CN100482067C - Fast tissue culture propagation process for azalea and camellia - Google Patents
Fast tissue culture propagation process for azalea and camellia Download PDFInfo
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- CN100482067C CN100482067C CNB2006101614665A CN200610161466A CN100482067C CN 100482067 C CN100482067 C CN 100482067C CN B2006101614665 A CNB2006101614665 A CN B2006101614665A CN 200610161466 A CN200610161466 A CN 200610161466A CN 100482067 C CN100482067 C CN 100482067C
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Abstract
The fast tissue culture propagation process for azalea and camellia includes the following steps: cutting tender stem segment from the maternal plant in April or May; soaking in alcohol solution, sterilizing in HgCl2 solution and cutting the stem tip; inoculating the stem tip in primary culture medium to grow cespitose bud and cutting single bud seedling after the bud grows to 2-3cm length; culturing the single bud seedling in secondary culture medium inside a sealed container to proliferate test tube seedling repeatedly; culturing the test tube seedling in rooting culture medium inside sealed container to grow new root to 0.3-0.5cm long, and transplanting. The process can provide seedling of azalea and camellia continuously.
Description
Technical field
The present invention relates to a kind of camellia tissue culture and rapid propagation method.Be specifically related to a kind of fast tissue culture propagation process for azalea and camellia.Platymiscium propagation technique field.
Background technology
Camellia azalea originates in China; be found in provincial nature reserve, goose phoenix screen-like mountain peak, Chinese Guangdong province county in spring in 1986; the first-grade state protection plant, cultivation " four seasons cuckoo tea " or " cuckoo tea " by name is Theaceae Camellia Red Hill tea group light arnotto camellia subgroup plant.
Camellia azalea is a kind of very rare Woody flower, perpetual bloom, and blade is more more smooth than common camellia, does not have sawtooth, even under the condition of 38 ℃ of temperature, still completely sets safflower, has very high ornamental value and as the germ plasm resource value.Because it is high that its ecological condition to existence requires, natural storage is less, only is grown in a narrow and small zone in the original producton location, have 2000 during discovery surplus strain.At present many enterprises and R﹠D institution are carrying out introducing a fine variety of camellia azalea and are breeding test." Guangdong forestry science and technology " 03 phase in 2003 has been reported " tests of camellia azalea cutting propagation " such as Zhang Yan, " Guangdong forestry science and technology " 03 phase in 2004 has been reported " tests of camellia azalea grafting and breeding " such as Li Yunshu, and the extra large gardening Co., Ltd of cloud peak, Wenzhou City camellia research institute, Jinhua city flower etc. has also carried out the research of camellia azalea grafting and breeding.By the R﹠D institution over 20 years tackling key problem, graft survival rate can reach more than 90% at present, but cuttage survival rate only about 40%.So at present during the artificial propagation camellia azalea, grafting has become the main path of camellia azalea breeding, and propagation by grafiting is subjected to many influences such as scion, operant level and grafting season, the camellia azalea limited amount of breeding, because wild resource is very limited, also be difficult to commercialize again.Have only to have solved manually numerously soon, enlarge the planting number of camellia azalea, set up the business development planting base, just can make this precious resources obtain the most effective protection and exploitation, realize the true value of its resource.Patent publication No. 85102805,2003124233.2 discloses Camellia nitidissima tissue-culturing rapid propagation medium.And fast tissue culture propagation process for azalea and camellia yet there are no report.
Summary of the invention
The objective of the invention is to overcome above-mentioned deficiency, a kind of fast tissue culture propagation process for azalea and camellia is provided, have deficiency, both protected the original producton location resource, produce the camellia azalea seedling again continuously to overcome above-mentioned propagation method.
The object of the present invention is achieved like this: a kind of fast tissue culture propagation process for azalea and camellia is characterized in that it comprises following processing step:
Step 1, explant selection
April is to May, and the clip camellia azalea has just been extracted tender tip stem section soon out from the maternal plant;
Step 2, explant are handled
Alcoholic solution with 70～75% soaked 30～40 seconds, and aseptic water washing repeatedly drains the water, and puts into 0.1%～0 again) .15%HgCl
2Sterilized in the solution 8～10 minutes, sterile water repeatedly washes, and cuts stem apex again, and is standby;
Step 3, initial culture
The stem apex that step 2 is got, be seeded on the initial culture base, carry out initial culture, initial culture intensity of illumination 1000～15001x, 23 ± 2 ℃ of cultivation temperature, incubation time 40 ± 5 days, form the bud of growing thickly like this around the stem apex base portion gradually, when the bud clump grows to 2～3cm, be cut into single bud, standby;
Step 4, successive transfer culture
The single bud immigration that step 3 is downcut is equipped with in the airtight container of subculture medium, carries out successive transfer culture, expand numerous test-tube plantlet, 21 ± 2 ℃ of cultivation temperature, light application time 14 ± 1 hours/day, medium pH value 5.8～6.0 was cultivated 40～45 days, form the long budling of 5～6cm with 4～5 stipes, for the propagation clone, be cut into the segment of band stipes, each budling is divided into the 2-3 section, per 40～45 days subcultures like this once expand numerous test-tube plantlet later on.
Step 5, culture of rootage
Step 4 is expanded numerous test-tube plantlet takes out, immigration is equipped with in the closed container of root media, carry out culture of rootage, test-tube plantlet is intensity of illumination 1500～20001x in root media, 23 ± 2 ℃ to 25～35 days of cultivation temperature, when test-tube plantlet root in medium grew to the new root of 0.3～0.5cm, bottle outlet was transplanted.
The tissue culture and rapid propagation method of camellia azalea of the present invention, the described initial culture based component of step 3 is: MS+ZT
0.2～0.8+ BA
1-2+ NAA
0.5～1+ PVP2～3g/1+2～3% sucrose; The described successive transfer culture based component of step 4 is MS+ZT
0.2～0.8+ BA
1-2+ 2ip
1～2+ IBA
0.05～0.1+ PVP2～3g/1+2～3% sucrose; The described culture of rootage based component of step 5 is 1/2MS+NAA
0.7+ IBA
0.5+ BA
0.01+ 15% sucrose+AC0.8g/l.
It is low that the tissue-culturing rapid propagation seedling-cultivating method of camellia azalea of the present invention has solved original reproduction coefficient, grafting speed is slow, and be subjected to the difficult problem of grafting seasonal effect, test-tube plantlet monthly increment coefficient is reached about 2～3, can reach test-tube plantlet about 2～3 strains from explant propagation in 1 year.So both protect maternal plant, can satisfy the large-scale production demand of camellia azalea again.
Embodiment
The present invention relates to a kind of camellia azalea tissue-culturing rapid propagation seedling-cultivating method, it comprises that explant is drawn materials and processing, initial culture, successive transfer culture, culture of rootage processing step, and its concrete steps are as follows:
Step 1, explant selection
April is to the continuous sunny in May, and the clip camellia azalea has just been extracted tender tip stem section soon out from the maternal plant.
Step 2, explant are handled
The stem section of choosing in the step 1 is shaken washing 6～8 minutes earlier in 1% washing powder water, flowing water washed 30 minutes under water tap again, gauze with cleaning blots water, and then in aseptic superclean bench, be placed in 70% the alcoholic solution soaking disinfection 30 seconds, aseptic water washing drains the water for 2～3 times, changes 0.1%HgCl again over to
2Sterilized in the solution 8～10 minutes, aseptic water washing 5～6 times, each 3～5 minutes, blot the moisture on surface with sterile gauze, cut stem apex again, standby.
Step 3, initial culture
Stem apex with step 2 gets is seeded in MS+ZT
0.5+ BA
2+ NAA
0.8On the initial culture base of+PVP3g/1+2% sucrose, carry out initial culture, initial culture intensity of illumination 1000～15001x, 23 ℃ of cultivation temperature, incubation time 40～45 days forms the bud of growing thickly so gradually around the stem apex base portion, when the long 2～3cm of bud clump, be cut into single bud, standby.
Step 4, successive transfer culture
The single bud immigration that step 3 is downcut is equipped with in the airtight container of subculture medium, carries out successive transfer culture, expand numerous test-tube plantlet, expanding numerous test-tube plantlet subculture medium is MS+ZT
0.5+ BA
1+ 2ip
2+ IBA
0.1+ PVP3g/l+3% sucrose, 21 ± 2 ℃ of cultivation temperature, light application time 14 ± 1 hours/day, medium pH value 5.8～6.0, cultivated 40～45 days, and can form the long budling of 5～6cm, be the propagation clone with 4～5 left and right sides stipes, can be cut into the segment of band stipes, each budling may be partitioned into the 2-3 section.Per 40～45 days subcultures like this once expand numerous test-tube plantlet later on.
Step 5, culture of rootage
Treat that step 4 expands numerous test-tube plantlet and breeds after some, choose the high test-tube plantlet of 1.5～2cm, move into and be equipped with in the closed container of root media, carry out culture of rootage.The culture of rootage based component is 1/2MS+NAA
0.7+ IBA
0.5+ BA
0.01+ 15% sucrose+AC0.8g/l.Test-tube plantlet is intensity of illumination 1500～20001x in root media, 23 ℃ of cultivation temperature, and root original hase projection appears in the seedling root after 20 days, to about 35 days, when test-tube plantlet root in medium grows to the new root of the white of 0.3-0.5cm, carries out test-tube seedling transplanting.The test-tube seedling transplanting administrative skill is identical with conventional woody tissue cultivating seedling.
Annotate:
ZT---zeatin
BA---6-benzyladenine
NAA---α-Nai Yisuan
IBA---indolebutyric acid
2ip---6-γ, γ-dimethylallyladenine
PVP---polyvinylpyrrolidone
AC---active carbon
ZT
0.2～0.8Expression ZT0.2 ~ 0.8mg/l.All the other measurement units are identical.
Claims (1)
1, a kind of fast tissue culture propagation process for azalea and camellia is characterized in that it comprises following processing step:
Step 1, explant selection
April is to May, and the clip camellia azalea has just been extracted tender tip stem section soon out from the maternal plant;
Step 2, explant are handled
Alcoholic solution with 70～75% soaked 30～40 seconds, and aseptic water washing 2～3 times drains the water, and puts into 0.1%～0.15%HgCl again
2Sterilized in the solution 8～10 minutes, aseptic water washing 5～6 times cuts stem apex again, and is standby;
Step 3, initial culture
The stem apex that step 2 is got, be seeded on the initial culture base, carry out initial culture, initial culture intensity of illumination 1000～1500lx, 23 ± 2 ℃ of cultivation temperature, incubation time 40 ± 5 days, form the bud of growing thickly like this around the stem apex base portion gradually, when the bud clump grows to 2～3cm, be cut into single bud, standby;
Step 4, successive transfer culture
The single bud immigration that step 3 is downcut is equipped with in the airtight container of subculture medium, carries out successive transfer culture, expand numerous test-tube plantlet; 21 ± 2 ℃ of cultivation temperature, light application time 14 ± 1 hours/day, medium pH value 5.8～6.0 was cultivated 40～45 days, formed the long budling with 4～5 stipes of 5～6cm, for the propagation clone, was cut into the segment of band stipes, and each budling is divided into the 2-3 section; Per 40～45 days subcultures like this once expand numerous test-tube plantlet later on;
Step 5, culture of rootage
Step 4 is expanded numerous test-tube plantlet takes out, immigration is equipped with in the closed container of root media, carry out culture of rootage, test-tube plantlet is intensity of illumination 1500～2000lx in root media, 23 ± 2 ℃ of cultivation temperature are cultured to 25～35 days, when test-tube plantlet root in medium grows to the new root of 0.3～0.5cm, bottle outlet is transplanted
The described initial culture based component of step 3 is: MS+ZT 0.2～0.8mg/l+BA 1～2mg/l+NAA 0.5～1mg/l+PVP 2～3g/l+2～3% sucrose;
The described successive transfer culture based component of step 4 is MS+ZT 0.2～0.8mg/l+BA 1～2mg/l+2ip 1～2mg/l+IBA 0.05～0.1mg/l+PVP2～3g/l+2～3% sucrose;
The described culture of rootage based component of step 5 is 1/2MS+NAA 0.7mg/l+IBA 0.5mg/l+BA 0.01mg/l+15% sucrose+AC0.8g/l.
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| CN100482067C true CN100482067C (en) | 2009-04-29 |
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| CN103535282B (en) * | 2013-11-01 | 2014-12-31 | 重庆文理学院 | Tissue culture medium of camellia azalea and propagation method thereof |
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