CN102960250B - Method for efficiently forming seedlings in vitro by utilizing stem segments of rhododendron plants in Changbai Mountains - Google Patents

Abstract

The invention relates to plant propagation and in particular relates to a method for efficiently forming seedlings in vitro by utilizing stem segments of rhododendron plants in the Changbai Mountains. Taking rhododendron aureum as example, the method comprises the steps of: (1) collecting branches of the rhododendron aureum in late October, carrying out low-temperature dormancy on the branches, carrying out water planting to promote the germination and the growth of lateral buds and cutting in a form of one segment with one leaf after the treatment to be used as explants for later use, wherein the culture medium for the rooting of the stem segments and the germination and the growth of the lateral buds comprises the components of: the basic culture medium+ 0.13-0.14mg.L-1 of KT (kinetin)+ 0.05-0.08mg.L-1 of IBA (Indole Butyric Acid)+ 0.02-0.05mg.L-1 of NAA (Naphthyl Acetic Acid)+ 2.70-2.90mg.L-1 of GA(Gibberellin)3. According to the method, the process is simple, the cost is low, the operability is strong and the capability of improving the seedling producing efficiency is high, so that the method can be directly applied to industrial seedling cultivation of the rhododendra.

Description

A kind of in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section that utilizes

technical field

The present invention relates to a kind ofly, utilize in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section.

Background technology

In the prior art, Rhododendron in Changbai platymiscium comprise Rhododendron aureum georgi ( rhododendron chrysanthumpall.), short fruit cuckoo ( rhododendron brachycarpumd. Don), according to white cuckoo ( rhododendron micranthumturcz.), capitate rhododendron branchlet and leaf ( rhododendron parvifoliumadams.), large word cuckoo ( rhododendron schlippenbachiimaxim.), rhododendron dauricum ( rhododendron dauricuml.), korean rhododendron ( rhododendron mucronulatumturcz.), felt cuckoo ( rhododendron confertissimumnakai) and Rhododendron redowskianum ( rhododendron redowskianummaxim.) etc. have 9 kinds; be Jilin Province and lay special stress on protecting plant; wherein Rhododendron aureum georgi and Rhododendron redowskianum are the Precious And Rare Plants of China country three-level protective; short fruit cuckoo is Changbaishan area Precious And Rare Plants; Rhododendron aureum georgi, felt cuckoo, the white cuckoo of photograph and capitate rhododendron branchlet and leaf are evergreen shrubss; short fruit cuckoo is evergreen dungarunga, and rhododendron dauricum is half evergreen shrubs.Rhododendron aureum georgi, Rhododendron redowskianum and felt cuckoo distribution height above sea level reach 2500 meters high, belong to alpine rose.Rhododendron aureum georgi, large word cuckoo, higher according to white cuckoo, capitate rhododendron branchlet and leaf, korean rhododendron and rhododendron dauricum complete stool volatile oil content are essential oil and medicinal plant.These 9 kinds of cuckoos can be all garden plants by artificial introduction and acclimatization.

Above 9 kinds of cuckoos are to water and soil conservation and maintain the ecological balance and play an important role, and are the germ plasm resource of azalea breeding.Dan Qi China distributes very narrow, only on Changbai Mountain, has larger population to distribute, and other area is only fragmentary distribution, and because people disorderly adopt disorderly and dig, wild resource suffers great threat.These 9 kinds of Rhododendron seeds are tiny, and germination rate is low, and the seedling survival rate forming after sprouting is extremely low; Cottage propagation needs a large amount of Rhododendron simsii Planch branches, wild resource is destroyed greatly, and rooting rate and transplanting survival rate extremely low, development and utilization is extremely restricted.

At present, the in-vitro propagate report of 9 kinds of azaleas is more, by callus again the approach such as Bud Differentiation seedling, Axillary shoot proliferation and Direct Regeneration there is growth coefficient advantages of higher, but still have that sugarcane explants through callus induction breaks up again, Axillary shoot proliferation and the Direct Regeneration cycle longer, further rooting rate slowly again, step is complicated, cost is high, poor operability to obtain indefinite bud, and the shortcomings such as degeneration and variation very easily occur long-term propagation continuously.

Summary of the invention

The object of the invention is to provide for above-mentioned deficiency that a kind of method is feasible, step is simple and direct, cost is low and reproduction speed is fast utilizes in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section.

Technical solution of the present invention is: a kind of in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section that utilizes, and its step is as follows:

(1) late October gathers Rhododendron aureum georgi branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of Rhododendron aureum georgi stem section is: minimal medium+KT 0.13～0.14 mgL ^-1+ IBA 0.05～0.08 mgL ^-1+ NAA 0.02～0.05 mgL ^-1+ GA ₃2.70～2.90 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

(1) late October gathers short fruit cuckoo branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of short fruit cuckoo stem section is: minimal medium+KT 0.09～0.12 mgL ^-1+ IBA 0.03～0.07 mgL ^-1+ NAA 0.04～0.06 mgL ^-1+ GA ₃2.80～3.00 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

(1) late October gathers according to white cuckoo branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) according to the take root medium of simultaneously axillary bud sprouting growth of white cuckoo stem section, be: minimal medium+KT 0.17～0.25 mgL ^-1+ IAA 0.20～0.25 mgL ^-1+ NAA 0.02～0.05 mgL ^-1+ GA ₃2.30～2.60 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

(1) late October gathers capitate rhododendron branchlet and leaf branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of capitate rhododendron branchlet and leaf stem section is: minimal medium+KT 0.08～0.12 mgL ^-1+ IBA 0.15～0.20 mgL ^-1+ NAA 0.03～0.05 mgL ^-1+ GA ₃2.00～2.30 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

(1) late October gathers large word cuckoo branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) the take root medium of axillary bud sprouting growth simultaneously of large word cuckoo stem section is: minimal medium+KT 0.07～0.10 mgL ^-1+ IAA 0.20～0.25 mgL ^-1+ NAA 0.01～0.03 mgL ^-1+ GA ₃2.20～2.50 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

(1) late October gathers rhododendron dauricum branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of rhododendron dauricum stem section is: minimal medium+KT 0.04～0.07 mgL ^-1+ NAA 0.02～0.04 mgL ^-1+ GA ₃1.00～1.50 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

(1) late October gathers korean rhododendron branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of korean rhododendron stem section is: minimal medium+KT 0.03～0.06 mgL ^-1+ NAA 0.03～0.07 mgL ^-1+ GA ₃0.09～1.30 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

(1) late October gathers felt cuckoo branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of felt cuckoo stem section is: minimal medium+KT 0.08～0.12 mgL ^-1+ IAA 0.12～0.15 mgL ^-1+ NAA 0.05～0.07 mgL ^-1+ GA ₃1.80～2.00 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

(1) late October gathers Rhododendron redowskianum branch, and branch is carried out to athermobiosis, and temperature is controlled at-10～-5 ℃, takes out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid after 60 d; When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol of volume fraction, rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of Rhododendron redowskianum stem section is: minimal medium+KT 0.17～0.20 mgL ^-1+ IAA 0.14～0.18 mgL ^-1+ GA ₃1.30～1.80 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

Advantage of the present invention is: 1 and with respect to the callus approach such as Bud Differentiation seedling, Axillary shoot proliferation and Direct Regeneration again, not only seedling speed is fast, reproduction coefficient is high for the modes of reproduction that tender stem-root and axillary bud sprouting growth are synchronously carried out, and inheritance stability, step is simple and direct, cost is low, workable, can greatly improve seedling production efficiency, belong to forming seedling through one step culture, can directly apply to azalea factorial seedling growth.2, calculate by statistics, 9 kinds of azalea plantlet survival rates can reach 90.3%～98.5%.

Below in conjunction with embodiment, embodiments of the present invention are described in further detail.

Embodiment

Utilize in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, its step is as follows:

The experiment that the Rhododendron aureum georgi forming seedling through one step culture of take is carried out is example:

1 materials and methods

1.1 materials and processing

Late October; in Changbai Mountain National Nature Reserve, adopt Rhododendron aureum georgi branch, and put into refrigerator-freezer athermobiosis after branch is wrapped with plastic sack, temperature is controlled at-10～-5 ℃; after 60 d, take out and terminal bud is removed, short its sprouting of lateral bud growth of water planting in the Gibberellins solution of 300 times of liquid.When sprouting of lateral bud grows to 2.50 cm, fresh and tender lateral bud is cut, remove tender leaf and petiole, with 75% ethanol (volume fraction), rinse and wash 60 s, move to and in saturated liquor natrii hypochloritis, soak 8 min, aseptic water washing 10 times, aseptic filter paper blots surface moisture, removes that after the tissue of ethanol and clorox poison wound, to be cut into 1 section, 1 leaf standby as explant.

1.2 Rhododendron aureum georgi stem sections are taken root, and axillary bud sprouting is simultaneously grown, high-frequency plant is regenerated and hardening

Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride (VB ₁), 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.Medium supplemented kinetin KT (0.03～0.010 mgL ^-1), indolebutyric acid IBA (0.10～0.45 mgL ^-1), methyl α-naphthyl acetate NAA (0.06～0.13 mgL ^-1) and gibberellin GA ₃(1.40～2.60 mgL ^-1), agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, the tender stem section of Rhododendron aureum georgi is at periodicity of illumination 9 hd ^-1, cultivate under intensity of illumination 1000 lx and temperature (23 ± 2) ℃ condition.Speed and rooting rate and axillary bud sprouting growth rate and the length of for improving the tender stem section of Rhododendron aureum georgi, taking root, test by Uniform Design method, selects U ₁₃(13 ⁴) evenly show, each processes 10 tender stem sections of inoculation, repeats screening Rhododendron aureum georgi stem section is taken root and axillary bud sprouting is grown kinetin, indolebutyric acid, methyl α-naphthyl acetate and gibberellin best concentration ratio 3 times.Stem section is cultivated growth length after 35 d statistics rooting rates and axillary bud sprouting.Rooting rate=(the stem section sum of the stem hop count/inoculation of taking root) * 100%, stem segment with axillary buds length is in 1 cultivation cycle process, the length after stem segment with axillary buds germination and growth (not comprising former stem segment length).

Treat that stem section is taken root and axillary bud sprouting grows to 3.50 cm, when axillalry bud sends 6～8 leaves, on superclean bench, open blake bottle, stay 1 leaf to cut seedling axillalry bud dry, cut into after 1 section, 1 leaf seedling is dry again, again little stem section is transferred to stem section is taken root and axillary bud sprouting growth medium in, calculate every bottle in each stem section take root and average propagation multiple and cycle of axillary bud sprouting growth.

When Rhododendron aureum georgi test-tube plantlet, breed while growing to 2.00～2.50 cm to the quantity of needs and plantlet, plantlet is taken out from blake bottle, (concentration is 2 mgL to be placed on Sandofan solution ^-1) in clean the agar on root, plantlet is planted in the mixed-matrix of the turfy soil of sterilizing through carbendazim (150 times of liquid), rot pine needle and river sand (2:3:1), what covering light transmission was good carries out moisture-heat preservation without dripping a plastic film, controlling temperature is (18 ± 2) ℃, keep relative moisture between 70%～75%, 13 hd ^-1natural lighting, when humidity temperature at too high or noon raises, film is opened to the osculum ventilation of take a breath, during humidity reduction, can spray in the morning proper amount of clear water.

2 results and analysis

2.1 KT, IBA, NAA and GA ₃the impact that concentration intersection proportioning is taken root on Rhododendron aureum georgi stem section

Data (table 1) can obtain regression equation after uniform Design software analysis is processed y=81.8+221 x ₁-17.5 x ₂-111 x ₃, significance α=0.05, multiple correlation coefficient r=0.9536, residual standard deviation s=2.0600, test value f _t =30.12 ﹥ critical values f _(0.05,3,9)=3.863, regression equation has significance, and kinetin, indolebutyric acid and methyl α-naphthyl acetate have significance to the Rooting effect of stem section, and gibberellin is not remarkable to the Rooting effect of stem section.It is known to the contribution margin of taking root and contribution rate by calculating kinetin, indolebutyric acid and methyl α-naphthyl acetate, u ₁=256, u ₁/ u=66.4%; u ₂=29.5, u ₂/ u=7.66%; u ₃=48.7, u ₃/ u=12.6%, but from contribution rate numerical value, the impact that kinetin is taken root on stem section much larger than indolebutyric acid and methyl α-naphthyl acetate, indolebutyric acid and methyl α-naphthyl acetate gap are little, illustrate that indolebutyric acid and methyl α-naphthyl acetate are all in the Rhododendron aureum georgi stem section indispensable important function of having taken root.Because kinetin and rooting rate are proportionate, and indolebutyric acid and methyl α-naphthyl acetate and rooting rate are negative correlation.Conjecture mass concentration is higher than 0.10 mgL ^-1kinetin, indolebutyric acid and methyl α-naphthyl acetate mass concentration are respectively lower than 0.10 mgL ^-1with 0.06 mgL ^-1time, rooting rate may be higher.For verifying the possibility of this conjecture, take again kinetin as 0.10,0.11,0.12,0.13,0.14 and 0.15 mgL ^-1, indolebutyric acid mass concentration is 0.10,0.09,0.08,0.07,0.06,0.05,0.04,0.03,0.02,0.01 and 0.00 mgL ^-1, methyl α-naphthyl acetate mass concentration is 0.06,0.05,0.04,0.03,0.02,0.01 and 0.00 mgL ^-1carry out the demonstration test of 11 levels, repeat 3 times.1.1 and 1.2 methods are pressed in concrete operations, found that, kinetin mass concentration is (0.12～0.14 mgL ^-1), indolebutyric acid mass concentration is (0.03～0.08 mgL ^-1) and methyl α-naphthyl acetate mass concentration be (0.02～0.05 mgL ^-1) time, the tender stem section of Rhododendron aureum georgi rooting rate is the highest, and average rooting rate reaches 99.9%.All higher than the rooting rate of listed 13 processing of table 1.

Table 1 the U that the tender stem section of Rhododendron aureum georgi is taken root and axillary bud sprouting somatotropin screens ₁₃(13 ⁴) experimental scheme and result

Table1 U ₁₃(13 ⁴)test design and result of hormone for rooting and axillary bud growing of stems of Rhododendron chrysanthum

2.2 KT, IBA, NAA and GA ₃the impact of concentration intersection proportioning on Rhododendron aureum georgi stem segment with axillary buds germination and growth

Data (table 1) can obtain regression equation after uniform Design software analysis is processed y=-0.270+13.7 x ₁--1.27 x ₂-0.729 x ₄, significance α=0.05, multiple correlation coefficient r=0.8996, residual standard deviation s=0.1860, test value f _t =12.73 ﹥ critical values f _(0.05,3,9)=3.863, regression equation has significance, and kinetin, indolebutyric acid and gibberellin have significance to the impact of the tender stem segment with axillary buds germination and growth of Rhododendron aureum georgi, and the impact of methyl α-naphthyl acetate stem segment with axillary buds germination and growth is not remarkable.In like manner, calculate kinetin, indolebutyric acid and gibberellin known to the contribution margin of stem segment with axillary buds germination and growth and contribution rate, u ₁=0.947, u ₁/ u=71.5%; u ₂=0.193, u ₂/ u=14.5%; u ₄=0.779, u ₄/ u=58.8%, illustrate that kinetin is greater than gibberellin, and the contribution much larger than indolebutyric acid to stem segment with axillary buds germination and growth, and indolebutyric acid is the indispensable key factor of Rhododendron aureum georgi stem segment with axillary buds germination and growth, because kinetin and gibberellin and stem segment with axillary buds germination and growth are proportionate, and indolebutyric acid and stem segment with axillary buds germination and growth are negative correlation.Infer thus, the mass concentration of kinetin and gibberellin is respectively higher than 0.10 mgL ^-1with 2.60 mgL ^-1, and lower than 0.10 mgL ^-1indolebutyric acid may stem segment with axillary buds germination and growth better.For checking, infer, take again kinetin as 0.10,0.11,0.12,0.13,0.14 and 0.15 mgL ^-1, indolebutyric acid mass concentration is 0.10,0.09,0.08,0.07,0.06,0.05,0.04,0.03,0.02,0.01 and 0.00 mgL ^-1, gibberellin is 2.60,2.70,2.80,2.90 and 3.00 mgL ^-1carry out the demonstration test of 11 levels, repeat 3 times.Found that, through the cultivation of 35 d, kinetin mass concentration is at 0.13～0.15 mgL ^-1, indolebutyric acid mass concentration is at 0.05～0.09 mgL ^-1with gibberellin mass concentration at 2.70～2.90 mgL ^-1in scope, Rhododendron aureum georgi stem segment with axillary buds germination and growth is all better, and the average growth length after axillary bud sprouting is 3.06 cm, all larger than the axillalry bud length of listed 13 processing of table 1.

From above experimental result, owing to affecting factor and the content range that the tender stem section of Rhododendron aureum georgi takes root, be that minimal medium, kinetin mass concentration are (0.12～0.14 mgL ^-1), indolebutyric acid mass concentration is (0.03～0.08 mgL ^-1) and methyl α-naphthyl acetate mass concentration be (0.02～0.05 mgL ^-1); And stem segment with axillary buds germination and growth major influence factors and content range be medium, kinetin (0.13～0.15 mgL ^-1), indolebutyric acid mass concentration is (0.05～0.09 mgL ^-1) and gibberellin mass concentration be (2.70～2.90 mgL ^-1).

Therefore, can determine that the take root medium of simultaneously axillary bud sprouting growth of Rhododendron aureum georgi stem section is: minimal medium+KT (0.13～0.14 mgL ^-1)+IBA (0.05～0.08 mgL ^-1)+NAA (0.02～0.05 mgL ^-1)+GA ₃(2.70～2.90 mgL ^-1).The average rooting rate 99.9% of tender stem section, the average growth length after axillary bud sprouting is 3.06 cm.

In like manner, can obtain the take root medium of simultaneously axillary bud sprouting growth of other 8 kinds of cuckoo stem sections as follows respectively:

The take root medium of simultaneously axillary bud sprouting growth of short fruit cuckoo stem section is: minimal medium+KT (0.09～0.12 mgL ^-1)+IBA (0.03～0.07 mgL ^-1)+NAA (0.04～0.06 mgL ^-1)+GA ₃(2.80～3.00 mgL ^-1).The average rooting rate 99.7% of tender stem section, the average growth length after axillary bud sprouting is 3.17 cm.

According to the take root medium of axillary bud sprouting growth simultaneously of white cuckoo stem section, be: minimal medium+KT (0.17～0.25 mgL ^-1)+IAA (0.20～0.25 mgL ^-1)+NAA (0.02～0.05 mgL ^-1)+GA ₃(2.30～2.60 mgL ^-1).The average rooting rate 99.4% of tender stem section, the average growth length after axillary bud sprouting is 3.22 cm.

The take root medium of simultaneously axillary bud sprouting growth of capitate rhododendron branchlet and leaf stem section is: minimal medium+KT (0.08～0.12 mgL ^-1)+IBA (0.15～0.20 mgL ^-1)+NAA (0.03～0.05 mgL ^-1)+GA ₃(2.00～2.30 mgL ^-1).The average rooting rate 99.5% of tender stem section, the average growth length after axillary bud sprouting is 2.96 cm.

The take root medium of axillary bud sprouting growth simultaneously of large word cuckoo stem section is: minimal medium+KT (0.07～0.10 mgL ^-1)+IAA (0.20～0.25 mgL ^-1)+NAA (0.01～0.03 mgL ^-1)+GA ₃(2.20～2.50 mgL ^-1).The average rooting rate 99.9% of tender stem section, the average growth length after axillary bud sprouting is 2.87 cm.

The take root medium of simultaneously axillary bud sprouting growth of rhododendron dauricum stem section is: minimal medium+KT (0.04～0.07 mgL ^-1)+NAA (0.02～0.04 mgL ^-1)+GA ₃(1.00～1.50 mgL ^-1).The average rooting rate 99.5% of tender stem section, the average growth length after axillary bud sprouting is 3.30 cm.

The take root medium of simultaneously axillary bud sprouting growth of korean rhododendron stem section is: minimal medium+KT (0.03～0.06 mgL ^-1)+NAA (0.03～0.07 mgL ^-1)+GA ₃(0.09～1.30 mgL ^-1).The average rooting rate 99.0% of tender stem section, the average growth length after axillary bud sprouting is 3.37 cm.

The take root medium of simultaneously axillary bud sprouting growth of felt cuckoo stem section is: minimal medium+KT (0.08～0.12 mgL ^-1)+IAA (0.12～0.15 mgL ^-1)+NAA (0.05～0.07 mgL ^-1)+GA ₃(1.80～2.00 mgL ^-1).The average rooting rate 99.6% of tender stem section, the average growth length after axillary bud sprouting is 2.80 cm.

The take root medium of simultaneously axillary bud sprouting growth of Rhododendron redowskianum stem section is: minimal medium+KT (0.17～0.20 mgL ^-1)+IAA (0.14～0.18 mgL ^-1)+GA ₃(1.30～1.80 mgL ^-1).The average rooting rate 99.8% of tender stem section, the average growth length after axillary bud sprouting is 2.77 cm.

2.3 9 kinds of cuckoo high frequency plant regenerations are set up

By 1.2 methods, 9 kinds of cuckoos are on average by 12 stem sections of every bottle graft kind, it within average 28 days, is 1 cycle, each stem section on average can on average cut into 7 sections of calculating in 1 cultivation cycle, have every year n the cycle (9 kinds of cuckoos are 12 cycles every year) to expand numerous, a year production cuckoo seedling number is: ∑ is produced test-tube plantlet=12 * 7 per year ⁿstrain, visible the present invention can meet the needs of the 9 kinds of azalea factorial seedling growths in Changbai Mountain.

The hardening of 2.4 9 kinds of cuckoo plantlets and transplanting

According to the method in 1.2, when seeding propagation is after needs quantity, the healthy and strong plantlet of all choosing more than 3.00 cm carries out acclimatization and transplants, calculates by statistics, and 9 kinds of azalea plantlet survival rates can reach 85.3%～88.5%.

Claims (9)

1. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using the tender stem section of Rhododendron aureum georgi bough water planting lateral bud standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of Rhododendron aureum georgi stem section is: minimal medium+KT 0.13～0.14 mgL ^-1+ IBA 0.05～0.08 mgL ^-1+ NAA 0.02～0.05 mgL ^-1+ GA ₃2.70～2.90 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

2. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using the tender stem section of short fruit cuckoo bough water planting lateral bud standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of short fruit cuckoo stem section is: minimal medium+KT 0.09～0.12 mgL ^-1+ IBA 0.03～0.07 mgL ^-1+ NAA 0.04～0.06 mgL ^-1+ GA ₃2.80～3.00 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

3. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using according to white cuckoo bough water planting lateral bud tender stem section standby as explant;

(2) according to the take root medium of simultaneously axillary bud sprouting growth of white cuckoo stem section, be: minimal medium+KT 0.17～0.25 mgL ^-1+ IAA 0.20～0.25 mgL ^-1+ NAA 0.02～0.05 mgL ^-1+ GA ₃2.30～2.60 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

4. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using the tender stem section of capitate rhododendron branchlet and leaf bough water planting lateral bud standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of capitate rhododendron branchlet and leaf stem section is: minimal medium+KT 0.08～0.12 mgL ^-1+ IBA 0.15～0.20 mgL ^-1+ NAA 0.03～0.05 mgL ^-1+ GA ₃2.00～2.30 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

5. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using the tender stem section of large word cuckoo bough water planting lateral bud standby as explant;

(2) the take root medium of axillary bud sprouting growth simultaneously of large word cuckoo stem section is: minimal medium+KT 0.07～0.10 mgL ^-1+ IAA 0.20～0.25 mgL ^-1+ NAA 0.01～0.03 mgL ^-1+ GA ₃2.20～2.50 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

6. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using the tender stem section of rhododendron dauricum bough water planting lateral bud standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of rhododendron dauricum stem section is: minimal medium+KT 0.04～0.07 mgL ^-1+ NAA 0.02～0.04 mgL ^-1+ GA ₃1.00～1.50 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

7. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using the tender stem section of korean rhododendron bough water planting lateral bud standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of korean rhododendron stem section is: minimal medium+KT 0.03～0.06 mgL ^-1+ NAA 0.03～0.07 mgL ^-1+ GA ₃0.09～1.30 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

8. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using the tender stem section of felt cuckoo bough water planting lateral bud standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of felt cuckoo stem section is: minimal medium+KT 0.08～0.12 mgL ^-1+ IAA 0.12～0.15 mgL ^-1+ NAA 0.05～0.07 mgL ^-1+ GA ₃1.80～2.00 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.

9. utilize an in vitro efficient seedling establishment method of Rhododendron in Changbai platymiscium stem section, it is characterized in that step is as follows:

(1) using the tender stem section of Rhododendron redowskianum bough water planting lateral bud standby as explant;

(2) the take root medium of simultaneously axillary bud sprouting growth of Rhododendron redowskianum stem section is: minimal medium+KT 0.17～0.20 mgL ^-1+ IAA 0.14～0.18 mgL ^-1+ GA ₃1.30～1.80 mgL ^-1; Medium supplemented agar powder 7.8 gL ^-1, add sucrose 10.0 gL ^-1, regulating pH value to 5.6, tender stem section is at periodicity of illumination 9 hd ^-1, cultivate stem section 35 d under intensity of illumination 1000 lx and 23 ± 2 ℃ of conditions of temperature; Minimal medium composition and content: 87 mgL ^-1(NH ₄) ₂sO ₄, 375 mgL ^-1nH ₄nO ₃, 180 mgL ^-1kNO ₃, 320 mgL ^-1caCl ₂2H ₂o, 245 mgL ^-1mgSO ₄7H ₂o, 436 mgL ^-1kH ₂pO ₄; 18.4 mgL ^-1feSO ₄7H ₂o, 24.9 mgL ^-1na ₂eDTA2H ₂o; 12.4 mgL ^-1mnSO ₄4H ₂o, 7.2 mgL ^-1znSO ₄7H ₂o, 5.7 mgL ^-1h ₃bO ₃, 0.32 mgL ^-1kI, 0.05 mgL ^-1na ₂moO ₄2H ₂o; 0.3 mgL ^-1thiamine hydrochloride VB ₁, 0.25 mgL ^-1nicotinic acid, 75 mgL ^-1inositol, 0.25 mgL ^-1glycine.