CN107750950B - Efficient breeding method for Tilia miqueliana strains - Google Patents
Efficient breeding method for Tilia miqueliana strains Download PDFInfo
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Abstract
The invention discloses a method for efficiently breeding Tilia miqueliana strains, which comprises the steps of shearing stem segments with axillary buds as explants, sterilizing the surfaces of the stem segments, and inoculating the stem segments into a primary culture medium for culture; cutting axillary buds, transferring the axillary buds into a proliferation culture medium until adventitious buds are induced from the axillary buds; separating adventitious buds into single buds, and then transferring the buds to an elongation culture medium; moving the extended adventitious bud out of the tissue culture room, and hardening the seedling under the natural illumination condition; after washing the elongated buds after hardening, cutting the elongated buds into micro-cut sections with proper sizes, and then carrying out rooting pretreatment; and (3) planting the morphological lower end of the stem section treated by the rooting promoting liquid in a nutrient medium, and performing seedling culture on Tilia miqueliana Maxim plants in a greenhouse. The propagation method combines the extended seedling propagation process, the rooting process and the domestication process of Tilia miqueliana Maxim, expands the propagation coefficient of Tilia miqueliana Maxim, improves the survival rate of tissue culture seedlings, accelerates the seedling propagation process, and is favorable for realizing the industrialized seedling culture of Tilia miqueliana Maxim.
Description
Technical Field
The invention relates to a rapid propagation technology of plants, in particular to a method for efficiently breeding Tilia miqueliana Maxim strains.
Background
Tilia miqueliana Maxim is a Tiliaceae Tilia deciduous tree, a basswood, and a unique tree species in China. The tree shape is beautiful, the posture is ambitious, the tree is full of yellow flowers in summer, the flowers are fragrant and gloomy, the plant diseases and insect pests are few, the resistance to smoke dust and harmful gas is strong, and the tree is an excellent garden ornamental tree species and honey source plant; in addition, the Tilia miqueliana Maxim has excellent wood material, and both flowers and barks can be used as medicines, so that the Tilia miqueliana Maxim is an excellent tree species which integrates the functions of garden appreciation, honey source plants, wood species, medicinal use and the like. Tilia miqueliana is produced in Jiangsu, Zhejiang, Anhui, Jiangxi and other provinces in China, but the population quantity and the individual quantity of Tilia miqueliana are gradually reduced and even in the state of scarce wild resources due to the difficult reproduction and the damaged habitat. Therefore, the research on the propagation technology of Tilia miqueliana is in the forefront.
At present, tilia miqueliana propagation mainly comprises seeds and cutting propagation, but due to the low yield of tilia miqueliana seeds, hard seed shells and deep dormant seeds, the seed germination rate is low, and the seed collection is very difficult, so that the application of the seed propagation in the production of tilia miqueliana is greatly limited. In addition, related researches show that the cutting propagation of tilia miqueliana is very difficult, cuttings are easy to rot, the cutting survival rate is low, and no cutting propagation technology can be directly applied to the production of tilia miqueliana at present. The plant tissue culture technology has the advantages of short breeding period, high breeding coefficient, less needed explant material and the like, and plays an important role in plant breeding. At present, the research on tissue culture breeding of tilia miqueliana has been reported, but most of the stem sections of the seedlings of tilia miqueliana of one to three years are used as materials, the problems of serious pollution, low breeding efficiency, complicated domestication and transplantation process of the tissue culture seedlings in the later period, low survival rate and the like exist in the breeding process, and the requirement of breeding of tilia miqueliana is difficult to meet. Aiming at a plurality of problems in the breeding of tilia miqueliana, the development of a high-efficiency breeding method of a tilia miqueliana strain is urgently needed.
Disclosure of Invention
The invention aims to overcome the defects of the prior art, provides a method for efficiently breeding Tilia miqueliana Maxim strains, and can effectively improve the breeding efficiency and shorten the production period.
The invention is realized by the following technical scheme, and the method comprises the following steps:
(1) taking a branch of perennial Tilia miqueliana as a material, cutting a stem section with axillary buds as an explant, sterilizing the surface, inoculating the explant into a primary culture medium, and culturing for 5-10 days until the axillary buds germinate to be 0.5-1.0 cm in length;
(2) cutting the axillary buds obtained in the step (1), transferring the axillary buds into a proliferation culture medium, and culturing for 20-30 days until adventitious buds with the height of 1-2 cm are induced from the axillary buds;
(3) separating the adventitious buds obtained in the step (2) into single buds, then transferring the single buds to an elongation culture medium, and culturing for 15-20 days until the height of the adventitious buds is 3-5 cm;
(4) moving the adventitious buds stretched in the step (3) out of a tissue culture room, and hardening seedlings for 5-7 days under the condition of natural illumination;
(5) flushing the elongated buds hardened in the step (4) with running water, cutting into micro-cut segments with proper size, and then placing the micro-cut segments in rooting promoting liquid for rooting pretreatment;
(6) and (3) fixedly planting the morphological lower end of the stem section treated by the rooting promoting liquid in the step (5) in a nutrient medium sprayed with the MS basic culture solution, and performing seedling culture on Tilia miqueliana plants in a greenhouse on the principle that the lower end is not overlapped with the lower end.
In the step (1), the stem section with axillary buds of tilia miqueliana is obtained from the non-lignified stem section of the current-year young branch of the wild perennial tilia miqueliana plant.
In a preferred embodiment of the present invention, in the step (1), the primary medium is an MS medium supplemented with 0.1 to 1.0mg/LTDZ, 0.05 to 0.5mg/L ZT, 30g/L sucrose and 7.0g/L agar.
In a preferred embodiment of the present invention, in the step (2), the multiplication medium is MS medium supplemented with 0.5 to 2.0mg/LTDZ, 0.1 to 1.0mg/L ZT, 0.1 to 2.0mg/L IAA, 30g/L sucrose and 7.0g/L agar.
In a preferred embodiment of the present invention, in the step (3), the adventitious bud elongation medium is 1/2MS medium supplemented with 0.2 to 1.0mg/LTDZ, 0.1 to 0.5mg/L IBA, 0.05 to 0.5mg/L GA3, 20g/L sucrose and 7.0g/L agar.
In a preferred embodiment of the present invention, in the steps (1) to (3), the cultivation conditions are 24 ± 2 ℃ at an illumination intensity of 2500 to 3000lx and an illumination period of 13 to 15 hours of illumination/9 to 11 hours of darkness.
In a preferred embodiment of the present invention, in the step (5), the micro-cutting of an appropriate size is performed by cutting the acclimated elongated bud into a stem segment having a length of 0.5 to 2.0cm and having at least 1 axillary bud or terminal bud.
As one preferable mode of the present invention, in the step (5), the rooting pretreatment comprises: and (3) soaking the morphological lower end of the cut segment in rooting promoting liquid containing 200-2000 mg/L spermidine, 100-500 mg/L LIAA and 250-1000 mg/L VC for 10-80 s.
In a preferred embodiment of the present invention, in the step (6), the nutrient medium components are nutrient soil, vermiculite and river sand mixed in a ratio of 4:1: 1.
In a preferred embodiment of the present invention, in the step (6), the greenhouse culture conditions are: the temperature is 24-28 ℃, and the humidity is 80-90%.
Compared with the prior art, the invention has the following advantages: the method takes the current-year branches of wild perennial tilia miqueliana plants as explants for tissue culture regeneration, has convenient material acquisition and rich materials, and can maintain the excellent properties of the parent strain of the tilia miqueliana;
the method directly transplants the elongated Tilia miqueliana tissue culture seedlings into a matrix of a greenhouse for rooting and seedling formation after the tissue culture seedlings are treated by the rooting promoting liquid, simplifies the whole breeding link, improves the quality of the tissue culture seedlings, shortens the production time, saves the tissue culture space and reduces the production cost;
the propagation method combines the extended seedling propagation process, the rooting process and the domestication process of Tilia miqueliana Maxim, expands the propagation coefficient of Tilia miqueliana Maxim, improves the survival rate of tissue culture seedlings, accelerates the seedling propagation process, and is favorable for realizing the industrialized seedling culture of Tilia miqueliana Maxim.
Drawings
FIG. 1 is a diagram of a Tilia miqueliana stem section inoculated in an axillary bud germination medium;
FIG. 2 is a diagram of axillary bud germination of Tilia miqueliana stem;
FIG. 3 is a diagram of axillary bud proliferation of Tilia miqueliana Maxim;
FIG. 4 is a drawing of adventitious bud elongation of Tilia miqueliana Maxim;
FIG. 5 is a diagram of tissue culture micro-stem of Tilia miqueliana Maxim for rooting and breeding;
FIG. 6 is a diagram of micro-stem segments colonized in a nutrient medium;
FIG. 7 is a diagram of micro-stem in vitro rooting and seedling.
Detailed Description
The following examples are given for the detailed implementation and specific operation of the present invention, but the scope of the present invention is not limited to the following examples.
Example 1
The method for efficient breeding of tilia miqueliana strains comprises the following specific steps:
(1) as shown in fig. 1, a current-year branch of wild perennial Tilia miqueliana is taken as a material, and a semi-lignified stem section is cut as an explant; sterilizing the surface, cutting into 1.0 cm-sized pieces with axillary buds, inoculating in primary culture medium, and inducing germination of axillary buds in a constant temperature culture room with temperature of 25 deg.C, illumination intensity of 2500lx, illumination period of 15 h/9 h dark; after culturing for 10 days, the axillary buds germinate to about 1.0cm, and as shown in figure 2, the germination rate of the axillary buds is 100%; wherein the primary culture medium is MS culture medium supplemented with 0.5mg/L TDZ, 0.25mg/L ZT, 30g/L sucrose and 7.0g/L agar.
(2) Cutting the axillary buds obtained in the step (1), transferring the axillary buds to a proliferation culture medium, and performing proliferation culture on the axillary buds in a constant-temperature culture room with the temperature of 25 ℃, the illumination intensity of 2500lx and the illumination period of 15h illumination/9 h darkness; after 30 days of culture, the axillary buds induce adventitious buds with the height of about 2cm, the rate of inducing the adventitious buds is 94.7%, and each explant averagely generates 4.8 adventitious buds as shown in figure 3; wherein the proliferation culture medium is MS culture medium supplemented with 1.0mg/L TDZ, 0.6mg/L ZT, 0.5mg/L IAA, 30g/L sucrose and 7.0g/L agar.
(3) Separating the adventitious buds obtained in the step (2) into single buds, then transferring the single buds to an elongation culture medium, and performing elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 25 ℃, the illumination intensity of 2500lx and the illumination period of 15h illumination/9 h darkness; after 20 days of culture, the buds were elongated to 3-5 cm with 3-5 intact leaves, as shown in FIG. 4, wherein the adventitious bud elongation medium was 1/2MS medium supplemented with 0.75mg/L TDZ, 0.25mg/L IBA, 0.2mg/L GA3, 20g/L sucrose and 7.0g/L agar.
(4) Moving the bud elongated in the step (3) out of a tissue culture room, and hardening the seedling for 7d under the condition of natural illumination;
(5) washing the domesticated elongated bud in the step (4) with flowing water, shearing the domesticated elongated bud into a micro stem section which is at least provided with 1 axillary bud or terminal bud and is about 0.5cm long, as shown in figure 5, and placing the morphological lower end of the cut section in rooting promoting liquid containing 1000mg/L spermidine, 300mg/L IAA and 500mg/L VC for 30s for rooting pretreatment;
(6) planting the morphological lower end of the stem section treated by the rooting promoting liquid in the step (5) in a mixed nutrient substrate (nutrient soil: vermiculite: river sand 4:1:1) sprayed with MS basic culture solution, and performing seedling culture on Tilia miqueliana plants in a greenhouse with the temperature of 26 ℃ and the humidity of 80% on the principle that the lower end is not overlapped with the MS basic culture solution as shown in figure 6; after 4 weeks of culture, the Tilia miqueliana regenerated plant with strong root system is obtained, and the rooting rate of the buds in the nutrient medium is as high as 100 percent, as shown in figure 7.
Example 2
The method for efficient breeding of tilia miqueliana strains comprises the following specific steps:
(1) taking the current-year branch of wild perennial Tilia miqueliana Maxim as a material, and shearing a semi-lignified stem section as an explant; sterilizing the surface, cutting into 1.0 cm-sized sections with axillary buds, inoculating in primary culture medium, and inducing germination of axillary buds in a constant temperature culture room with temperature of 26 deg.C, illumination intensity of 3000lx, and illumination period of 14h illumination/10 h dark; after 8 days of culture, the axillary buds germinate to about 1.0cm, and the germination rate of the axillary buds is 100%; wherein the primary culture medium is MS culture medium supplemented with 1.0mg/LTDZ, 0.5mg/L ZT, 30g/L sucrose and 7.0g/L agar.
(2) Cutting the axillary buds obtained in the step (1), transferring the axillary buds to a proliferation culture medium, and performing proliferation culture on the axillary buds in a constant-temperature culture room with the temperature of 26 ℃, the illumination intensity of 3000lx and the illumination period of 14h illumination/10 h darkness; after culturing for 25 days, inducing adventitious buds with the height of about 2cm by the axillary buds, wherein the induction rate of the adventitious buds is 93.6 percent, and each explant averagely generates 4 adventitious buds; wherein the proliferation culture medium is MS culture medium supplemented with 2.0mg/L TDZ, 1.0mg/L ZT, 2.0mg/L IAA, 30g/L sucrose and 7.0g/L agar.
(3) Separating the adventitious buds obtained in the step (2) into single buds, then transferring the single buds to an elongation culture medium, and performing elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 26 ℃, the illumination intensity of 3000lx and the illumination period of 14h illumination/10 h darkness; after 18 days of culture, the bud is elongated to 3-5 cm and has 3-5 complete leaves, wherein the adventitious bud elongation culture medium is 1/2MS culture medium added with 1.0mg/L TDZ, 0.5mg/L IBA, 0.5mg/L GA3, 20g/L sucrose and 7.0g/L agar.
(4) Moving the bud elongated in the step (3) out of a tissue culture room, and hardening the seedling for 7d under the condition of natural illumination;
(5) washing the domesticated elongated buds in the step (4) with flowing water, shearing the domesticated elongated buds into micro stem sections which are at least provided with 1 axillary bud or terminal bud and about 2cm long, and placing the morphological lower ends of the cut sections in rooting promoting liquid containing 2000mg/L spermidine, 500mg/L IAA and 1000mg/L VC for soaking for 80s for rooting pretreatment;
(6) planting the morphological lower end of the stem section treated by the rooting promoting liquid in the step (5) in a mixed nutrient substrate (nutrient soil: vermiculite: river sand 4:1:1) sprayed with MS basic culture solution, and performing seedling culture on Tilia miqueliana plants in a greenhouse with the temperature of 28 ℃ and the humidity of 90% on the principle of no overlapping; after 4 weeks of culture, the Tilia miqueliana regenerated plant with strong root system is obtained, and the rooting rate of buds in the nutrient medium is up to 100%.
Example 3
The method for efficient breeding of tilia miqueliana strains comprises the following specific steps:
(1) taking the current-year branch of wild perennial Tilia miqueliana Maxim as a material, and shearing a semi-lignified stem section as an explant; sterilizing the surface of the axillary bud, cutting into 1.0 cm-sized sections with axillary buds, inoculating the sections into a primary culture medium, and performing germination induction on the axillary buds in a constant-temperature culture room with the temperature of 23 ℃, the illumination intensity of 2700lx and the illumination period of 13h illumination/11 h darkness; after 5 days of culture, the axillary buds germinate to about 1.0cm, and the germination rate of the axillary buds is 100%; wherein the primary culture medium is MS culture medium supplemented with 0.1mg/L TDZ, 0.05mg/L ZT, 30g/L sucrose and 7.0g/L agar.
(2) Cutting the axillary buds obtained in the step (1), transferring the axillary buds into a proliferation culture medium, and performing proliferation culture on the axillary buds in a constant-temperature culture room with the temperature of 23 ℃, the illumination intensity of 2700lx and the illumination period of 13h illumination/11 h darkness; after culturing for 20 days, inducing adventitious buds with the height of about 2cm by the axillary buds, wherein the induction rate of the adventitious buds is 92.1 percent, and each explant averagely generates 3.8 adventitious buds; wherein the proliferation culture medium is MS culture medium supplemented with 0.5mg/L TDZ, 0.1mg/L ZT, 0.1mg/L IAA, 30g/L sucrose and 7.0g/L agar.
(3) Separating the adventitious buds obtained in the step (2) into single buds, then transferring the single buds to an elongation culture medium, and performing elongation culture on the adventitious buds in a constant-temperature culture room with the temperature of 23 ℃, the illumination intensity of 2700lx and the illumination period of 13h illumination/11 h darkness; after culturing for 15 days, the bud is elongated to 3-5 cm and has 3-5 complete leaves, wherein the adventitious bud elongation culture medium is 1/2MS culture medium added with 0.2mg/L TDZ, 0.1mg/L IBA, 0.2mg/L GA3, 20g/L sucrose and 7.0g/L agar.
(4) Moving the bud elongated in the step (3) out of a tissue culture room, and hardening the seedling for 7d under the condition of natural illumination;
(5) washing the domesticated elongated buds in the step (4) with flowing water, shearing the domesticated elongated buds into micro-stem sections which are at least provided with 1 axillary bud or terminal bud and about 1.0cm long, and soaking the morphological lower ends of the cut sections in rooting promoting liquid containing 200mg/L spermidine, 100mg/L IAA and 250mg/L VC for 20s for rooting pretreatment;
(6) planting the morphological lower end of the stem section treated by the rooting promoting liquid in the step (5) in a mixed nutrient substrate (nutrient soil: vermiculite: river sand 4:1:1) sprayed with MS basic culture solution, and performing seedling culture on Tilia miqueliana plants in a greenhouse with the temperature of 24 ℃ and the humidity of 85% on the principle of no overlapping; after 4 weeks of culture, the Tilia miqueliana regenerated plant with strong root system is obtained, and the rooting rate of buds in the nutrient medium is up to 100%.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.
Claims (6)
1. A method for efficiently breeding Tilia miqueliana strains is characterized by comprising the following steps:
(1) taking a branch of perennial Tilia miqueliana as a material, cutting a stem section with axillary buds as an explant, sterilizing the surface, inoculating the explant into a primary culture medium, and culturing for 5-10 days until the axillary buds germinate to be 0.5-1.0 cm in length;
(2) cutting the axillary buds obtained in the step (1), transferring the axillary buds into a proliferation culture medium, and culturing for 20-30 days until adventitious buds with the height of 1-2 cm are induced from the axillary buds;
(3) separating the adventitious buds obtained in the step (2) into single buds, then transferring the single buds to an elongation culture medium, and culturing for 15-20 days until the height of the adventitious buds is 3-5 cm;
(4) moving the adventitious buds stretched in the step (3) out of a tissue culture room, and hardening seedlings for 5-7 days under the condition of natural illumination;
(5) flushing the elongated buds hardened in the step (4) with running water, cutting into micro-cut segments with proper size, and then placing the micro-cut segments in rooting promoting liquid for rooting pretreatment;
(6) planting the morphological lower end of the stem section treated by the rooting promoting liquid in the step (5) in a nutrient medium sprayed with MS basic culture solution, and performing seedling culture on Tilia miqueliana plants in a greenhouse on the principle that the lower end is not overlapped with the root promoting liquid;
in the step (1), the primary culture medium is an MS culture medium added with 0.1-1.0 mg/L TDZ, 0.05-0.5 mg/L ZT, 30g/L sucrose and 7.0g/L agar;
in the step (2), the multiplication culture medium is an MS culture medium added with 0.5-2.0 mg/L TDZ, 0.1-1.0 mg/L ZT, 0.1-2.0 mg/L IAA, 30g/L sucrose and 7.0g/L agar;
in the step (3), the adventitious bud elongation culture medium is a 1/2MS culture medium added with 0.2-1.0 mg/L TDZ, 0.1-0.5 mg/L LIBA, 0.05-0.5 mg/L GA3, 20g/L sucrose and 7.0g/L agar;
in the step (5), the rooting pretreatment comprises the following steps: and (3) soaking the morphological lower end of the cut segment in rooting promoting liquid containing 200-2000 mg/L spermidine, 100-500 mg/L LIAA and 250-1000 mg/L VC for 10-80 s.
2. The method for efficient breeding of tilia miqueliana strain according to claim 1, wherein in the step (1), the stem segment with axillary bud of tilia miqueliana is obtained from non-lignified stem segment of current-year young shoot of field perennial tilia miqueliana plant.
3. The method for efficient breeding of tilia miqueliana strain according to claim 1, wherein in the steps (1) to (3), the culture conditions are 24 ± 2 ℃ of temperature, 2500-3000 lx of illumination intensity and 13-15 h of illumination/9-11 h of darkness of illumination period.
4. The method for efficient breeding of tilia miqueliana strain according to claim 1, wherein in the step (5), the micro-cutting with suitable size means that the domesticated elongated bud is cut into stem segments with at least 1 axillary bud or terminal bud and a length of 0.5-2.0 cm.
5. The method for efficient breeding of tilia miqueliana strain according to claim 1, wherein in the step (6), the nutrient substrate is prepared by mixing nutrient soil, vermiculite and river sand according to a ratio of 4:1: 1.
6. The method for efficient breeding of tilia miqueliana strain according to claim 1, wherein in the step (6), the greenhouse culture conditions are as follows: the temperature is 24-28 ℃, and the humidity is 80-90%.
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