CN102217539A - Isolated rooting culture method for fir clone - Google Patents
Isolated rooting culture method for fir clone Download PDFInfo
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Abstract
本发明公开了一种杉木无性系离体生根培养方法，该方法包括以下步骤：选取杉木优良无性系原株的树干的基部萌条为组织培养材料，接入继代增殖培养基进行继代增殖培养，诱导分化出不定芽，然后将不定芽转入MS培养基上进行伸长培养；不定芽长到2～3cm时，转入诱导生根培养基培养，得生根试管苗；移栽生根试管苗，选用黄土、泥炭土3：1比例混合后用作移栽基质。与现有技术中的杉木组培方法相比，本发明的杉木无性系离体生根培养方法优势在于：有效解决了杉木不同优良无性系在组织培养条件下组培苗的生根困难问题，实现建立杉木稳定高效的组培体系，生根率最高可达到100％（洋020），为加快杉木组培苗产业化发展提供技术支撑。
The invention discloses a method for in vitro rooting culture of a Chinese fir clone, which comprises the following steps: selecting the basal shoots of the trunk of the original plant of an excellent Chinese fir clone as a tissue culture material, and inserting it into a subculture proliferation medium for subculture multiplication Cultivate, induce and differentiate adventitious buds, and then transfer the adventitious buds to MS medium for elongation culture; when the adventitious buds grow to 2-3 cm, transfer to the rooting induction medium for cultivation to obtain rooting test-tube plantlets; transplant rooting test-tube plantlets , use loess and peat soil in a ratio of 3:1 and use it as a transplanting substrate. Compared with the Chinese fir tissue culture method in the prior art, the method for in vitro rooting culture of Chinese fir clones of the present invention has the advantages of effectively solving the difficult problem of rooting of tissue culture seedlings of different excellent Chinese fir clones under tissue culture conditions, and realizing the establishment of The stable and efficient tissue culture system of Chinese fir has a rooting rate of up to 100% (Yang 020), which provides technical support for accelerating the industrialization of Chinese fir tissue culture seedlings.
Description
Technical field
The present invention relates to the China fir vegetative propagation technique, be specifically related to the stripped culture of rootage method of a kind of China fir clone.
Background technology
China fir (Cunninghamia lanceolata) is the evergreen megaphanerophyte of gymnosperm Taxodiaceae (Taxodiaceae), property happiness warm and moist weather, and growth is rapid, and trunk is perfectly straight, up to 30m, the diameter of a cross-section of a tree trunk 1.3 meters above the ground 2.5～3m.The China fir material is light and soft, careful, texture is straight, easily processing; For usefulness such as building, bridge, shipbuilding, electric welding, mine timber, wooden Chinese toons, and can make raw materials such as papermaking, weaving; Bark and root, leaf are used as medicine, and can dispel pathogenic wind and remove dampness, astringing to arrest bleeding; The seed oil-containing is about 20%, for soap system.
China fir is China's main reproducting tree species in south and most important commodity commerical tree species, and cultivation history is long, and its afforestation area and stand all occupy the first place of China forest plantation.Along with the improving constantly of management level, breeding obtains paying much attention to strong sprout in recent years.South China fir producing region seed selection a large amount of excellent Chinese fir clones, and utilize the tissue culture rapid propagating technology that these choiceness have been carried out asexual grow seedling afforestation, obtained significant effect of increasing production.But because different excellent Chinese fir clones are in the otherness of character inheritance and physiological foundation of taking root, cause different China fir clones under same group of training prescription condition success rate and efficient aspect have very big difference, greatly influenced good China fir clonal tissue culture seedling and cultivated and apply.
Summary of the invention
Goal of the invention: at the deficiencies in the prior art, the purpose of this invention is to provide the stripped culture of rootage method of a kind of China fir clone, and then the difficult problem of taking root of the different choiceness of solution China fir tissue cultivating seedling under conditions of tissue culture, the group training system of China fir stability and high efficiency is set up in realization, provides technical support for accelerating the industrialized development of China fir tissue cultivating seedling.
Technical scheme: in order to realize the foregoing invention purpose, the technical solution used in the present invention is as follows:
A kind of China fir clone culture of rootage method that exsomatizes may further comprise the steps:
(1) the base portion coppice shoot of choosing the trunk of excellent Chinese fir ortet is the tissue culture material, inserts the shoot proliferation medium and carries out shoot proliferation and cultivate, and induces to differentiate indefinite bud, indefinite bud is changed on the MS medium extend cultivation then; The shoot proliferation culture medium prescription is 3/4MS+6BA0.3mgL
-1+ NAA0.02mgL
-1+ sucrose 3%;
When (2) indefinite bud of step (1) is elongated to 2～3cm, change the root induction medium culture over to, get rooting tube plantlet; The root induction medium is a minimal medium with 1/2MS or 1/4MS, adds plant growth regulator and 2% sucrose therein; Plant growth regulator is IBA0.05 ~ 0.4mgL
-1, NAA0.05 ~ 0.4mgL
-1, IAA0.1 ~ 1.0mgL
-1, or IBA0.05 ~ 0.4mgL
-1With NAA0.05 ~ 0.4mgL
-1Mixing;
(3) transplantation rooting test-tube plantlet selects for use loess, peat soil 3:1 ratio to mix the back as transplanting medium.
Preferably: in the step (2), the root induction medium is 1/2MS, and plant growth regulator is IBA0.1 ~ 0.4mgL
-1Or NAA0.1 ~ 0.2mgL
-1
Preferably: in rapid (2), the root induction medium is 1/4MS, and plant growth regulator is IBA0.1 ~ 0.2mgL
-1Or NAA0.05 ~ 0.2mgL
-1
Preferably: in the step (2), the root induction medium is 1/2MS, and plant growth regulator is IBA0.05 ~ 0.2mgL
-1With NAA0.05 ~ 0.4mgL
-1Mixing.
Preferably: in the step (2), the root induction medium is 1/4MS, and plant growth regulator is IBA0.05 ~ 0.2mgL
-1With NAA0.05 ~ 0.4mgL
-1Mixing.
Preferably: in the step (3),, rooting tube plantlet put into carry out hardening under the natural lighting, unclamp blake bottle day by day and seal film until removing at last in transplantation rooting test-tube plantlet the last week.
Preferably: in the step (3), the root length of test-tube plantlet is not less than 1cm.
Preferably: in the step (3), the root length of test-tube plantlet is 1～3cm.
In group training process, rooting of vitro seedling is subjected to influence of various factors such as medium, hormone and condition of culture.Generally speaking, rooting of vitro seedling all need carry out in low salt culture medium.This is cause medium osmotic pressure to change because salinity changes, thus influence test-tube plantlet to nutrient absorption and in medium h substance.And the variation of salinity also makes, and nitrogen concentration drops to a favourable level that is fit to take root in the plant body.Among the application, test-tube plantlet rooting efficiency on the medium of 1/4MS in ocean 062, ocean 061, ocean 021, ocean 023 is better, its rooting rate is the highest can to reach 93.3%, 56.7%, 70%, 90% respectively, but the test-tube plantlet in ocean 020 is taken root on the minimal medium of 1/2MS better, and the highest rooting rate is 100%.
The test-tube seedling transplanting survival rate just is to identify root of hair quality and the most effective foundation of root of hair technology, the transplanting survival rate of the application's China fir test-tube plantlet reaches more than 70%, can illustrate that the China fir test-tube plantlet adventive root that the stripped culture of rootage method of China fir clone is cultivated is high-quality.
Beneficial effect: compare with China fir tissue culture method of the prior art, the stripped culture of rootage method advantage of China fir clone of the present invention is: the difficult problem of taking root that efficiently solves the different choiceness of China fir tissue cultivating seedling under conditions of tissue culture, the group training system of China fir stability and high efficiency is set up in realization, rooting rate can reach 100%(ocean 020), provide technical support for accelerating the industrialized development of China fir tissue cultivating seedling, can produce good social benefit, have good economic outlook.
Description of drawings
Fig. 1 is the figure of taking root in ocean 062;
Fig. 2 is the figure of taking root in ocean 020;
Fig. 3 is the figure of taking root in ocean 061;
Fig. 4 is the figure of taking root in ocean 021;
Fig. 5 is the figure of taking root in ocean 023;
Fig. 6 is that test-tube seedling transplanting survives figure.
Embodiment
The present invention is described further below in conjunction with drawings and Examples.
Following examples adopt Yang Kou forest farm, the Fujian excellent Chinese fir clone aseptic seedling of Nanjing Forestry University and Fujian cooperation seed selection, and the application abbreviates each sample respectively as: ocean 062, ocean 020, ocean 061, ocean 023 and ocean 021.These materials originate from the defect individual in the superior families that the second generation genetic improvement of Nan Lin-Fujian cooperation development selects in the works, have carried out the clonal test in more than 10 year after breeding into clone, select the back as the choiceness accelerates multiplication again.All tissue culture materials all originate from the base portion coppice shoot of the trunk of ortet.
Below the condition of culture (removing special indicating) that uses of each embodiment be: all additional agar 0.6% in the medium, pH5.8, illumination (2000lx) 14h/d, about 25 ℃ of cultivation temperature.
The differentiation and the elongation of embodiment 1 bud
Each tissue culture material (ocean 062, ocean 020, ocean 061, ocean 023 and ocean 021) is put into the shoot proliferation medium respectively cultivate, induce to differentiate indefinite bud, the shoot proliferation culture medium prescription adopts 3/4MS+6BA0.3 mgL
-1+ NAA0.02mgL
-1+ sucrose 3%.Will indefinite bud change on the MS medium of no any growth regulator and extend cultivation through inducing differentiation.
Embodiment 2 root inductions
When each indefinite bud length to 2 of embodiment 1～3cm, change it over to root media and cultivate it and take root.In root media, add plant growth regulator and sucrose 2%, observe statistical data.The data statistics index comprises: the induction frequency of adventive root (%)=grow indefinite bud number/inoculation indefinite bud number * 100% of adventive root; The sum of average indefinite radical=adventive root/the induce indefinite bud number of adventive root; The induction frequency of formation ability (the RFC)=average indefinite radical * adventive root of adventive root.
In cultivated in ocean 062, the experimental result of the situation of choosing of root media and plant growth regulator and correspondence thereof was as shown in table 1, and as seen from the table, it is best to take root with the medium of additional NAA, as NAA concentration＜0.2mgL
-1The time, rooting rate becomes positive correlation with NAA concentration; When NAA concentration〉0.2mgL
-1The time, be negative correlation; Equal 0.2mgL and work as NAA concentration
-1The time, rooting rate is up to 46.7%.The medium effect of additional IAA is the poorest.Under the NAA of same concentrations level, rooting rate is apparently higher than the 1/2MS medium in the 1/4MS medium; Under the IBA of same concentrations level, rooting rate is also apparently higher than the 1/2MS medium in the 1/4MS medium.Wherein with the additional NAA0.2mgL of 1/4MS
-1The medium effect is best, and rooting rate is 56.7%, and explant grows fine.
The situation of choosing of foreign 062 root media of table 1 and plant growth regulator and corresponding experimental result table thereof
| Medium and plant growth regulator | Inoculation explant number | Rooting rate | Mean elements (bar) | Adventive root forms ability |
| 1/2MS+NAA0.05 mg·L -1 | 30 | 20 | 2.5 | 0.5 |
| 1/2MS+NAA0.1 mg·L -1 | 30 | 30 | 5.5 | 1.65 |
| 1/2MS+NAA0.2 mg·L -1 | 30 | 46.7 | 4 | 1.87 |
| 1/2MS+NAA0.4 mg·L -1 | 30 | 6.7 | 1.5 | 0.10 |
| 1/2MS+IBA0.05 mg·L -1 | 30 | 3.3 | 1 | 0.03 |
| 1/2MS+IBA0.1 mg·L -1 | 30 | 10 | 1.3 | 0.13 |
| 1/2MS+IBA0.2 mg·L -1 | 30 | 13.3 | 2 | 0.26 |
| 1/2MS+IBA0.4 mg·L -1 | 30 | 6.7 | 1.5 | 0.10 |
| 1/2MS+IAA0.1 mg·L -1 | 30 | 0 | 0 | 0 |
| 1/2MS+IAA0.2 mg·L -1 | 30 | 0 | 0 | 0 |
| 1/2MS+IAA0.5 mg·L -1 | 30 | 26.7 | 3.5 | 0.93 |
| 1/2MS+IAA1.0 mg·L -1 | 30 | 0 | 0 | 0 |
| 1/4MS+NAA0.05mg·L -1 | 30 | 43.3 | 3.1 | 1.34 |
| 1/4MS+NAA0.1mg·L -1 | 30 | 50 | 3.9 | 1.95 |
| 1/4MS+NAA0.2mg·L -1 | 30 | 56.7 | 4 | 2.27 |
| 1/4MS+NAA0.4mg·L -1 | 30 | 13.3 | 2 | 0.26 |
| 1/4MS+IBA0.05mg·L -1 | 30 | 3.3 | 2 | 0.06 |
| 1/4MS+IBA0.1mg·L -1 | 30 | 13.3 | 1.8 | 0.24 |
| 1/4MS+IBA0.2mg·L -1 | 30 | 20.0 | 2.3 | 0.46 |
| 1/4MS+IBA0.4mg·L -1 | 30 | 10 | 1.7 | 0.17 |
| 1/4MS+IAA0.1 mg·L -1 | 30 | 0 | 0 | 0 |
| 1/4MS+IAA0.2 mg·L -1 | 30 | 13.3 | 1.8 | 0.24 |
| 1/4MS+IAA0. 5 mg·L -1 | 30 | 33.3 | 2.7 | 0.90. |
In cultivate in ocean 062, the experimental result of the situation of choosing of root media and plant growth regulator (2 kinds) and correspondence thereof is as shown in table 2, as seen from the table, and when in the 1/2MS medium, adding NAA and IBA simultaneously, China fir adventitious bud rooting rate generally rises, and average rooting rate is 53.9%.Be grown in additional 0.1mgL
-1NAA and 0.4 mgL
-1The rooting rate of the tissue cultivating seedling on the medium of IBA can reach 73.3%, has improved the tissue cultivating seedling rooting rate greatly, and along with the prolongation of time, rooting rate also rises to some extent, and the explant growth conditions is good.Hence one can see that, and NAA and IBA synergy more help the China fir adventitious bud rooting.After indefinite bud inserts root media 20d, there is adventive root to grow on the root media of additional IBA of 1/2MS and NAA, adventive root length to 1 ~ 1.5cm behind the 23d.On the root media of additional separately IBA of 1/2MS, 1/4MS and NAA, the root induction time is wanted 3 ~ 5d in evening, and this explanation NAA and IBA are used, and can obviously shorten rootage duration.
Under the same hormone combination, in the 1/4MS medium, the average rooting rate of explant reaches 82.2%, 53.9% average rooting rate under the 1/2MS medium.At additional 0.05mgL
-1NAA and 0.05 mgL
-1The explant rooting rate is the highest on the medium of IBA, is 93.3%; At additional 0.1mgL
-1NAA and 0.2 mgL
-1Rooting rate is 90% on the medium of IBA, and it is higher that adventive root forms ability, and the plant strain growth state is better than the former.In NAA concentration is 0.05 mgL
-1Average rooting rate is 84.2%; NAA concentration is 0.1mgL
-1The Shi Pingjun rooting rate is 85%, and is in rising trend with the increase rooting rate of IBA concentration, reaches the highest during IBA0.2; NAA concentration is 0.2 mgL
-1The Shi Pingjun rooting rate is 77.5%, reaches the highest when IBA0.2.Hence one can see that, and the best in ocean 062 is taken root for filling a prescription and is 1/4MS+NAA0.1mgL
-1+ IBA0.2mgL
-1, take root figure as shown in Figure 1.
The situation of choosing and the experimental result of foreign 062 root media of table 2 and plant growth regulator (2 kinds)
| Medium and plant growth regulator | Inoculation explant number | Rooting rate | Mean elements (bar) | Adventive root forms ability |
| 1/2MS+NAA0.05mg·L -1+IBA0.05mg·L -1 | 30 | 43.3 | 2.4 | 1.04 |
| 1/2MS+NAA0.05mg·L -1+IBA0.1mg·L -1 | 30 | 53.3 | 1.7 | 0.91 |
| 1/2MS+NAA0.05mg·L -1+IBA0.2mg·L -1 | 30 | 60 | 2.6 | 1.59 |
| 1/2MS+NAA0.05mg·L -1+IBA0.4mg·L -1 | 30 | 56.7 | 2.7 | 1.53 |
| 1/2MS+NAA0.1mg·L -1+IBA0.05mg·L -1 | 30 | 56.7 | 3.8 | 2.15 |
| 1/2MS+NAA0.1mg·L -1+IBA0.1mg·L -1 | 30 | 40 | 1.9 | 0.76 |
| 1/2MS+NAA0.1mg·L -1+IBA0.2mg·L -1 | 30 | 56.7 | 2.8 | 1.59 |
| 1/2MS+NAA0.1mg·L -1+IBA0.4mg·L -1 | 30 | 73.3 | 2.5 | 1.83 |
| 1/2MS+NAA0.2mg·L -1+IBA0.05mg·L -1 | 30 | 33.3 | 3.2 | 1.07 |
| 1/2MS+NAA0.2mg·L -1+IBA0.1mg·L -1 | 30 | 50 | 4.9 | 2.45 |
| 1/2MS+NAA0.2mg·L -1+ IBA 0.2mg·L -1 | 30 | 66.7 | 2.8 | 1.87 |
| 1/2MS+NAA0.2mg·L -1+IBA0.4mg·L -1 | 30 | 56.7 | 1.9 | 1.07 |
| 1/4MS+NAA0.05mg·L -1+IBA0.05mg·L -1 | 30 | 93.3 | 2.3 | 2.15 |
| 1/4MS+NAA0.05mg·L -1+IBA0.1mg·L -1 | 30 | 80 | 2 | 1.6 |
| 1/4MS+NAA0.05mg·L -1+IBA0.2mg·L -1 | 30 | 80 | 3.3 | 2.64 |
| 1/4MS+NAA0.05mg·L -1+IBA0.4mg·L -1 | 30 | 83.3 | 3.4 | 2.83 |
| 1/4MS+NAA0.1mg·L -1+IBA0.05mg·L -1 | 30 | 80 | 2.8 | 2.24 |
| 1/4MS+NAA0.1mg·L -1+IBA0.1mg·L -1 | 30 | 83.3 | 2.5 | 2.08 |
| 1/4MS+NAA0.1mg·L -1+IBA0.2mg·L -1 | 30 | 90 | 2.8 | 2.52 |
| 1/4MS+NAA0.1mg·L -1+IBA0.4mg·L -1 | 30 | 86.7 | 2.6 | 2.25 |
| 1/4MS+NAA0.2mg·L -1+IBA0.05mg·L -1 | 30 | 80 | 2.7 | 2.16 |
| 1/4MS+NAA0.2mg·L -1+IBA0.1mg·L -1 | 30 | 73.3 | 2.4 | 1.76 |
| 1/4MS+NAA0.2mg·L -1+IBA0.2mg·L -1 | 30 | 86.7 | 2.9 | 2.51 |
| 1/4MS+NAA0.2mg·L -1+IBA0.4mg·L -1 | 30 | 70 | 3 | 2.1 |
In cultivate in ocean 020, the situation of choosing of root media and plant growth regulator and corresponding experimental result thereof are as shown in table 3, and be as seen, best with the situation of taking root of explant in the medium that adds IAA from table 2, average rooting rate is 95%, and rhizome junction callus is few; At IAA1.0 mgL
-1Rooting rate reaches 100%, and the bar number of adventive root is many, and the formation ability of adventive root is the highest, is 8.6.The average rooting rate of explant is 70% in the medium of additional NAA, is 0.2 mgL in concentration
-1With 0.4 mgL
-1Two prescriptions in the explant base portion expand, callus is many, the root that bears is thinner and more delicate.At the medium of additional IBA, average rooting rate only is 25.8%.Hence one can see that, and the best root media of foreign 020 genotype is: 1/2MS+IAA 1.0mgL
-1Change ocean 020 indefinite bud of inducing over to 1/4MS+NAA0.1mgL
-1+ IBA0.2mgL
-1In, rooting rate is 100%, 5.3 of mean elements, and the seedling growth conditions is good, as shown in Figure 2.
The situation of choosing of foreign 020 root media of table 3 and plant growth regulator and corresponding experimental result thereof
| Medium and plant growth regulator | Inoculation explant number | Rooting rate | Mean elements (bar) | Adventive root forms ability |
| 1/2MS+NAA0.05mg·L -1 | 30 | 70 | 5.6 | 3.92 |
| 1/2MS+NAA0.1 mg·L -1 | 30 | 80 | 2.7 | 2.16 |
| 1/2MS+NAA0.2 mg·L -1 | 30 | 66.7 | 4.5 | 3.00 |
| 1/2MS+NAA0.4 mg·L -1 | 30 | 63.3 | 5.4 | 3.41 |
| 1/2MS+IBA0.05mg·L -1 | 30 | 13.3 | 2.3 | 0.31 |
| 1/2MS+IBA0.1 mg·L -1 | 30 | 13.3 | 2.3 | 0.31 |
| 1/2MS+IBA0.2 mg·L -1 | 30 | 40 | 3 | 1.2 |
| 1/2MS+IBA0.4 mg·L -1 | 30 | 36.7 | 1.6 | 0.59 |
| 1/2MS+IAA0.1 mg·L -1 | 30 | 86.7 | 5.8 | 5.0 |
| 1/2MS+IAA0.2 mg·L -1 | 30 | 93.3 | 4.3 | 4.0 |
| 1/2MS+IAA0.5mg·L -1 | 30 | 100 | 4.5 | 4.5 |
| 1/2MS+IAA1.0mg·L -1 | 30 | 100 | 8.6 | 8.6 |
In cultivate in ocean 061, the experimental result of the situation of choosing of root media and plant growth regulator and correspondence thereof is as shown in table 4, as seen from the table, ocean 061 genotype is in the minimal medium of 1/2MS, NAA, IBA, the IAA of additional variable concentrations, its rooting rate is all lower, and the highest rooting rate is 36.7%, and average rooting rate only is 22.0%.When adding NAA and IBA simultaneously in the 1/4MS medium, foreign 061 genotype adventitious bud rooting rate rises to some extent, and average rooting rate reaches 50.3%.Being grown in concentration is 0.1mgL
-1NAA and 0.2 mgL
-1Adventitious bud rooting rate on the medium of IBA is up to 56.7%, and it is also the highest that adventive root forms ability, is 1.87, specifically takes root figure as shown in Figure 3.Therefore, ocean 061 genotypic the best is taken root for filling a prescription and is: 1/4MS+NAA0.1mgL
-1+ IBA0.2mgL
-1
The situation of choosing of foreign 061 root media of table 4 and plant growth regulator and corresponding experimental result thereof
| Medium and plant growth regulator | Inoculation explant number | Rooting rate | Mean elements (bar) | Adventive root forms ability |
| 1/2MS+NAA0.05mg·L -1 | 30 | 16.7 | 1.4 | 0.23 |
| 1/2MS+ NAA0.1 mg·L -1 | 30 | 20 | 4 | 0.8 |
| 1/2MS+ NAA0.2 mg·L -1 | 30 | 20 | 2 | 0.4 |
| 1/2MS+ NAA0.4 mg·L -1 | 30 | 13.3 | 2 | 0.26 |
| 1/2MS+IBA0.05mg·L -1 | 30 | 20 | 4.1 | 0.82 |
| 1/2MS+IBA0.1 mg·L -1 | 30 | 26.7 | 2 | 0.53 |
| 1/2MS+IBA0.2 mg·L -1 | 30 | 20 | 5 | 1.0 |
| 1/2MS+IBA0.4 mg·L -1 | 30 | 30 | 5 | 1.5 |
| 1/2MS+IAA0.1 mg·L -1 | 30 | 36.7 | 3.5 | 1.28 |
| 1/2MS+IAA0.2 mg·L -1 | 30 | 10 | 2 | 0.2 |
| 1/2MS+IAA0.5mg·L -1 | 30 | 33.3 | 2.5 | 0.83 |
| 1/2MS+IAA1.0mg·L -1 | 30 | 16.7 | 5.4 | 0.90 |
| 1/2MS+NAA0.05mg·L -1 | 30 | 16.7 | 1.4 | 0.23 |
| 1/4MS+NAA0.05mg·L -1+IBA0.05mg·L -1 | 30 | 46.7 | 2.3 | 1.07 |
| 1/4MS+NAA0.05mg·L -1+IBA0.1mg·L -1 | 30 | 50 | 2 | 1.0 |
| 1/4MS+NAA0.05mg·L -1+IBA0.2mg·L -1 | 30 | 53.3 | 2.8 | 1.49 |
| 1/4MS+NAA0.05mg·L -1+IBA0.4mg·L -1 | 30 | 50 | 3.4 | 1.7 |
| 1/4MS+NAA0.1mg·L -1+IBA0.05mg·L -1 | 30 | 50 | 2.6 | 1.3 |
| 1/4MS+NAA0.1mg·L -1+IBA0.1mg·L -1 | 30 | 53.3 | 2.5 | 1.33 |
| 1/4MS+NAA0.1mg·L -1+IBA0.2mg·L -1 | 30 | 56.7 | 3.3 | 1.87 |
| 1/4MS+NAA0.1mg·L -1+IBA0.4mg·L -1 | 30 | 56.7 | 2.8 | 1.59 |
| 1/4MS+NAA0.2mg·L -1+IBA0.05mg·L -1 | 30 | 46.7 | 2.7 | 1.26 |
| 1/4MS+NAA0.2mg·L -1+IBA0.1mg·L -1 | 30 | 43.3 | 2.4 | 1.04 |
| 1/4MS+NAA0.2mg·L -1+IBA0.2mg·L -1 | 30 | 53.3 | 2.9 | 1.55 |
| 1/4MS+NAA0.2mg·L -1+IBA0.4mg·L -1 | 30 | 43.3 | 3 | 1.30 |
In cultivate in ocean 021, the experimental result of the root media of optimizing and the situation of choosing of plant growth regulator and correspondence thereof is as shown in table 5, as seen, during with IBA, on average rooting rate can reach 59.9% to ocean 021 explant at the NAA of the additional variable concentrations of 1/4MS minimal medium from table 5.When NAA concentration is 0.1 mgL
-1, IBA concentration is 0.2 mgL
-1The time adventitious bud rooting rate be 70%, mean elements is 2.8, it is the strongest that average adventive root forms ability, is 1.96, specifically takes root figure as shown in Figure 4.Therefore, foreign 021 genotype the best is taken root for filling a prescription and is: 1/4MS+NAA0.1mgL
-1+ IBA0.2mgL
-1
The root media that table 5 ocean 021 is optimized and the situation of choosing of plant growth regulator and corresponding experimental result thereof
| Medium and plant growth regulator | Inoculation explant number | Rooting rate | Mean elements (bar) | Adventive root forms ability |
| 1/4MS+NAA0.1mg·L -1+IBA0.05mg·L -1 | 30 | 46 | 2.5 | 1.15 |
| 1/4MS+NAA0.1mg·L -1+IBA0.1mg·L -1 | 30 | 56.7 | 2.4 | 1.36 |
| 1/4MS+NAA0.1mg·L -1+IBA0.2mg·L -1 | 30 | 70 | 2.8 | 1.96 |
| 1/4MS+NAA0.1mg·L -1+IBA0.4mg·L -1 | 30 | 66.7 | 2.6 | 1.73 |
In cultivate in ocean 023, the experimental result of the root media of optimizing and the situation of choosing of plant growth regulator and correspondence thereof is as shown in table 6, as seen, foreign 023 genotype is when the NAA of the additional variable concentrations of 1/4MS minimal medium and IBA from table 6, and average rooting rate reaches 81.7%.At additional NAA0.1 mgL
-1With IBA0.2 mgL
-1Medium on, the adventitious bud rooting rate reaches 90%, mean elements 3.8, adventive root formation ability is 3.42, plant grows fine, and specifically takes root figure as shown in Figure 5.Therefore, ocean 023 genotypic the best is taken root for filling a prescription and is: 1/4MS+NAA0.1mgL
-1+ IBA0.2mgL
-1
The root media that table 6 ocean 023 is optimized and the situation of choosing of plant growth regulator and corresponding experimental result thereof
| Medium and plant growth regulator | Inoculation explant number | Rooting rate | Mean elements (bar) | Adventive root forms ability |
| 1/4MS+NAA0.1mg·L -1+IBA0.05mg·L -1 | 30 | 73.3 | 2.8 | 2.05 |
| 1/4MS+NAA0.1mg·L -1+IBA0.1mg·L -1 | 30 | 83.3 | 2.5 | 2.08 |
| 1/4MS+NAA0.1mg·L -1+IBA0.2mg·L -1 | 30 | 90 | 3.8 | 3.42 |
| 1/4MS+NAA0.1mg·L -1+IBA0.4mg·L -1 | 30 | 80 | 3 | 2.4 |
Embodiment 3
Ocean 061 is inoculated in 1/4MS+NAA0.1mgL
-1+ IBA0.2mgL
-1In the medium, add variable concentrations active carbon (table 7) in medium, cultural method is observed statistics with embodiment 2 behind the 35d.After inserting the active carbon medium, the aseptic seedling growing way a little less than, when 26d, found that just adventive root grows.As shown in Table 7, rooting rate is 46.7 on the medium of active carbon 0.02% adding, constantly descend with the increase rooting rate of concentration of activated carbon, and be 0 at active carbon greater than 0.2% o'clock rooting rate; And the medium rooting rate of non-activity charcoal is 56.7%.As can be known, active carbon is unfavorable for the China fir adventitious bud rooting.
Table 7 active carbon is to ocean 061 influence of taking root
| Active carbon (mg/L) | Inoculation explant number | The explant number of taking root | Rooting rate (%) |
| 0.2 | 15 | 7 | 46.7 |
| 0.5 | 15 | 6 | 40 |
| 1.0 | 15 | 3 | 20 |
| 2.0 | 15 | 0 | 0 |
| 5.0 | 15 | 0 | 0 |
The domestication and the transplanting of embodiment 4 regeneration plants
The rooting tube plantlet of embodiment 2, transplant to put into the last week and carry out hardening under the natural lighting, day by day unclamp blake bottle and seal film until removing at last, in culturing room, unclamp earlier lid, place 2d, move on to indoor then, place 3d about 25 ℃, flush away base portion medium is transplanted in the matrix, covers with Polypropylence Sheet, spray water every day 2 ~ 3 times, keeping humidity, 7d after this will note the temperature and humidity that keeps certain preventing the bad root phenomenon that occurs because of humidity is excessive simultaneously again, open Polypropylence Sheet about 14d, keep certain humidity, spray water every day 1 ~ 2 time, will note simultaneously letting in air.Select for use loess, peat soil 3:1 ratio to mix the back as transplanting medium.(1～3cm) and three types of long root (more than the 3cm), each type is got 20 strains and is tested by the length of root test-tube plantlet to be divided into short root (1cm is following), middle root.Plant into matrix after test-tube plantlet being taken out and cleans the medium of root, keep ventilating and 80 ~ 90% ambient humidity.Routine observation, the survival rate and the growing state of statistics test-tube plantlet behind the 30d, concrete outcome is as shown in table 8.In the table 8, the transplanting survival rate of the rooting tube plantlet that the group training obtains is more than 70%, and root length plantlet of transplant effect when 1～3cm is best, have the new root of part to take place, plant normal growth, basal part of stem have sprouting to take out life, the proof test-tube seedling transplanting survives and begins growth, and concrete the transplanting grown figure as shown in Figure 6.
Table 8 a long table as a result that influences to transplant survival
| Handle | The root type | Handle the strain number | Survive the strain number | The root growth situation | The plant strain growth situation |
| 1 | <1cm | 20 | 14 | Elongation growth | Robust growth |
| 2 | 1~3cm | 20 | 17 | Elongation growth, new root takes place | Basal part of stem grows sprouting, riotous growth |
| 3 | >3cm | 20 | 12 | New root takes place, and part is rotted | Growth is normal |
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