CN115885844A - Autumn flame rapid propagation method after northern domestication - Google Patents
Autumn flame rapid propagation method after northern domestication Download PDFInfo
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Abstract
The invention relates to a northern domesticated autumn flame rapid propagation method. Mainly solves the problem that the prior American red maple variety is not suitable for being directly planted or propagated by utilizing the original variety because the American red maple variety is frozen or slightly frozen when being planted in the arctic-arctic region. Comprises the following steps: (1) explant selection, pretreatment and disinfection treatment; (2) starting culture; (3) subculture multiplication culture; (4) strong seedling culture; (5) rooting induction culture; and (6) hardening seedlings and transplanting and culturing. According to the growth characteristics of the American red maple autumn flame, a start culture medium suitable for American red maple autumn flame tissue culture, subculture multiplication culture, strong seedling culture and rooting induction culture are configured on the basis of an MS culture medium and an improved MS culture medium, and an American red maple tissue culture system is successfully established to obtain an American red maple regeneration plant which is domesticated to be suitable for northeast. The method lays a foundation for further developing the research on tissue culture of cold-resistant American red maples in northern areas.
Description
The technical field is as follows:
the invention relates to the technical field of rapid propagation of plants, in particular to a northern domesticated autumn flame rapid propagation method.
The background art comprises the following steps:
the "autumn flame" is also called American autumn flame red maple, american red maple, autumn flame maple, etc., belonging to Acer of Aceraceae. The Acer rubrum is a plant of Anacardia of Aceraceae, and has the advantages of rapid growth, strong adaptability, drought resistance, moisture resistance, and cold resistance. The leaves in autumn are colorful and are plants represented by colorful leaf trees. In recent years, the planting application range in south is wide, the main propagation mode is cuttage, and because the original American red maple variety is frozen or slightly frozen in the extremely cold regions in northeast, the original American red maple variety is not suitable for being directly planted or propagated by utilizing the original variety, so that the American red maple is still blank for propagation in the arctic-east regions.
The invention content is as follows:
the invention provides a rapid autumn flame propagation method after northern domestication, aiming at overcoming the problem that the prior American red maple variety is frozen or slightly frozen in the arctic-arctic cold area and is not suitable for direct planting or propagation by utilizing the original variety in the background art. The autumn flame rapid propagation method after northern domestication can establish an American red maple tissue culture system to obtain an American red maple regeneration plant which is domesticated to be suitable for northeast, and fills the blank that the American red maple is not bred in extremely cold northeast regions.
The invention can solve the problems by the following technical scheme: the rapid autumn flame propagation method after northern domestication comprises the following steps:
(1) Explant selection and pretreatment:
collecting strongly growing autumn flame annual semi-lignified twig as an explant, keeping the explant moist, cleaning stem sections with axillary buds, cutting the stem sections into a plurality of stem sections, cleaning the stem sections, and moving the stem sections to a super clean workbench for later use; each stem section is ensured to have axillary buds;
(2) Sterilizing the explants;
(3) Starting culture
The sterilized explant needs to be cut off the surface wound part, the lower end of a bud is reserved with 1.2-1.5cm, the upper end of the bud is reserved with 0.5-0.7cm, and then the explant is vertically inoculated in a blank culture medium; transferring to axillary bud starting culture medium for induction culture 7-10 days later; directly inoculating the explant subjected to induced culture into an axillary bud starting culture medium after disinfection treatment, and transferring a tissue culture bottle into a tissue culture room after inoculation;
(4) Subculture of multiplication
Carrying out subculture proliferation on the strong tissue culture seedlings in the tissue culture room in the step (3), shearing stem sections with terminal buds, inoculating each stem section with 2-3 internodes and 2.5-3cm high to a subculture proliferation culture medium; carrying out subculture for 40 days for one period;
(5) Strong seedling culture
The seedlings after the subculture proliferation culture are placed in a strong seedling culture medium for strong seedling culture by subtracting basal leaves, and are cultured for 40 days for one period;
(6) Root induction culture
After strong seedling culture, the tissue culture seedling grows to 5cm for rooting induction culture, and 3-4 pairs of leaves are reserved; a rooting induction test can be carried out in a rooting induction culture medium in a tissue culture bottle; selecting well-growing tissue culture seedlings with few branches from the tissue culture seedlings of the acer rubrum after seedling strengthening for rooting culture for 30 days for one period;
(7) Hardening off and transplanting culture
Selecting the tissue culture seedlings with good rooting for transplanting and hardening; moving to indoor for placement, performing closed-bottle seedling hardening under natural illumination, placing for 5d, performing half-open-cover treatment, and transplanting after two days; and after hardening the seedlings, lightly taking out the tissue culture seedlings from the tissue culture bottle, cleaning roots, ensuring that the roots have no residual culture medium, transplanting the tissue culture seedlings into a matrix, fully irrigating bottom water for the first transplanting, then placing the tissue culture seedlings into an illumination incubator, and placing the tissue culture seedlings into a greenhouse for field planting after 1 week.
Preferably, the collection time of the autumn flame in the step (1) is morning on a sunny day; washing stem of axillary bud under running water for 60-70min when cleaning; cleaning the stem segments with axillary buds, cutting into a plurality of stem segments, cutting into 4-5 cm, keeping 0.8-1cm at the upper end of the axillary buds and 1.6-2.0cm at the lower end of the axillary buds, ensuring that each stem segment has axillary buds, and removing leaves on the stem segments.
Preferably, the explant sterilization treatment process in the step (2) comprises the following steps: after the axillary bud stem segments of the American red maple are pretreated, the axillary bud stem segments are disinfected in a sterile operating platform, firstly, the explants are put into 2% Jieermie to be soaked for 9-10min and washed in sterile water for several times, then, the explants are immediately taken out to be put into the sterile water to be washed for 2-3 times after being put into 75% alcohol to be disinfected for 20-30 s, and then, the explants are put into 10% sodium hypochlorite for 4-5min and washed in the sterile water.
Preferably, the axillary bud initiation medium in step (3) is: taking improved MS as a basic culture medium, adding ZT0.1mg/L (promoting callus germination whiskers) and IBA1.0mg/L (promoting germination) to perform axillary bud starting culture; adding 5g-7g of agar and 30g-35g of sucrose into each liter of culture medium, wherein the pH value is 5.8-6.0; the axillary bud starting culture conditions are as follows: adjusting the temperature to 22-24 deg.C, humidity to 65-70%, illumination time of 14 h/day, and illumination intensity of 1400-1500lx.
Preferably, the subculture multiplication medium in the step (4) is: taking improved MS as a basic culture medium, adding four plant growth regulators of 0.005mg/L of TDZ (novel plant growth regulator with strong cytokinin activity), 0.1mg/L of 6-BA (promoting cell division), 30.1mg/L of GA (promoting plant germination and accelerating plant growth process) and 0.1mg/L of IBA, adding 5g-7g of agar and 30g-35g of cane sugar into each liter of culture medium, and adjusting the pH value to 5.8-6.0; the subculture conditions for proliferation are: the temperature is 23-24 ℃, the humidity is 70%, the illumination time is 14 h/day, and the illumination intensity is 1500lx.
Preferably, the improved MS culture medium is specifically: NH 4 NO 3 1.3g/L,KNO 3 1.9g/L,CaCl 2 ·2H 2 O 0.44g/L,MgSO 4 ·7H 2 O 0.74g/L,KH 2 PO 4 0.17g/L,KI 0.00083g/L,H 3 BO 3 0.0062g/L,MnSO 4 ·4H 2 O 0.0223g/L,ZnSO 4 ·7H 2 O 0.0086g/L,Na 2 MoO 4 ·2H 2 O 0.00025g/L,CuSO 4 ·5H 2 O 0.0000025g/L,CoCl 2 ·6H 2 O 0.0000025g/L,Na 2 ·EDTA 0.0373g/L,FeSO 4 ·7H 2 O0.0278 g/L, nicotinic acid 0.5mg/L, thiamine hydrochloride (VB) 1 ) 0.5mg/L, pyridoxine hydrochloride (VB) 6 ) 0.4mg/L, inositol 100mg/L and glycine 2mg/L.
Preferably, the strong seedling culture medium in the step (5) is: MS is taken as a basic culture medium, plant growth regulators GA 3.0 mg/L +6-BA 1.0mg/L + IBA0.2mg/L are added, 30g/L of sucrose is added into the culture medium, 5g/L of agar is added, and the pH value is 5.8-6.0; the strong seedling culture conditions are as follows: the culture temperature is 23-24 ℃, the humidity is 60-70%, the illumination time is 14 h/day, and the illumination intensity is 1400-1500lx. Two explants per flask were cultured for 40 days for one cycle.
Preferably, the rooting induction culture medium in the step (6) is: adopting 1/2MS culture medium as the basic culture medium of the root culture stage. GA 3.0 mg/L +2,4-D2 mg/L + activated carbon 1g/L was added. Adding 30g-35g/L of sucrose into the culture medium, adding 5g-7g/L of q agar, and adjusting the pH value to 5.8; the rooting induction culture conditions are as follows: the culture temperature is 23-24 ℃, the humidity is 60-70%, the illumination time is 14 h/day, and the illumination intensity is 1400-1500lx. Two explants per flask were cultured for 30 days for one cycle.
Preferably, the seedling exercising and transplanting culture conditions in the step (7) are as follows: setting the temperature at 23-24 deg.c and humidity at 60-70%; the illumination time is 14 h/day, and the seedlings are placed in a greenhouse for permanent planting after 1 week; the selected substrates in the seedling hardening and transplanting culture are imported turfy soil and perlite, and the volume ratio of the imported turfy soil to the perlite is 2:1.
although the adaptability of the red maples domesticated for over ten (more) years is enhanced, the red maples basically adapt to the growth of the northeast, the cuttage in the propagation method is not feasible, the domesticated quantity is less, the sowing is also not feasible, and the autumn flame is a hybrid, so in order to propagate the variety of the autumn flame quickly, the tissue culture technology is designed to propagate the autumn flame quickly.
Compared with the background technology, the invention has the following beneficial effects:
the autumn flame nursery stock suitable for growing in northeast regions is increased through rapid propagation, the seedling using amount of a nursery land can be met, the blank that no American red maple exists in arctic-arctic regions can be filled, and a large number of seedlings can be rapidly supplied and subsequent cold-resistant varieties and domestication research can be rapidly carried out. The method takes domesticated American red maple autumn flame as a material, researches and screens the best explant and a disinfection mode, is suitable for a culture medium of primary induction culture, subculture multiplication culture, strong seedling culture and rooting culture, and researches culture conditions and transplanting and seedling hardening. Successfully establishes an American red maple tissue culture system to obtain an American red maple regeneration plant which is domesticated and adapted to northeast regions. The method lays a foundation for further developing the research on tissue culture of cold-resistant American red maples in northern areas. Provides technical support for industrial seedling culture of the American red maples.
In the traditional tissue culture experimental research of the American red maple, the axillary bud germination of the American red maple is less influenced by exogenous hormones, the main influencing factor is a basic culture medium, and in recent years, a WPM (woody plant medium) is also commonly used for tissue culture of woody plants. In the experiment, improved MS, MS and WPM are mainly adopted for screening a basic culture medium in primary culture, the prophase WPM culture medium induces the explant to sprout faster but the later leaf to yellow, and after the MS culture medium induces the explant to sprout, the plant grows weaker than other two varieties. And improving the strong growth of the MS induced plants. The research recombines and improves the proportion of major elements in MS, adjusts the proportion of exogenous hormones suitable for each tissue culture stage of the American red maple, determines the optimal proportion combination, greatly improves the multiplication coefficient, establishes a tissue culture rapid propagation system and expands the production of cold-resistant American red maple seedlings suitable for northern culture.
The autumn flame rapid propagation method after northern domestication of the invention has the tissue culture bottle seedlings of about 10000 red maples at present.
Drawings
FIG. 1 is a photograph of explants in priming culture according to an embodiment of the present invention;
FIG. 2 is a photograph of a subculture of plantlets according to the embodiment of the present invention;
FIG. 3 is a photograph of a strong seedling cultured seedling according to an embodiment of the present invention;
FIG. 4 is a photograph of autumn flame seedlings cultured by rooting induction according to an embodiment of the present invention:
FIG. 5 is a photograph of autumn flame seedlings transplanted after acclimatization in accordance with an embodiment of the present invention.
The specific implementation mode is as follows:
in order to make the objects, technical solutions and advantages of the present invention clearer, the following will describe in further detail the process of the fast autumn flame propagation method after northern domestication.
The northern domesticated autumn flame rapid propagation method comprises the following steps:
(1) Explant selection and pretreatment:
the method comprises the steps of collecting annual twigs of American red maples which grow robustly and do not have diseases and insect pests in the morning on a sunny day, spraying water in time on the way of being brought back to a laboratory, keeping the twigs wet, and preventing test materials from being dehydrated, dried and inactivated. The stem segments with axillary buds are cleaned up and washed under running water for 1h. When branches are treated, stem segments with axillary buds are cut into about 4cm long, 1cm is left at the upper ends of the axillary buds, and about 2cm is left at the lower ends of the axillary buds. The axillary buds of each stem section are ensured, and the leaves on the stem sections are removed. The part with axillary buds needs to use a soft hair light brush and can be dipped with a small amount of liquid detergent. And after cleaning, moving the workpiece to a position below the superclean workbench for later use.
(2) Sterilizing the explants; after the axillary bud stem segments of the American red maples are pretreated, the axillary bud stem segments are disinfected in a sterile operation table, the explants are firstly placed in 2% Jieer to be soaked for 9-10min and washed in sterile water for 2-3 times, the explants are immediately taken out after being placed in 75% alcohol to be disinfected for 20-30 s and then are placed in the sterile water to be washed for 2-3 times, 10% sodium hypochlorite is placed for 4-5min, and the explants are washed in the sterile water for 2-3 times. The beaker is shaken continuously during the disinfection period to make the explant fully contact with the disinfection solvent.
(3) Starting culture:
when the culture is started, the improved MS is used as a basic culture medium, and 0.1mg/L ZT and 1.0mg/L IBA are added to carry out axillary bud starting culture. 5g of agar and 30g of sucrose are added per liter of medium, and the pH value is 5.8-6.0.
The sterilized explant needs to be cut off a wound part on the surface, the lower end of a bud is reserved with 1.2cm-1.5cm, the upper end of the bud is reserved with 0.5cm-0.7cm, the purpose is to reserve an effective part of the bud which can grow a parent body and provide nutrition, and then the bud is vertically inoculated in a blank culture medium; transferring to axillary bud initiation culture medium for induction culture 7-10 days later. Taking improved MS as a basic culture medium, and adding ZT0.1mg/L and IBA1.0mg/L to perform axillary bud starting culture; adding 5g-7g of agar and 30g-35g of sucrose into each liter of culture medium, wherein the pH value is 5.8-6.0; wherein ZT is added for promoting callus germination; IBA acts to promote germination; and (3) directly inoculating the explant after the induction culture in an axillary bud starting culture medium after the disinfection treatment. After inoculation, the tissue culture bottle is transferred to a tissue culture room, the temperature is adjusted to be 22-24 ℃, the humidity is 65-70%, the illumination time is 14 h/day, and the illumination intensity is 1400-1500lx.
The improved MS culture medium comprises the following specific steps: NH 4 NO 3 1.3g/L，KNO 3 1.9g/L，CaCl 2 ·2H 2 O 0.44g/L，MgSO 4 ·7H 2 O 0.74g/L，KH 2 PO 4 0.17g/L，KI 0.00083g/L，H 3 BO 3 0.0062g/L，MnSO 4 ·4H 2 O 0.0223g/L，ZnSO 4 ·7H 2 O 0.0086g/L，Na 2 MoO 4 ·2H 2 O 0.00025g/L，CuSO 4 ·5H 2 O 0.0000025g/L，CoCl 2 ·6H 2 O 0.0000025g/L，Na 2 ·EDTA 0.0373g/L，FeSO 4 ·7H 2 O0.0278 g/L, nicotinic acid 0.5mg/L, thiamine hydrochloride (VB) 1 ) 0.5mg/L of pyridoxine hydrochloride (VB) 6 ) 0.4mg/L, inositol 100mg/L and glycine 2mg/L.
The axillary bud initiation culture medium is: the improved MS is a basic culture medium, and 0.1mg/L ZT and 1.0mg/L IBA are added. The components of the starting culture medium can ensure the normal growth of the explant and the normal extension and leaf expansion of the axillary buds.
(4) Subculture of multiplication
Taking the strong tissue culture seedling for subculture multiplication, cutting stem sections with terminal buds, inoculating each stem section with 2-3 internodes and 2.5-3cm high into a subculture multiplication medium, and aiming at multiplying the buds. Modified MS is used as a basic culture medium, four plant growth regulators of TDZ 0.005mg/L, 6-BA0.1mg/L, GA30.1mg/L and IBA0.1mg/L are added, 5g-7g of agar and 30g-35g of cane sugar are added to each liter of culture medium, and the pH value is 5.8-6.0. Wherein, TDZ is a novel plant growth regulator and has strong cytokinin activity; 6-BA can promote cell division; GA can promote plant germination and accelerate the plant growth process; IBA is a plant growth regulator. The culture temperature is 23-24 ℃, the humidity is 70%, the illumination time is 14 h/day, and the illumination intensity is 1500lx. Two explants per flask were cultured for 40 days for one cycle.
The subculture proliferation culture comprises the following steps: the improved MS is a basic culture medium, four plant growth regulators of TDZ 0.005mg/L, 6-BA0.1mg/L and GA30.1mg/L, IBA are added, and 5g to 7g of agar and 30g to 35g of cane sugar are added to each liter of culture medium.
(5) Strong seedling culture
The seedlings after subculture multiplication are placed in a strong seedling culture medium for strong seedling culture after the basal leaves are removed, the strong seedling culture medium takes MS as a basic culture medium as a basis, plant growth regulators GA 3.0 mg/L +6-BA 1.0mg/L + IBA0.2mg/L are added, 30g/L of sucrose is added in the culture medium, 5g/L of agar is added, the pH value is 5.8-6.0, the culture temperature is 23-24 ℃, the humidity is 60-70%, the illumination time is 14 h/day, and the illumination intensity is 1400lx-1500lx. Two explants per flask were cultured for 40 days for one cycle.
The strong seedling culture medium comprises: MS is a basic culture medium, plant growth regulators GA 3.0 mg/L,6-BA 1.0mg/L and IBA0.2mg/L are added, sucrose is added into the culture medium at a ratio of 30g/L, and agar is added at a ratio of 5g/L.
(6) Root induction culture
When the tissue culture seedling grows to 5cm, 3-4 pairs of leaves are reserved, so that the nutrition supply to the upper part is reduced, and rooting is facilitated. Rooting induction tests can be carried out in tissue culture bottles. And selecting the tissue culture seedlings with good growth vigor and few branches from the tissue culture seedlings of the acer rubrum after strong seedling culture for rooting culture. Adopting 1/2MS culture medium as the basic culture medium of the root culture stage. GA 3.0 mg/L +2,4-D2 mg/L + activated carbon 1g/L was added. 30g-35g/L of sucrose, 5g-7g/L of q-agar, 5.8 of PH value, 23-24 ℃ of culture temperature, 60-70% of humidity, 14 h/day of illumination time and 1400-1500lx of illumination intensity. Two explants per flask were cultured for 30 days for one cycle.
The rooting induction culture comprises the following steps: the 1/2MS culture medium is a basic culture medium, and 1.0mg/L GA3+ 2,4-D2 mg/L activated carbon + 1g/L activated carbon is added. 30g-35g/L of sucrose and 5g-7g/L of q agar are added into the culture medium.
(7) Hardening off and transplanting culture
Transplanting and hardening the tissue culture seedlings which have rooted in the bottles. Moving to indoor for placing, performing bottle closing and seedling hardening under natural illumination, placing for 5d, performing half-opening cover treatment, and transplanting after two days. After hardening the seedling, gently taking out the tissue culture seedling from the tissue culture bottle, cleaning the root, and transplanting the seedling into the matrix of the imported turfy soil and the perlite after ensuring that no culture medium is left on the root, wherein the volume ratio of the imported turfy soil to the perlite is 2:1. the first transplantation is carried out by thoroughly watering the bottom water, and then the seedlings are placed in an illumination incubator with the temperature of 23-24 ℃ and the humidity of 60-70%. The illumination time is 14 h/day, and the seedlings are placed in a greenhouse for permanent planting after 1 week.
Example 1
The autumn flame rapid propagation method after northern domestication of the invention is further explained by taking the explant of the several trees as an example, wherein the explant is used as a female parent and is used for large continuous introduction:
(1) Explant selection and pretreatment: the American red maple which is introduced from the female parent as the Dalian acer palmatum and grows robustly and has no disease and pest is collected in the morning on a sunny day, and the annual red maple is taken back to a laboratory to be sprayed with water in time to keep moist and prevent the test material from being dehydrated, dried and inactivated. The stem segments with axillary buds are cleaned up and washed under running water for 1h. When branches are treated, stem segments with axillary buds are cut into about 4cm long, 1cm is left at the upper ends of the axillary buds, and about 2cm is left at the lower ends of the axillary buds. The axillary buds of each stem section are ensured, and the leaves on the stem sections are removed. The part with axillary buds needs to use a soft hair light brush and can be dipped with a small amount of liquid detergent. And after cleaning, moving to the position below the superclean bench for later use.
(2) Disinfection treatment of explants
After the axillary bud stem segments of the American red maple are pretreated, the axillary bud stem segments are disinfected in a sterile operating platform, the explants are firstly put into 2% Jieer to be soaked for 10min and washed in sterile water for 2-3 times, the explants are immediately taken out to be put into the sterile water to be washed for 2-3 times after being put into 75% alcohol for disinfection treatment for 30S, 10% sodium hypochlorite is put into the sterile water for 5min and washed in the sterile water for 2-3 times, and the explants are washed in the sterile water for 2-3 times. Shaking the beaker during the sterilization process to allow the explant to contact the sterilization solvent sufficiently.
(3) Starting culture
When the first treatment and the initial culture are carried out, the improved MS is used as a basic culture medium, and 6-BA1.0mg/L and 0.2mg/L of IBA are added for carrying out the axillary bud initial culture. 5g of agar and 30g of sucrose were added per liter of medium, pH 5.8.
The treated explants were required to excise the surface wound portion to form a fresh incision. The lower end of the bud is 1.5cm, the upper end is 0.5cm, and then the bud is vertically inoculated in a blank culture medium. After 10 days, the cells were transferred to an axillary bud initiation medium for induction culture. The improved MS is used as a basic culture medium, and 6-BA1.0mg/L and 0.2mg/L of IBA are added for axillary bud starting culture. 5g of agar and 30g of sucrose were added per liter of medium, pH 5.8. Directly inoculating the disinfected explant into an axillary bud starting culture medium, transferring a tissue culture bottle into a tissue culture room after inoculation, adjusting the temperature to 23 ℃, the humidity to 70%, the illumination time to 14 h/day and the illumination intensity to 1500lx.
And secondly, during the starting culture, the WPM is used as a basic culture medium, and 6-BA1.0mg/L and 0.2mg/L of IBA are added for axillary bud starting culture. 5g of agar and 30g of sucrose are added per liter of medium, and the pH value is 5.8-6.0. The other culture environments were identical to those of treatment 1.
And thirdly, when the culture is started, the improved MS is used as a basic culture medium, and 6-BA1.0mg/L and 0.2mg/L of IBA are added for axillary bud starting culture. 5g of agar and 30g of sucrose are added per liter of medium, and the pH value is 5.8-6.0. The other culture environments were identical to those of treatment 1.
And fourthly, when the culture is started, the improved MS is used as a basic culture medium, and 0.1mg/L ZT and 0.1mg/L IBA are added to carry out axillary bud starting culture. 5g of agar and 30g of sucrose are added per liter of medium, and the pH value is 5.8-6.0. The other culture environments were identical to those of treatment 1.
And fifthly, starting culture by taking improved MS as a basic culture medium and adding ZT0.1mg/L and IBA1.0mg/L to perform axillary bud starting culture. 5g of agar and 30g of sucrose are added per liter of medium, and the pH value is 5.8-6.0. The other culture environments were identical to those of treatment 1.
And sixthly, performing axillary bud starting culture by taking the improved MS as a basic culture medium and adding ZT0.1mg/L and IBA 1.5mg/L during starting culture. 5g of agar and 30g of sucrose are added per liter of medium, and the pH value is 5.8-6.0. The other culture environments were identical to those of treatment 1.
The specific culture scheme and the effect of different minimal medium and hormone ratios on the axillary bud initiation culture are shown in Table 1.
TABLE 1
As can be seen from Table 1, the improvement of MS basal medium has a significant effect on the initiation of explants based on the addition of 6-BA and IBA at the same hormone concentration. The improved MS is used as a basic culture medium, and ZT and IBA are added for induction culture, and test results show that the growth induction rate of tissue culture seedlings with 0.1mg/L ZT and 1.0mg/L IBA added in the five treatment schemes is the highest, and the axillary bud induction rate reaches 93.33%. The photographs of the explants in the priming culture are shown in FIG. 1.
(4) Subculture of multiplication
When the first step and the secondary multiplication culture are processed, an improved MS culture medium is used as a basic culture medium, four plant growth regulators are added for secondary multiplication culture, and the secondary multiplication culture medium is improved MS +0.005mg/L TDZ +0.1 mg/L6-BA +0.1mg/L GA3+0.1mg/L IBA.
And carrying out subculture proliferation on the explants obtained by the culture scheme of the fifth treatment in the start culture.
And (3) carrying out subculture proliferation on the strong tissue culture seedlings obtained by the five schemes of starting culture treatment, shearing stem sections with terminal buds, inoculating the stem sections into a subculture proliferation culture medium, wherein the stem sections are about 3 internodes and 3cm high. The improved MS is used as a basic culture medium, four plant growth regulators of 0.005mg/L of TDZ and 0.1mg/L, GA 30.1.1 mg/L, IBA 0.1.1 mg/L of 6-BA are added, 5g of agar and 30g of cane sugar are added into each liter of the culture medium, and the pH value is 5.8. The culture temperature is 23 ℃, the humidity is 70%, the illumination time is 14 h/day, and the illumination intensity is 1500lx. Two explants per flask were cultured for 40 days for one cycle.
And the secondary multiplication culture medium is modified MS +0.005mg/L TDZ +0.5 mg/L6-BA +0.5mg/L GA3+0.2mg/L IBA. The other culture environments were identical to those of treatment 1.
And step three, the subculture multiplication medium is modified MS +0.005mg/L TDZ +1 mg/L6-BA +1mg/L GA3+0.5mg/L IBA. The other culture environments were identical to those of treatment 1.
And the treatment IV and the subculture multiplication medium are modified MS +0.01mg/L TDZ +0.1 mg/L6-BA +0.5mg/L GA3+0.5mg/L IBA. The other culture environments were identical to those of treatment 1.
And fifthly, the subculture multiplication medium is modified MS +0.01mg/L TDZ +0.5 mg/L6-BA +1mg/L GA3+0.1mg/L IBA. The other culture environments were identical to those of treatment 1.
And sixthly, the secondary multiplication culture medium is modified MS +0.01mg/L TDZ +1 mg/L6-BA +0.1mg/L GA3+0.2mg/L IBA. The other culture environments were identical to those of treatment 1.
Seventhly, the subculture multiplication medium is modified MS +0.1mg/L TDZ +0.1 mg/L6-BA +1mg/L GA3+0.2mg/L IBA. The other culture environments were identical to those of treatment 1.
And eighthly, the subculture multiplication medium is modified MS +0.1mg/L TDZ +0.5 mg/L6-BA +0.1mg/L GA3+0.5mg/L IBA. The other culture environments were identical to those of treatment 1.
Ninth, the subculture multiplication medium is modified MS +0.1mg/L TDZ +1 mg/L6-BA +0.5mg/L GA3+0.1mg/L IBA. The other culture environments were identical to those of treatment 1.
The specific culture scheme and the effect of different phytohormones on the subculture are shown in Table 2.
TABLE 2
As can be seen from table 2, the test results show that the concentration of TDZ has a significant difference in the proliferation effect of acer rubrum. When the TDZ concentration was increased to 0.1ml/L, the proliferation factor became significantly small. The culture medium suitable for tissue culture subculture proliferation of the acer rubrum is treated nine: improved MS +0.005mg/L TDZ +0.1mg/L GA3+0.1mg/L IBA +0.1 mg/L6-BA mg/L. The photograph of the subcultured plantlets is shown in FIG. 2.
(5) Strong seedling culture
When the first step and the second step are carried out, MS is used as a basic culture medium for strong seedling culture, 1.0mg/L of plant growth regulator GA3, 1.0mg/L of 6-BA and 0.2mg/L of IBA0 are added, 30g/L of cane sugar is added into the culture medium, 5g/L of agar is added, and the pH value is 5.8.
And (4) performing strong seedling culture on the explant obtained by the culture scheme of the nine treatments in the subculture proliferation culture.
Subtracting basal leaves, performing strong seedling culture by taking MS as a basic culture medium, adding 1.0mg/L of plant growth regulator GA3+ 6-BA1.0mg/L + IBA0.2mg/L, adding 30g/L of sucrose and 5g/L of agar in the culture medium, wherein the pH value is 5.8, the culture temperature is 23 ℃, the humidity is 70%, the illumination time is 14 h/day, and the illumination intensity is 1500lx. Two explants per flask were cultured for 40 days for one cycle.
And in the second treatment and the second strong seedling culture, MS is used as a basic culture medium for strong seedling culture, a plant growth regulator GA 3.6 mg/L +6-BA 1.0mg/L + IBA0.2mg/L is added, 30g/L of sucrose is added into the culture medium, 5g/L of agar is added, the pH value is 5.8, and other culture environments are completely the same as those of the first treatment.
And in the third treatment and the fourth treatment, MS is used as a basic culture medium for strong seedling culture, a plant growth regulator GA 3.2 mg/L,6-BA 1.0mg/L and IBA0.2mg/L are added, sucrose is added into the culture medium by 30g/L, agar is added by 5g/L, the pH value is 5.8, and other culture environments are completely the same as those of the treatment 1.
The results of the experiments on the effects of the specific culture protocol and GA3 concentration on the strong seedlings of the tissue culture seedlings are shown in Table 3.
TABLE 3
Test results show that GA3 with different concentrations has obvious difference on the influence of the plant height of the tissue culture seedlings, and the GA3 added in 1mg/L in the first treatment is suitable for strong seedling culture of American red maple, the plant height is high, the stem is thick, and the plant grows more robustly. The photographs of the strong seedling culture plantlets are shown in FIG. 3.
(6) Root induction culture
When the first treatment and the rooting induction culture are carried out, a 1/2MS culture medium is adopted as a basic culture medium in a rooting culture stage, and 2,4-D0.5 mg/L is added. The culture medium was supplemented with 30g/L sucrose, 5g/L q agar, and a pH of 5.8.
And (4) carrying out rooting induction culture on the tissue culture seedling obtained by the first culture scheme in the strong seedling culture.
And when the tissue culture seedling grows to 5cm, keeping 3-4 pairs of leaves. Rooting induction tests can be carried out in tissue culture bottles. And selecting the tissue culture seedlings with good growth vigor and few branches from the tissue culture seedlings of the acer rubrum after strong seedling culture for rooting culture. Adopting 1/2MS culture medium as the basic culture medium of the root culture stage. 2,4-D0.5 mg/L was added. 30g/L of sucrose, 5g/L of q-agar, 5.8 of PH value, 23 ℃ of culture temperature, 70% of humidity, 14 h/day of illumination time and 1500lx of illumination intensity. Two explants per flask were cultured for 30 days for one cycle.
And in the second treatment and rooting induction culture, a 1/2MS culture medium is adopted as a basic culture medium in the rooting culture stage, and 2,4-D1 mg/L is added. Sucrose addition 30g/L, q agar 5g/L, pH 5.8, and other culture conditions were identical to those of treatment 1.
And thirdly, when rooting induction culture is carried out, adopting a 1/2MS culture medium as a basic culture medium in a rooting culture stage, and adding 2,4-D2 mg/L. Sucrose addition 30g/L, q agar 5g/L, pH 5.8, and other culture conditions were identical to those of treatment 1.
And fourthly, when the rooting induction culture is carried out, adopting a 1/2MS culture medium as a basic culture medium in the rooting culture stage. GA 3.0 mg/L +2,4-D0.5 mg/L + activated carbon 1g/L was added. Sucrose addition 30g/L, q agar 5g/L, pH 5.8, and other culture conditions were identical to those of treatment 1.
And fifthly, adopting a 1/2MS culture medium as a basic culture medium in the rooting culture stage when performing rooting induction culture. GA 3.0 mg/L +2,4-D1.0 mg/L + activated carbon 1g/L was added. Sucrose addition 30g/L, q agar 5g/L, pH 5.8, and other culture conditions were identical to those of treatment 1.
And sixthly, when rooting induction culture is carried out, adopting a 1/2MS culture medium as a basic culture medium in a rooting culture stage. GA 3.0 mg/L +2,4-D2 mg/L + activated carbon 1g/L was added. Sucrose addition 30g/L, q agar 5g/L, pH 5.8, and other culture conditions were identical to those of treatment 1.
The results of comparing the influences of specific culture schemes and hormone ratios on rooting of tissue culture seedlings of Acer rubrum are shown in Table 4.
TABLE 4
As shown in Table 4, the research shows that the rooting effect of the tissue culture seedling treated by adding 2 mg/L2,4-D is better, the activated carbon can adsorb metabolic toxin to promote rooting, and the rooting rate is compared, so that the induction culture medium suitable for tissue culture rooting of the acer rubrum is a scheme for treating six: 1/2MS + GA3.0mg/L +2,4-D2 mg/L + activated carbon 1g/L, and the rooting rate reaches 100%. Photographs of the rooting-induced cultured autumn flame seedlings are shown in FIG. 4.
(7) Hardening off and transplanting culture
When the first treatment, seedling hardening and transplanting culture are carried out, the seedling hardening time is 3 days, and the matrix proportion is as follows: grass carbon: perlite =1:1, namely the transplanting and seedling exercising mode is that after half-opening and exercising for 3d, the seedling is transplanted in a mode that the ratio of turf to perlite is 1:1, the other medium components and the culture environment were exactly the same as those in example 4.
And (4) carrying out hardening and transplanting culture on the tissue culture seedling obtained by the culture scheme of treating the sixth step in the root induction culture.
Transplanting and hardening off the tissue culture seedlings after rooting in the bottle. Move to indoor place, pay attention to can not directly put under the intense illumination. And (4) carrying out bottle closing and seedling hardening under the natural illumination condition, carrying out half-open cover treatment after placing for 3d, and transplanting after two days. After hardening the seedlings, gently taking out the tissue culture seedlings from the tissue culture bottle, cleaning the roots, and ensuring that the ratio of the transplanted turf to the perlite after the roots are not residual is 1:1 in a matrix. The first transplantation is carried out, bottom water is thoroughly poured, and then the seedlings are placed in an illumination incubator, the temperature is set to be 23 ℃, and the humidity is set to be 70%. The illumination time is 14 h/day, and the seedlings are placed in a greenhouse for permanent planting after 1 week.
And II, when the seedlings are acclimatized and transplanted for culture, acclimatizing the seedlings for 3 days, importing turfy soil: perlite =1:1, the other culture environments were identical to those of treatment 1.
And thirdly, hardening seedlings for 3 days during hardening and transplanting culture, importing turfy soil: perlite =2:1, the other culture environments were identical to those of treatment 1.
And fourthly, hardening off the seedling for 5 days during seedling hardening and transplanting culture, wherein the grass carbon: perlite =1:1, other culture environments were identical to treatment 1.
And fifthly, hardening the seedling for 5 days when hardening the seedling and transplanting the seedling, and importing turfy soil: perlite =1:1, the other culture environments were identical to those of treatment 1.
And sixthly, when hardening seedlings and transplanting culture are carried out, the hardening seedling time is 5 days, and the imported turfy soil: perlite =2:1, the culture environment of the other media was exactly the same as that of treatment 1.
Seventhly, hardening seedlings and transplanting culture, wherein the hardening seedling time is 7 days, and the grass peat: perlite =1:1, and the other culture environments were identical to treatment 1.
Eighthly, hardening seedlings and transplanting culture, wherein the hardening seedling time is 7 days, and turfy soil is imported: perlite =1:1, the other culture environments were identical to those of treatment 1.
The results of comparing the effects of specific culture schemes, different hardening-off times and matrix combinations on the survival rate of transplanted seedlings are shown in Table 5.
TABLE 5
As can be seen from Table 5, the experimental results show that the survival rates of the tissue culture seedlings under different seedling exercising time and transplanting matrix conditions are different, which results in significant differences. After closing the cover for 5 days, carrying out half-open cover treatment, setting three gradients (3 d,5d and 7 d), observing and finding that the tissue culture seedling on the third day with the cover opened has the best state without water loss, the tissue culture seedling on the fifth day has the water loss phenomenon, and the leaves are slightly wilted. The tissue culture seedling at the seventh day has a large amount of bacteria, and white hair is formed at the root. The transplanting survival rate of the acclimatized seedling on the fifth day is obviously higher than that of the third day and the seventh day, and the survival rate trend rises firstly and then declines with the increase of time. The transplanting survival rate is influenced by different types of transplanting substrates, the survival rate of the tissue culture seedlings transplanted by the turfy soil is lower than that of imported turfy soil, and a large amount of bacteria are generated to influence the transplanting survival rate of the tissue culture seedlings. But the difference of the transplanting survival rate is not obvious in the matrixes with different proportions of imported turfy soil and perlite. Therefore, according to the test results, the optimal transplanting and seedling exercising mode is a scheme of six treatments: transplanting the half-uncovered hardened seedlings to imported turfy soil and perlite after 5d at a ratio of 2:1, the highest transplanting survival rate is 62.36 percent. The photograph of the autumn flame seedlings transplanted after acclimatization is shown in FIG. 5.
According to the research, domesticated American red maple autumn flame is used as a material, a starting culture medium suitable for American red maple autumn flame tissue culture, subculture multiplication culture, strong seedling culture and rooting induction culture are configured on the basis of an MS culture medium and an improved MS culture medium according to the growth characteristics of the American red maple autumn flame, and an American red maple tissue culture system is successfully established by using the preferred starting culture medium, subculture multiplication culture, strong seedling culture and rooting induction culture, so that a domesticated American red maple regeneration plant suitable for northeast is obtained. The method lays a foundation for further developing the research on tissue culture of cold-resistant American red maples in northern areas. Provides technical support for industrial seedling culture of the American red maples.
Claims (10)
1. A northern domesticated autumn flame rapid propagation method is characterized in that: the method comprises the following steps:
(1) Explant selection and pretreatment:
collecting annual semi-lignified twigs of strong autumn flame as explants, keeping the explants moist, cleaning stem segments with axillary buds, cutting the stem segments into a plurality of stem segments, cleaning the stem segments, and moving the stem segments to a clean bench for later use; each stem section is ensured to have axillary buds;
(2) Sterilizing the explants;
(3) Starting culture:
the sterilized explant needs to be cut off the wound part on the surface, the lower end of the bud is reserved with 1.2cm-1.5cm, the upper end is reserved with 0.5cm-0.7cm, and then the explant is vertically inoculated in a blank culture medium; transferring to axillary bud starting culture medium for induction culture 7-10 days later; directly inoculating the explant subjected to induced culture into an axillary bud starting culture medium after disinfection treatment, and transferring a tissue culture bottle into a tissue culture room after inoculation;
(4) Subculture multiplication culture:
carrying out subculture proliferation on the strong tissue culture seedlings in the tissue culture room in the step (3), shearing stem sections with terminal buds, inoculating each stem section with 2-3 internodes and 2.5-3cm high to a subculture proliferation culture medium; carrying out subculture for 40 days for one period;
(5) Strong seedling culture:
the seedlings after the subculture proliferation culture are placed in a strong seedling culture medium for strong seedling culture by subtracting basal leaves, and are cultured for 40 days for one period;
(6) Rooting induction culture:
after the strong seedling culture, the tissue culture seedling grows to 5cm for rooting induction culture, and 3~4 pairs of leaves are reserved; a rooting induction test can be carried out in a rooting induction culture medium in a tissue culture bottle; selecting well-growing tissue culture seedlings with few branches from the tissue culture seedlings of the acer rubrum after seedling strengthening for rooting culture for 30 days for one period;
(7) Hardening off and transplanting culture
Selecting the tissue culture seedlings with good rooting for transplanting and hardening; moving to indoor for placement, performing closed-bottle seedling hardening under natural illumination, placing for 5d, performing half-open-cover treatment, and transplanting after two days; and after hardening the seedlings, lightly taking out the tissue culture seedlings from the tissue culture bottle, cleaning roots, ensuring that the roots have no residual culture medium, transplanting the tissue culture seedlings into a matrix, fully irrigating bottom water for the first transplanting, then placing the tissue culture seedlings into an illumination incubator, and placing the tissue culture seedlings into a greenhouse for field planting after 1 week.
2. The northern domesticated autumn flame rapid propagation method according to claim 1, characterized in that: collecting autumn flame in the morning of fine days in step (1); washing stem of axillary bud under running water for 60-70min when cleaning; cleaning the stem segments with axillary buds, cutting into a plurality of stem segments, cutting into 4-5 cm, leaving 0.8-1cm at the upper end of the axillary bud and 1.6-2.0cm at the lower end of the axillary bud, ensuring that each stem segment has axillary buds, and removing leaves on the stem segments.
3. The northern domesticated autumn flame rapid propagation method according to claim 1, characterized in that: the explant sterilization treatment process in the step (2) comprises the following steps: after the axillary bud stem segments of the American red maples are pretreated, the axillary bud stem segments are disinfected in a sterile operation table, the explants are firstly placed in 2% Jieer to be soaked for 9-10min and washed in sterile water for several times, then placed in 75% alcohol to be disinfected for 20-30 s, then immediately taken out and placed in the sterile water to be washed for 2-3 times, and then placed in 10% sodium hypochlorite for 4-5min and washed in the sterile water.
4. The northern domesticated autumn flame rapid propagation method according to claim 1, characterized in that: the axillary bud starting culture medium in the step (3) is as follows: using improved MS as a basic culture medium, adding ZT0.1mg/L and IBA1.0mg/L to perform axillary bud starting culture; adding 5g-7g of agar and 30g-35g of sucrose per liter of culture medium, wherein the pH value is 5.8-6.0;
the axillary bud starting culture conditions are as follows: adjusting the temperature to 22-24 ℃, the humidity to 65-70%, the illumination time to 14 h/day and the illumination intensity to 1400-1500lx.
5. The northern domesticated autumn flame rapid propagation method according to claim 1, characterized in that: the subculture multiplication medium in the step (4) is as follows: taking improved MS as a basic culture medium, adding four plant growth regulators of 0.005mg/L TDZ and 0.1mg/L, GA 30.1.30.1 mg/L, IBA 0.1.1 mg/L6-BA, adding 5g-7g of agar and 30g-35g of cane sugar per liter of culture medium, and adjusting the pH value to 5.8-6.0;
the subculture conditions for proliferation are: the temperature is 23-24 ℃, the humidity is 70%, the illumination time is 14 h/day, and the illumination intensity is 1500lx.
6. The northern domesticated autumn flame rapid propagation method according to claim 4 or 5, characterized in that: the improved MS culture medium comprises the following specific steps: NH (NH) 4 NO 3 1.3g/L,KNO 3 1.9 g/L,CaCl 2 ·2H 2 O 0.44g/L,MgSO 4 ·7H 2 O 0.74g/L,KH 2 PO 4 0.17g/L,KI 0.00083g/L,H 3 BO 3 0.0062g/L,MnSO 4 ·4H 2 O0.0223g/L,ZnSO 4 ·7H 2 O 0.0086g/L, Na 2 MoO 4 ·2H 2 O 0.00025g/L, CuSO 4 ·5H 2 O 0.0000025g/L, CoCl 2 ·6H 2 O 0.0000025 g/L, Na 2 ·EDTA 0.0373g/L, FeSO 4 ·7H 2 O0.0278 g/L, nicotinic acid 0.5mg/L, thiamine hydrochloride (VB) 1 ) 0.5mg/L of pyridoxine hydrochloride (VB) 6 ) 0.4mg/L, inositol 100mg/L, glycine 2mg/L.
7. The northern domesticated autumn flame rapid propagation method according to claim 1, characterized in that: the strong seedling culture medium in the step (5) is as follows: MS is taken as a basic culture medium, plant growth regulators GA 3.0 mg/L +6-BA 1.0mg/L + IBA0.2mg/L are added, 30g/L of sucrose is added into the culture medium, 5g/L of agar is added, and the pH value is 5.8-6.0;
the strong seedling culture conditions are as follows: the culture temperature is 23-24 ℃, the humidity is 60-70%, the illumination time is 14 h/day, and the illumination intensity is 1400-1500 lx; two explants per flask were cultured for 40 days for one cycle.
8. The northern domesticated autumn flame rapid propagation method according to claim 1, characterized in that: the rooting induction culture medium in the step (6) is as follows: adopting a 1/2MS culture medium as a basic culture medium in a root culture stage; adding 1.0mg/L of GA3, 2,4-D2 mg/L and 1g/L of activated carbon; adding 30g-35g/L of sucrose into the culture medium, adding 5g-7g/L of q agar, and adjusting the pH value to 5.8; the rooting induction culture conditions are as follows: the culture temperature is 23-24 ℃, the humidity is 60-70%, the illumination time is 14 h/day, and the illumination intensity is 1400lx-1500lx; two explants per flask were cultured for 30 days for one cycle.
9. The northern domesticated autumn flame rapid propagation method according to claim 1, characterized in that: the seedling hardening and transplanting culture conditions in the step (7) are as follows: setting the temperature at 23-24 deg.c and humidity at 60-70%; the illumination time is 14 h/day, and the seedlings are placed in a greenhouse for permanent planting after 1 week.
10. The northern domesticated autumn flame rapid propagation method according to claim 1, characterized in that: and (3) selecting imported turfy soil and perlite as substrates in the seedling hardening and transplanting culture in the step (7), wherein the volume ratio of the imported turfy soil to the perlite is 2:1.
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