CN101485293B - Tissue culture, seedling cultivation and rooting method of Fructus schisandrae - Google Patents
Tissue culture, seedling cultivation and rooting method of Fructus schisandrae Download PDFInfo
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- CN101485293B CN101485293B CN2009103007497A CN200910300749A CN101485293B CN 101485293 B CN101485293 B CN 101485293B CN 2009103007497 A CN2009103007497 A CN 2009103007497A CN 200910300749 A CN200910300749 A CN 200910300749A CN 101485293 B CN101485293 B CN 101485293B
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Abstract
The invention relates to plant propagation technology, in particular to a schizandra chinensis tissue culture seedling raising and rootage method. The method comprises the following steps: obtaining clustered seedlings through the tissue culture technology; intercepting the stems of the seedlings and putting the stems in a rootage culture medium for pretreatment; then taking root in a substrate. The method is characterized by quick propagation, high propagation coefficient, convenient management, low production cost, high stocking percent, short culture cycle, anniversary production and industrialization promotion.
Description
Technical field
The present invention relates to a kind of plant propagation technology, i.e. tissue culture, seedling cultivation and rooting method of Fructus schisandrae.
Background technology
In prior art, fructus schisandrae (Schisandra chinensis (Turcz) Baill) another name Schisandra sphenanthera Rehd.et WilS, Fructus Zanthoxyli Plansipini is the perennial fallen leaves woody climber of Magnoliaceae Schizandra, fruit and seed are famous Chinese medicine, are used as medicine to be used for treating neurasthenia, weak, night sweat etc.; The stem micromicro is made condiment, spices, and fruit juice can be made beverage, fruit wine etc.Fructus schisandrae length can reach more than the 10m, stem skin taupe, and skin is obvious, the sprig brown, rib is arranged slightly, single leaf alternate, matter is thin, is with membranous slightly, handle is elongated, blade oval, long 5-11cm, wide 3-5cm, the anxious point of tip or gradually sharp, the base portion wedge shape, the edge is dredged and is given birth to little pointed tooth, top bottle green, glossy, there is not hair, pubescence, complete stool fragrance, flower unisexuality are arranged when tender on the following arteries and veins.Be distributed in China northeast, ground such as North China; Korea, Soviet Union the Far East Area also has.
Schisandra sphenanthera Rehd.et WilS is as fruit, medicine dual-purpose resource, and DEVELOPMENT PROSPECT is very wide.In " special-protection-by-the-State natural crude drugs species register ", Schisandra sphenanthera Rehd.et WilS is listed in the three-level protective species.Fructus schisandrae is genuine northern medicine, but mainly exists the provenance proterties various aborning, output and active constituent content instability, and problems such as difficult quality control, product can only be decided to be wild gradeless and uniformly-priced goods, cultivation gradeless and uniformly-priced goods.The seed propagation aberration rate is very high, is difficult to obtain the seedling of proterties unanimity, thereby can not be used for standardization production; Division propagation then exists breeding slow, easily degenerates and propagates disease, also is unsuitable for the large-scale production in modern times.
The fructus schisandrae mode of breeding comprises seed seedling-raising, cottage propagation and tissue culture, adopts the method for tissue culture to produce the basis that the Schisandra sphenanthera Rehd.et WilS seedling is realization batch production production, but does not have accomplished so far aborning.Wu Xiuju equals the tissue culture of reporting the medicinal plant fructus schisandrae in 1999, makes explant with the tender stem of band axillalry bud, is basal medium with the MS medium, at additional 6-BA
2.0+ ZT
0.1Mg/L, the induced bud effect is good; At additional 6-BA
2.0+ NAA
0.2+ ZT
0.1Mg/L, the rate of increase of bud is best; Rooting efficiency the best when adding 0.2mg/L NAA does not have reproducible results but provide data to experimentize with reference to related data.
Summary of the invention
Technical solution of the present invention is: tissue culture, seedling cultivation and rooting method of Fructus schisandrae is to utilize the tissue culture technique seedling that obtains to grow thickly, and intercepts its stem section carry out preliminary treatment in root media, takes root in matrix afterwards.
Described root media is 1/8MS+IAA0.8mg/L+KT0.50mg/L, preliminary treatment condition of culture: temperature 20-24 ℃, and dark place reason 48h.
Described matrix is any or the combination in peat soil, thin river sand, humus, perlite or the vermiculite.Be preferably peat soil and thin river sand (peat soil: thin river sand=4: 1).Matrix is selected to be not limited thereto, and can also be the conventional substrate that plant propagation is used.
Advantage of the present invention is: 1, the method for tissue culture of the present invention is bred Schisandra sphenanthera Rehd.et WilS, and breeding is fast, and reproduction coefficient is big, convenient management, and production cost is low.2, can access that proterties is stable, neat, consistent, the Schisandra sphenanthera Rehd.et WilS seedling of no damage by disease and insect, it is better to have genetic stability, and speed is fast, and the planting percent height is economical and practical, workable, characteristics such as simple and direct easy grasp.3, cultivation cycle is short, but produces in the anniversary, helps realizing batch production production and automation control.
Below in conjunction with embodiment embodiments of the present invention are described in further detail.
Embodiment
Tissue culture, seedling cultivation and rooting method of Fructus schisandrae:
One, utilizes conventional organization culture technique (as axillalry bud grow thickly differentiation or callus differentiation) to obtain the Schisandra sphenanthera Rehd.et WilS seedling of growing thickly, intercept its stem section.
Two, grow thickly seedling preliminary treatment and culture of rootage
In the 1/8MS medium intersection proportioning of variable concentrations plant growth regulating substance to the fructus schisandrae preliminary treatment after the influence of taking root of stem section
1 materials and methods
1.1 the screening of bud seedling rooting preliminary treatment medium
With 1/8MS is medium, IAA, IBA and the KT of additional variable concentrations.Sucrose 10g/L, agar 8.0g/L, pH6.5 screens the only preliminary treatment medium of taking root.
Condition of culture: temperature (22 ± 2) ℃, dark place reason 48h.
1.2 sand culture is taken root
To from blake bottle, take out through the tender stem section of 48h dark place reason, the residual agar of flush away stem segment base portion in containing 1.00mg/L Sandofan solution, implant then in the thin river sand that 15 times of Sandofan solution disinfections are crossed, cover with moisture-heat preservation with the good plastic film of printing opacity, humidity remains on 80%, and temperature is controlled at (18 ± 2) ℃, ventilation at noon every day 10min, throw off film behind the 10d, spray clear water every day sooner or later each 1 time, 30d (my god) back statistics rooting rate.
1.3 method
Employing 1/8MS is a minimal medium, plant growth regulating substance IAA, IBA and the KT of additional variable concentrations, and growth hormone IAA, IBA concentration all are controlled between the 0.10-1.00mg/L, and KT concentration is controlled between the 0.10-0.50mg/L.By trial test as can be known, auxin concentration surpasses the expansion of 1.00mg/L seedling base portion and produces the callus knurl, continuing to cultivate root sends from the callus knurl, such seedling is when transplanting, root comes off from the seedling base portion with the callus knurl, and transplanting survival rate is almost nil, may be root with the fiber of seedling stem dredge tissue be connected imperfect due to, the an amount of KT of medium supplemented more additional take root soon and many, may be that the KT of debita spissitudo helps that the root original hase forms rapidly and the quantity of formation is more.For the rooting rate that improves fructus schisandrae and the test efficiency of rooting rate, adopt uniform design, each handles inoculation seedling number is 30, selects U for use
10(10
8) even table (seeing Table 1).
Table 1U
10(10
3) factor and level design
2 data analyses
Data analysis is adopted uniform Design (Uniform Design) software with processing.Investigate IAA, IBA and KT concentration intersect proportioning to the influence of rooting rate (in table and equation, X
1Be IAA concentration, X
2Be IBA concentration, X
3Be KT concentration, Y is the rooting rate of sand culture 30d behind the preliminary treatment 48h, the results are shown in Table 2).
Table 2U
10(10
3) uniform Design experimental establishment and result
Data are handled through uniform Design software, get regression equation Y=33.5+46.6X
1+ 0.635X
2+ 24.7X
3, sample size N=10, significance α=0.01, coefficient of multiple correlation R=0.9989, test value Ft=876.3, critical value F
(0.01,3,6)=9.780, Ft＞F
(0.01,3,6), regression equation is remarkable.Each member of equation is carried out significance test as can be known: test value F
(2)=0.3447, critical value F
(0.01,1,6)=13.75, F
(2)≤ F
(0.01,1,6), this member of equation is not remarkable, needs to reject, and rejects significantly to experimentize behind the member of equation, will test the gained data through uniform Design software handle regression equation Y=34.1+46.4X
1+ 24.3X
3, coefficient of multiple correlation R=0.9988, test value F
t=1450, critical value F
(0.01,2,7)=9.547, F
t＞F
(0.01,2,7), regression equation is remarkable.In like manner, each member of equation is carried out significance test as can be known: each member of equation is all remarkable.The optimum combination of obtaining Y according to regression equation is: X
1=1.00, X
3=0.50, on this combination foundation, try to achieve optimal solution: y=92.7, by formula Y=y ± u
a.S calculate the optimal value interval and be estimated as Y=92.7 (± 2.93), i.e. 89.77-95.63%.Do demonstration test once more with optimum combination, with the tender shoots base portion directly the bud seedling of regeneration cut into one section on a leaf and be inoculated on the 1/8MS medium of additional 1.00mg/LIAA and 0.50mg/L KT and carry out from blake bottle, taking out behind the preliminary treatment 48h, carrying out sand culture by 1.2 methods takes root, 30d (my god) after will dig out gently in the stem Duan Miaocong sand, the statistics rooting rate, show that by the result of taking root the stem section more than 93% has produced root, the generation that has lateral root and root development all normal.Make discovery from observation, there are three kinds of situations at the position that root takes place: first kind of situation is that the root of 60% stem section directly sends from stem section tangent plane, and radical reaches more than 4～5, and root length does not wait between 0.50～2.50cm; Second kind of situation is that 30% stem Duan Gencong seedling is done nearly base portion place and sent, and radical mostly is 2 greatly, and root is long for about 1.00cm, but root is sturdy and lateral root is more; The third situation is that 5% shoot root sends from the callus knurl that stem segment base portion produces, and radical mostly is 4～5 greatly, and root is long for about 2.00cm.The root that the third situation takes place is when transplanting, and root comes off from the seedling base portion with the callus knurl, and transplanting survival rate is almost nil, should not be selected to transplanting, and this may be the too high generation of auxin concentration.Therefore, the optimal value that experiment obtains is improved, promptly with the IAA of concentration 0.10,0.20,0.30,0.40,0.50,0.60,0.70,0.80,0.90,1.00mg/L, additional 0.50mg/LKT carries out 10 combinations and tests once more, experimental result proves, combination IAA0.80mg/L+KT0.50mg/L pretreated shoot root is all done nearly base portion from stem section tangent plane or seedling and is sent, be auxin concentration too high due to, rooting rate reaches more than 95%.In the estimation interval scope, and all bigger than all experiment Y values.As seen, the optimum preliminary treatment medium of taking root of fructus schisandrae is: 1/8MS+IAA0.8mg/L+KT0.50mg/L.
3. conclusion
Experimental result shows that the trans ZT 2.20mg/L of medium ER+ induces effect better to the direct regeneration bud seedling of fructus schisandrae tender shoots base portion, and trans ZT is in 1.50～2.20mg/L concentration range, and bud seedling inductivity raises along with the increase of trans ZT concentration.The fructus schisandrae plant regeneration is taked the stem section of the direct regeneration bud seedling of tender shoots base portion to carry out carrying out conventional method after the preliminary treatment of 48h in the medium of 1/8MS+IAA0.8mg/L+KT0.50mg/L to take root, it is better to have genetic stability, speed is fast, the planting percent height, economical and practical, workable, advantages such as simple and direct easy grasp have reached the re-set target of high efficiency quick breeding.Simultaneously, this experimental applications uniform Design is handled, is analyzed data and tests and shortened groping the cycle of medium greatly, and the factorial seedling growth that result of study can be the fructus schisandrae fine quality provides foundation.
Three, the Schisandra sphenanthera Rehd.et WilS test-tube plantlet forms the administrative skill of finished product seedling and the biological characteristic research of finished product seedling growth
Because the test-tube plantlet long term growth is in culture vessel, isolate with external environment, formed a unique ecosystem, this ecosystem compare with external environmental condition have high temperature, high humidity, the low light level, aseptic four big-differences.As long as effectively make test-tube plantlet adapt to this species diversity, just can make it transplant survival.For improving the adaptability of test-tube plantlet, short its stalwartness finally reaches and improves the test-tube seedling transplanting survival rate.We are from temperature, light, humidity and have or not environmental factor such as assorted bacterium to consider to carry out hardening.Domestication begins in a couple of days, with the culture environment conditional likelihood; Later stage is similar to the expectation cultivation condition, progressively adapts to.
1. materials and methods
1.1 experiment material
Carry out preliminary treatment 48h through last method and carry out young plant that sand culture takes root for observing material.
1.2 method
Take out pretreated budlet seedling from test tube, wash the medium that root is adhered off, all remove, in case residual medium grows assorted bacterium with running water.Plant into filling matrix and (select the turfy soil or the humus soil of the certain fertility of sand and tool for use, water permeable in advance) seedling-cultivating tray in, in matrix, insert an aperture with a thick waddy of chopsticks when planting, then seedling is inserted, notice that seedling is tenderer, prevent to injure, plant the back matrix compacting around the seedling, plant the light degenerate water in back, the fastener shed guarantees that air humidity reaches more than 90%.After in seedling-cultivating tray, cultivating 30d, with digging out gently in the stem Duan Miaocong sand, in conjunction with field planting in nutritive cube, the statistics rooting rate.All plant that directly takes root in bud seedling base portion and bud seedling stem are colonizated in the nutritive cube of 20 * 15cm, the alms bowl mesostroma is the peat soil and the thin river sand (4: 1) of sterilizing through 400 times of 50% carbendazim wettable powder, with obscurity is 50% black sunshade net shading, regularly pouring.Treat that seedling is tending towards throwing off when obviously growing the sunshade net, random labelling 5 strains, per 10～15d carries out one-shot measurement, comprises plant height, stem foot diameter, labeled leaf length of a film and wide etc., averages.
Transplant the place: greenhouse by solar heat, plastic tunnel, culturing room.
2, result: see Table the per hundred strain bud seedling rooting condition survey tables of 3 tables 3
| Condition of culture | Otch expands to take root counts (individual) | Otch is directly taken root and is counted (individual) | Basal part of stem is taken root and is counted (individual) | Planting percent (%) |
| Greenhouse by solar heat | 5 | 80 | 12 | 97 |
| Plastic tunnel | 3 | 52 | 41 | 90 |
| Culturing room | 7 | 48 | 38 | 93 |
The bud seedling that the tender shoots base portion is directly regenerated cuts into one section on a leaf, is inoculated on the 1/8MS medium of additional 1.00mg/L IAA and 0.50mg/LKT, carries out sand culture again behind the cultivation 48h, and planting percent is up to more than 90%.Since less for the examination material quantity, be easy to management, three significant differences that sound does not have.With the greenhouse by solar heat is best.
Table 4 becomes seedling growing state application form
Plant season of growth of growing in nutritive cube can reach the commercial seedling standard, and root system is flourishing fully, plant height 15-25cm, and stem foot diameter 0.2-0.3cm can be used for Schisandra sphenanthera Rehd.et WilS and builds the garden.But the semi-finished product seedling of breeding after the early July culture of rootage, because the decline of environmental temperature, can continued growth though root system then occurs, immature, based on the water root, be difficult to survive the winter, on the other hand, growth rate is slow behind the axillary bud sprouting, the young sprout cripetura, the branch children is tender.
Claims (4)
1. tissue culture, seedling cultivation and rooting method of Fructus schisandrae is characterized in that utilizing tissue culture technique to obtain to grow thickly seedling, and intercepts its stem section carry out preliminary treatment in root media, takes root in matrix afterwards; Root media is 1/8MS+IAA0.8mg/L+KT0.50mg/L; Preliminary treatment condition of culture: temperature 20-24 ℃, dark place reason 48h.
2. according to the described tissue culture, seedling cultivation and rooting method of Fructus schisandrae of claim 1, it is characterized in that described matrix is any or the combination in peat soil, thin river sand, humus, perlite or the vermiculite.
3. according to the described tissue culture, seedling cultivation and rooting method of Fructus schisandrae of claim 2, it is characterized in that described matrix is peat soil and thin river sand.
4. according to the described tissue culture, seedling cultivation and rooting method of Fructus schisandrae of claim 3, it is characterized in that described matrix is peat soil: thin river sand=4: 1.
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