin zeatin
IBA indolebutyic acid indolebutyric acid
Summary of the invention
A kind of tissue culture quick propagation culturing method that the purpose of this invention is to provide big seed kiwi fruit.
The step of method is as follows:
1) explant selection and processing: get the branch that big seed kiwi fruit is given birth to semi-lignified then, flowing water is rinsed well, is cut into the 1-2cm stem with bud, and 75% alcohol is handled 30-35sec, sterile water washes down back with behind 0.1% the mercuric chloride sterilization 10-12min, aseptic water washing 4-5 time;
2) inducing culture: above-mentioned aseptic stem with bud is vertically inserted on the inducing culture, cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-1, cultivate 10-15d, axillary bud sprouting obtains 3-5cm and sprouts branch;
3) enrichment culture: above-mentioned sprouting branch is downcut, is divided into the 1-2cm stem with bud, change in the proliferated culture medium, cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-1, cultivate 15-20d, obtain the unrooted tissue cultivating seedling;
4) culture of rootage: above-mentioned unrooted tissue cultivating seedling is downcut 4-5cm, changes in the root media, cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-1, cultivate 15-20d, the plant that obtains taking root;
5) hardening and transplanting: the plant that will take root is taken out, move into phytotron, behind the natural daylight 15-20d in following strong sprout, open container ware lid, allow seedling adapt to natural conditions gradually, behind the hardening 5-7d,, then tissue cultivating seedling is colonizated in the peat soil of high-temperature sterilization: in perlite=2: 1 cultivation matrix with the medium of the careful flush away group of clear water Baconic portion, noting shades preserves moisture, especially 1-10d will accomplish that whole day shades after transplanting, notes water spray when fine, and plant survives after one month.
Described inducing culture is MS+BA 5 μ mol/L+NAA 1 μ mol/L, sucrose concentration 3%, pH value 5.8, agar 0.68%.Proliferated culture medium is MS+BA 2 μ mol/L+GA 1.5 μ mol/L+ZT 4 μ mol/L, sucrose concentration 3%, and pH value 5.8, agar 0.68%, 35d reproduction coefficient are 9-10.Root media is 1/2MS+IBA 2 μ mol/L, and rooting rate can reach 100%, cultivates 35d, average height of seedling 6.28cm, and average transplanting survival rate is 91%.
Advantage of the present invention: the production of (1) big seed kiwi fruit can be carried out under the manual control condition, is not subjected to the restriction of factors such as season, weather conditions and soil, can get rid of the invasion and attack of damage by disease and insect and the influence of residue of pesticide, the quality of the big seed kiwi fruit of strict control.(2) growth rate is fast, and is with short production cycle, and equipment is simple, and floor space is few, can save human and material resources etc., is convenient to batch production production.(3) carry out detoxification treatment in group training process, improve big seed quality of Chinese gooseberries, effectively solve wild big seed quality of Chinese gooseberries instability, the problem that outward appearance and active ingredient are irregular is road, grown place medicinal material and processing sell in the postpartum condition that provides safeguard.(4) this technical method has solved big seed kiwi fruit fast breeding and stable important technical links of taking root thereof, and has reached consistent, and the requirement that reproduction coefficient is high can be applicable to the purpose of suitability for industrialized production.(5) the big seed kiwi fruit seedling of using group culturation rapid propagating technology provided by the invention to obtain carries out artificial cultivation, and it is little to obtain individual difference, and the big seed kiwi fruit finished product of quality homogeneous can provide the raw material of stay in grade for producing cancer therapy drug.(6) can protect the germ plasm resource of big seed kiwi fruit, help protecting big seed kiwi fruit wild resource simultaneously, reduce destruction natural resources.
Embodiment
A kind of tissue culture quick propagation culturing method of big seed kiwi fruit comprises explant selection and processing, inducing culture, and enrichment culture, culture of rootage is transplanted steps such as hardening.In the concrete operations, 1) gets the branch that big seed kiwi fruit is given birth to semi-lignified then, flowing water is rinsed well, be cut into the 1-2cm stem with bud, 75% alcohol is handled 30-35sec, sterilizes behind the 10-12min aseptic water washing 4-5 time behind the aseptic water washing with 0.1% mercuric chloride, cut the part of explant variable color, be inoculated in the MS+BA 5 μ mol/L+NAA 1 μ mol/L medium and carry out inducing culture.Cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-12) cultivate 10-15d, axillary bud sprouting, and obtain 3-5cm and sprout branch, then the sprouting branch that obtains in every bottle is downcut, be divided into the 1-2cm stem with bud, change in the MS+BA 2 μ mol/L+GA1.5 μ mol/L+ZT 4 μ mol/L medium and carry out enrichment culture, cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-1, cultivate 15-20d, can obtain the green healthy and strong unrooted tissue cultivating seedling of 3-5 strain in each blake bottle; 3) again with the unrooted tissue cultivating seedling, be cut into long being transferred on the 1/2MS+IBA 2 μ mol/L medium of 4-5cm and carry out culture of rootage, cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-1, cultivate 15-20d, obtaining the plant 4 that takes root) and the plant that will take root takes out, and moves into artificial greenhouse, behind the natural daylight 15-20d in following strong sprout, opens container ware lid, allows seedling adapt to natural conditions gradually.Behind the hardening 5-7d,, then tissue cultivating seedling is colonizated in the peat soil of high-temperature sterilization: in perlite=2: 1 cultivation matrix with the medium of the careful flush away group of clear water Baconic portion.Note shading and preserves moisture, especially 1-10d will accomplish that whole day shades after transplanting, notes water spray when fine, after plant survives after about one month, can move into the plantation of purpose planting site, and average height of seedling is 6.28cm, and average transplanting survival rate is 91%.
Inducing culture in the above-mentioned technology (MS+BA 5 μ mol/L+NAA 1 μ mol/L), proliferated culture medium (MS+BA 2 μ mol/L+GA 1.5 μ mol/L+ZT 4 μ mol/L) and the pH value of root media (1/2MS+IBA 2 μ mol/L) are 5.8 ± 0.2, agar 0.68%, 20min sterilizes under an atmospheric pressure.
Use the present invention that the stem section of big seed kiwi fruit is inoculated on the MS+BA 5 μ mol/L+NAA 1 μ mol/L medium, 10-15d can sprout axillalry bud, and germination rate is 83.3%.To sprout the stem with bud that the sprouting branch that is cut into 1-2cm, change proliferated culture medium MS+BA 2 μ mol/L+GA 1.5 μ mol/L+ZT4 μ mol/L over to, about every 20d subculture is once on average bred multiple and can be reached 9-10 doubly.When invisible spectro seedling during up to 4-5cm, it is changed in the 1/2MS+IBA 2 μ mol/L root medias, cultivate the whole plant that 30d can be formed with root.
Adopt the present invention of above-mentioned measure, grasped inducing and the technology of growth, two critical stages of strengthening seedling and rooting of axillalry bud in the big seed kiwi fruit tissue culture, successfully solved the technological difficulties in the big seed kiwi fruit tissue-culturing rapid propagation, made this rare important medicinal plant of big seed kiwi fruit realize that fast seedling growing, industrialization cultivation become possibility.Use the present invention can protect this rare wild plant resource simultaneously effectively, reduce the private benign cycle that coyoting, formation sustainable use and reasonable development are coordinated mutually of digging.
Superiority of the present invention is embodied in: the plant tissue quick propagating technology that big seed kiwi fruit is provided, the guardian technique of quick breeding and stable important step such as take root thereof is provided, for the commercialization production of this medicinal plant provides a cover practicable production technology route, can be applied to the technical scale production of big seed kiwi fruit.
The present invention adopts following non-limiting case study on implementation to be further described.
Embodiment 1
The selection of explant: the branch of getting the big seed kiwi fruit that picks up from Hangzhou, midsummer, clean with running water, after flowing water washes half an hour, be cut into the 1-2cm stem with bud, place triangular flask, 75% alcohol is handled 30sec, again with behind the aseptic water washing with behind 0.1% the mercuric chloride sterilization 12min, behind aseptic water washing 4-5 time, be inoculated in respectively under aseptic condition in the inducing culture, pollution rate is 38%.Get the branch that picks up from Hangzhou, winter, by after the same disinfection methods, pollution rate is up to 87%.
Embodiment 2
Get and pick up from mountain area, Fuyang, Hangzhou, the branch of the big seed kiwi fruit in midsummer, clean with running water, after flowing water washes half an hour, be cut into the long stem with bud of 1-2cm, 75% alcohol is handled 30sec, again with sterilizing behind the 12min with 0.1% mercuric chloride behind the aseptic water washing, behind aseptic water washing 4-5 time, under aseptic condition, be inoculated in respectively among the inducing culture MS+BA 5 μ mol/L+NAA 1 μ mol/L.Cultivation temperature 24-26 ℃, every day light application time 16h, intensity of illumination 35 μ molm
-2s
-1, cultivate 10-15d.The sprouting branch that obtains on the inducing culture is downcut, changes increment medium MS+BA 2 μ mol/L+GA3 μ mol/L+ZT 4 μ mol/L over to, cultivation temperature 24-26 ℃, every day light application time 16h, intensity of illumination 35 μ molm
-2s
-1, cultivate 15-20d.Bud propagation multiple reaches 11.6 times, and the plant average height reaches 9.28cm, but the leaf look yellowish green.
Case study on implementation 3
Get and pick up from Hangzhou, the branch and the blade of the big seed kiwi fruit in midsummer, clean with running water, flowing water is cut into stem with bud after washing half an hour, places triangular flask, 75% alcohol was handled 30 seconds, again with behind the aseptic water washing with the sterilization of 0.1% mercuric chloride after 12 minutes, behind aseptic water washing 4-5 time, under aseptic condition, be inoculated in respectively among the inducing culture MS+BA 5 μ mol/L+NAA 1 μ mol/L.Cultivation temperature 24-26 ℃, every day light application time 16h, intensity of illumination 35 μ molm
-2s
-1, cultivated 10-15 days.The sprouting branch that obtains on the inducing culture is downcut, changes proliferated culture medium MS+BA 6 μ mol/L+NAA1 μ mol/L over to, cultivation temperature 24-26 ℃, every day light application time 16h, intensity of illumination 35 μ molm
-2s
-1, cultivated 15-20 days.Bud propagation multiple is 4.27, average height of seedling 3.85cm.
Case study on implementation 4
Get and pick up from Hangzhou, the branch and the blade of the big seed kiwi fruit in midsummer, clean with running water, flowing water is cut into stem with bud after washing half an hour, places triangular flask, 75% alcohol was handled 30 seconds, again with behind the aseptic water washing with the sterilization of 0.1% mercuric chloride after 12 minutes, behind aseptic water washing 4-5 time, under aseptic condition, be inoculated in respectively among the inducing culture MS+BA 5 μ mol/L+NAA 1 μ mol/L.Cultivation temperature 24-26 ℃, every day light application time 16h, intensity of illumination 35 μ molm
-2s
-1, cultivated 10-15 days.The sprouting branch that obtains on the inducing culture is downcut, change proliferated culture medium MS+BA 2 μ mol/L+GA1.5 μ mol/L+ZT 4 μ mol/L over to.Cultivation temperature 24-26 ℃, every day light application time 16h, intensity of illumination 35 μ molm
-2s
-1, cultivated 15-20 days.Change root media 1/2MS+IBA 1 μ mol/L afterwards over to, rooting rate 91.6%, the long 0.9cm of average root.Cultivation temperature 24-26 ℃, every day light application time 16h, intensity of illumination 35 μ molm
-2s
-1, cultivated 15-20 days.The plant that has taken root is taken out, move into phytotron, behind the natural daylight 15-20d in following strong sprout, open container ware lid, allow seedling adapt to natural conditions gradually.Behind the hardening 7d, with the medium of the careful flush away group of clear water Baconic portion, then with seedling planting (peat soil: perlite=2: 1) in the cultivation matrix of the bacterium of going out.Note shading and preserves moisture, will accomplish in the 10d after transplanting that especially whole day shades, note water spray when fine, plant survives after about one month, and average transplanting survival rate is 89%.
Claims (4)
1. a kind of tissue culture quick propagation culturing method of big seed kiwi fruit, step is as follows:
1) explant selection and processing: get the branch that big seed kiwi fruit is given birth to semi-lignified then, flowing water is rinsed well, is cut into the 1-2cm stem with bud, and 75% alcohol is handled 30-35sec, sterile water washes down back with behind 0.1% the mercuric chloride sterilization 10-12min, aseptic water washing 4-5 time;
2) inducing culture: above-mentioned aseptic stem with bud is vertically inserted on the inducing culture, cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-1, cultivate 10-15d, axillary bud sprouting obtains 3-5cm and sprouts branch;
3) enrichment culture: above-mentioned sprouting branch is downcut, is divided into the 1-2cm stem with bud, change in the proliferated culture medium, cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-1, cultivate 15-20d, obtain the unrooted tissue cultivating seedling;
4) culture of rootage: above-mentioned unrooted tissue cultivating seedling is downcut 4-5cm, changes in the root media, cultivation temperature 24-26 ℃, every day light application time 16-18h, intensity of illumination 35 μ molm
-2s
-1, cultivate 15-20d, the plant that obtains taking root;
5) hardening and transplanting: the plant that will take root is taken out, move into phytotron, behind the natural daylight 15-20d in following strong sprout, open container ware lid, allow seedling adapt to natural conditions gradually, behind the hardening 5-7d,, then tissue cultivating seedling is colonizated in the peat soil of high-temperature sterilization: in perlite=2: 1 cultivation matrix with the medium of the careful flush away group of clear water Baconic portion, noting shades preserves moisture, especially 1-10d will accomplish that whole day shades after transplanting, notes water spray when fine, and plant survives after one month.
2. a kind of tissue culture quick propagation culturing method of big seed kiwi fruit according to claim 1 is characterized in that described inducing culture is MS+BA 5 μ mol/L+NAA 1 μ mol/L, sucrose concentration 3%, pH value 5.8, agar 0.68%.
3. a kind of tissue culture quick propagation culturing method of big seed kiwi fruit according to claim 1, it is characterized in that described proliferated culture medium is MS+BA 2 μ mol/L+GA 1.5 μ mol/L+ZT 4 μ mol/L, sucrose concentration 3%, pH value 5.8, agar 0.68%, 35d reproduction coefficient are 9-10.
4. a kind of tissue culture quick propagation culturing method of big seed kiwi fruit according to claim 1, it is characterized in that described root media is 1/2MS+IBA2 μ mol/L, rooting rate can reach 100%, cultivates 35d, average height of seedling 6.28cm, average transplanting survival rate is 91%.
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