CN109463281B - Standardized production process of acacia melanoxylon tissue culture seedlings - Google Patents
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Abstract
A standardized production process of acacia melanoxylon tissue culture seedlings comprises the production of propagation seedlings and the production of rooting seedlings, wherein the production content of the propagation seedlings relates to inoculation density, a transfer cycle in a propagation culture period and a transfer cycle in a seedling production period; the production content of the rooted seedlings comprises inoculation density, seedling cutting height, seedling cutting degree, standing time during rooted seedling culture and standing time of a seedling hardening greenhouse, and the method comprises the following steps: a) after obtaining the acacia melanoxylon tissue culture seedling, carrying out proliferation culture for 15-30 days to obtain a proliferation seedling; b) transferring the proliferated seedlings for inoculation; c) culturing the inoculated proliferation seedlings for 25-40 days; d) cutting a single bud with the length of more than 2.0cm in the proliferated seedling to serve as a rooting seedling; e) inoculating rooted seedlings, and f) culturing the rooted seedlings for 4-24 days; g) hardening seedlings for 6-26 days. The invention realizes the increase of the yield of the nursery stock by standardized production, improves the quality of the nursery stock, improves the production efficiency, reduces the production cost, promotes the large-scale production of the acacia melanoxylon excellent clone and realizes the high-efficiency cultivation of the acacia melanoxylon clone forestry.
Description
Technical Field
The invention belongs to the technical field of standardized production processes of acacia melanoxylon tissue culture seedlings.
Technical Field
Acacia melanoxylon (Acacia melanoxylon) is a species of Acacia genus of Mimosaceae family, one of the highest major trees of Acacia species, originally produced in Australia, Babuya New Asia and Indonesia, etc. The acacia melanoxylon has beautiful wood grain, good material quality, nitrogen-fixing root nodules, rich dried-falling matters and good soil improvement performance, and can be successfully planted in Guangdong, Fujian, Guangxi, Hainan and the like after the 20 th century and the 90 th introduction of China.
In the century, the unit overcomes the problems of many branches and unobvious main stems by clonal breeding, and the acacia melanoxylon has become a multipurpose tree species for fast growing and high yield, precious materials, ecological public welfare and the like in south China. Based on incomplete statistics, the area of forest establishment of acacia melanoxylon clones developed at a rate of more than 1 ten thousand acres per year since 2015. In order to meet the requirement of large-scale development of the acacia melanoxylon clone artificial forest, acacia melanoxylon tissue culture research is developed in succession in China since the century, but most tissue culture factories have the problems of low yield, high cost and the like of tissue culture seedlings due to long subculture period and large difference of proliferation rate and rooting rate, and industrial production cannot be realized basically. On the basis of early selection, 10 excellent clones are collected by the unit to carry out tissue culture research, wherein 7 clones have higher production efficiency and are subjected to large-scale production. In the research, the standardized production process in acacia melanoxylon tissue culture plays an important role in increasing the yield of nursery stocks and improving the quality of the nursery stocks. The method can improve the production efficiency of the acacia melanoxylon tissue culture seedlings, promote the large-scale production process of the acacia melanoxylon tissue culture seedlings and lay a foundation for acacia melanoxylon clonal forestry. The acacia melanoxylon tissue culture is mainly focused on basic researches such as culture medium formula at home and abroad, and the production process standardization research is not reported.
Disclosure of Invention
In order to solve the problems, the invention aims to provide a standardized production process of acacia melanoxylon tissue culture seedlings, which can increase the yield of seedlings, improve the quality of the seedlings, improve the production efficiency, reduce the production cost, promote the large-scale production process of excellent acacia melanoxylon clones and realize the efficient cultivation of acacia melanoxylon clones forestry.
In order to achieve the purpose, the invention adopts the following technical scheme:
a standardized production process of acacia melanoxylon tissue culture seedlings comprises the following steps: a) after obtaining the acacia melanoxylon tissue culture seedling, carrying out proliferation culture for 15-30 days to obtain a proliferation seedling; b) transferring the proliferated seedlings for inoculation; c) culturing the inoculated proliferation seedlings for 25-40 days; d) cutting a single bud with the length of more than 2.0cm in the proliferated seedling to serve as a rooting seedling; e) inoculating a rooting seedling; f) culturing the rooted seedlings for 4-24 days; g) hardening seedlings for 6-26 days;
further, a more excellent standardized production process of the acacia melanoxylon tissue culture seedlings is characterized in that after the acacia melanoxylon tissue culture seedlings are obtained in the step a), enrichment culture is carried out for 15-20 days; culturing the proliferated seedlings inoculated in the step c) for 35-40 days; culturing the rooted seedlings in the step f) for 4-8 days; hardening seedlings in the step g) for 22-26 days;
in the step b), the inoculation density of the proliferated seedlings takes a 200mL cylindrical jam bottle (the bottom diameter is 5.5cm, and the height is 9.5cm) as an inoculation container, the thickness of a culture medium is 1.5cm, 8-12 clusters of seedlings are inoculated to each bottle, and 4-6 seedlings are inoculated to each cluster;
the culture medium for the propagation seedling culture in the step c) takes MS as a basic culture medium, 0.4-0.6mg of 6BA, 25-30g of white sugar and 5-6g of agar are added into each liter of the basic culture medium, the pH value is adjusted to 5-6, and the culture medium is pre-packaged at 1.0-1.2kg/cm2Sterilizing at 121 deg.C under high pressure for 18 min;
the number of the cut rooted seedlings in the step d) is 25-50, seedling clusters are not damaged when the rooted seedlings are cut, seedlings are not damaged, and the cut seedling clusters can continue propagation culture according to the growth condition of the seedlings;
in the step e), the inoculation density of the rooted seedlings takes a 200mL cylindrical jam bottle (the bottom is 5.5cm in diameter and 9.5cm in height) as an inoculation container, the thickness of a culture medium is 1.5cm, and 10-25 seedlings are inoculated in each bottle;
the culture medium for culturing the rooting seedling in the step f) takes 1/2MS as a basic culture medium, 1.0-1.5mg of IBA, 25-30g of white sugar and 5-6g of agar are added into each liter of the basic culture medium, the pH value is adjusted to 5-6, and the culture medium is pre-packaged at 1.0-1.2kg/cm2Sterilizing at 121 deg.C under high pressure for 18 min;
the culture temperature of the rooted seedlings in the step f) is 23-27 ℃, and 1500-2000Lx artificial illumination is added for 10-11h in the daytime;
in the step g), the seedling exercising temperature is 25-35 ℃, the illumination is 7000-10000Lx, the seedling exercising room is a sunlight glass greenhouse, and a shading net facility and a cooling facility are arranged outside 70 percent.
The acacia melanoxylon nursery stock obtained by the production process has the rooting rate of 85 percent, the average height of the nursery stock of 2.5cm, developed nursery stock root system, extended leaves and green stems.
The invention brings the technical effects that: the standardized production of acacia melanoxylon tissue culture seedlings can realize the increase of the yield of seedlings, improve the quality of the seedlings, improve the production efficiency, reduce the production cost, promote the large-scale production process of the acacia melanoxylon excellent clone, realize the efficient cultivation of the acacia melanoxylon clone forestry, and the acacia melanoxylon seedlings obtained through standardized production have developed root systems, unfolded blades and green stems and can be directly used for nursery transplantation.
Detailed description of the preferred embodiments
The present invention is described in further detail below with reference to examples, which are intended to be illustrative only and not to limit the scope of the invention, and various modifications of equivalent forms of the present invention which are obvious to those skilled in the art after reading the present invention are intended to be limited by the claims appended hereto.
Example 1
a) After obtaining the acacia melanoxylon tissue culture seedlings, carrying out proliferation culture for 15 days to obtain proliferation seedlings;
b) transferring the proliferated seedlings for inoculation: taking a 200mL cylindrical jam bottle (the bottom diameter is 5.5cm, the height is 9.5cm) as an inoculation container, the thickness of a culture medium is 1.5cm, 20 bottles are inoculated, 8 seedlings are inoculated in each bottle, each seedling is 4-6, and the monthly proliferation rate of the seedlings is 3.35%;
c) after the propagation seedling is cultured for 40 days, entering a seedling production stage, selecting a seedling cutting single bud with the seedling height of more than 2.0cm from the propagation seedling cluster from high to low during seedling production as a raw root seedling, wherein the number of the cut root seedlings is 45-50, and the seedling cluster is required not to be damaged and the seedlings are not damaged when the root seedlings are cut;
d) inoculating and rooting seedlings: taking a 200mL cylindrical jam bottle (the bottom diameter is 5.5cm, the height is 9.5cm) as an inoculation container, wherein the thickness of a culture medium is 1.5cm, and each bottle is inoculated with 20 seedlings;
e) cultivating root seedlings: placing the seedling after rooting culture in a culture room for 4 days, setting the temperature of the culture room at 23 ℃, and adding 1500Lx artificial illumination for 11h in the daytime;
f) after 4 days, a small amount of seedling base parts are exposed to white root tips, at the moment, the transferred rooted seedlings are put into a sunlight greenhouse for hardening, the hardening temperature is 25 ℃, the illumination is 10000Lx, the time is 26 days, the rooting rate of the obtained seedlings is 98.3%, the seedling height is 2.71 +/-0.08 cm, and the number of roots is 7.0 +/-0.6.
Example 2
a) After obtaining the acacia melanoxylon tissue culture seedlings, performing enrichment culture for 20 days to obtain the enrichment seedlings;
b) transferring the proliferated seedlings for inoculation: taking 200mL cylindrical jam bottles (the bottom diameter is 5.5cm, the height is 9.5cm) as an inoculation container, the thickness of a culture medium is 1.5cm, inoculating 20 bottles, inoculating 9 bundles of seedlings in each bottle, wherein each bundle of seedlings is 4-6, and the monthly proliferation rate of the seedlings is 3.41%;
c) after the propagation seedling is cultured for 35 days, the seedling enters a seedling production stage, seedling cutting single buds with the seedling height of more than 2.0cm are selected from the propagation seedling clusters from high to low during seedling production to serve as the rooted seedlings, the number of the cut rooted seedlings is 45-50, and the seedling clusters are required not to be damaged and the seedlings are not damaged when the rooted seedlings are cut;
d) inoculating and rooting seedlings: taking a 200mL cylindrical jam bottle (the bottom is 5.5cm in diameter and 9.5cm in height) as an inoculation container, wherein the thickness of a culture medium is 1.5cm, and 15 seedlings are inoculated in each bottle;
e) cultivating root seedlings: placing the seedling after rooting culture in a culture room for 8 days, setting the temperature of the culture room at 24 ℃, and adding 2000Lx artificial light for 10 hours in the daytime;
f) after 8 days, part of the base parts of the seedlings are exposed to white root tips, then the transferred rooted seedlings are put into a sunlight greenhouse for hardening, the hardening temperature is 30 ℃, the illumination is 8000Lx, the time is 22 days, the rooting rate of the obtained seedlings is 91.3%, the height of the seedlings is 2.89 +/-0.09 cm, and the number of roots is 6.2 +/-0.4.
Example 3
a) After obtaining the acacia melanoxylon tissue culture seedlings, carrying out enrichment culture for 25 days to obtain the enrichment seedlings;
b) transferring the proliferated seedlings for inoculation: taking a 200mL cylindrical jam bottle (the bottom diameter is 5.5cm, the height is 9.5cm) as an inoculation container, the thickness of a culture medium is 1.5cm, 20 bottles are inoculated, 11 seedlings are inoculated in each bottle, each seedling is 4-6, and the monthly proliferation rate of the seedlings is 2.59%;
c) after the propagation seedling is cultured for 30 days, the seedling enters a seedling production stage, seedling cutting single buds with the seedling height of more than 2.0cm are selected from the propagation seedling clusters from high to low during seedling production to serve as the rooted seedlings, the number of the cut rooted seedlings is 30-35, and the seedling clusters are required not to be damaged and the seedlings are not damaged when the rooted seedlings are cut;
d) inoculating and rooting seedlings: taking a 200mL cylindrical jam bottle (the bottom is 5.5cm in diameter and 9.5cm in height) as an inoculation container, wherein the thickness of a culture medium is 1.5cm, and 25 seedlings are inoculated in each bottle;
e) cultivating root seedlings: placing the seedling after rooting culture in a culture room for 12 days, setting the temperature of the culture room to be 26 ℃, and adding 1800Lx artificial illumination for 11h in the daytime;
f) after 12 days, white roots grow out from the base parts of the seedlings, the transferred rooted seedlings are put into a sunlight greenhouse for hardening, the hardening temperature is 30 ℃, the illumination is 9000Lx, the time is 18 days, the rooting rate of the obtained seedlings is 84.4%, the height of the seedlings is 2.92 +/-0.10 cm, and the number of the roots is 4.8 +/-0.3.
Example 4
a) After obtaining the acacia melanoxylon tissue culture seedling, carrying out enrichment culture for 30 days to obtain an enrichment seedling;
b) transferring the proliferated seedlings for inoculation: taking a 200mL cylindrical jam bottle (the bottom diameter is 5.5cm, the height is 9.5cm) as an inoculation container, the thickness of a culture medium is 1.5cm, 20 bottles are inoculated, 12 seedlings are inoculated in each bottle, each seedling is 4-6, and the monthly proliferation rate of the seedlings is 2.50%;
c) after the propagation seedling is cultured for 25 days, the seedling enters a seedling production stage, seedling cutting single buds with the seedling height of more than 2.0cm are selected from the propagation seedling clusters from high to low during seedling production to serve as the rooted seedlings, the number of the cut rooted seedlings is 25-30, and the seedling clusters are required not to be damaged and the seedlings are not damaged when the rooted seedlings are cut;
d) inoculating and rooting seedlings: taking a 200mL cylindrical jam bottle (the bottom is 5.5cm in diameter and 9.5cm in height) as an inoculation container, wherein the thickness of a culture medium is 1.5cm, and 10 seedlings are inoculated in each bottle;
e) cultivating root seedlings: placing the seedling after rooting culture in a culture room for 24 days, setting the temperature of the culture room to be 27 ℃, and adding 2000Lx artificial light for 10 hours in the daytime;
f) after 24 days, white roots grow out from the base parts of the seedlings, the transferred rooted seedlings are put into a sunlight greenhouse for hardening, the hardening temperature is 35 ℃, the illumination is 7000Lx, the time is 6 days, the rooting rate of the obtained seedlings is 63.2%, the height of the seedlings is 2.13 +/-0.06 cm, and the number of the roots is 1.6 +/-0.2.
The culture medium for propagation seedling culture in the above examples uses MS as basic culture medium, and each liter of basic culture medium is added with 0.4-0.6mg of 6BA, 25-30g of white sugar and 5-6g of agar, pH value is adjusted to 5-6, and the culture medium is packaged in advance at 1.0-1.2kg/cm2Sterilizing at high pressure and high temperature of 120-.
The rooting seedling culture medium is prepared by using 1/2MS as basic culture medium, adding 0.4-0.6mg of 6BA, 25-30g of white sugar and 5-6g of agar into each liter of basic culture medium, adjusting pH to 5-6, and packaging the culture medium at 1.0-1.2kg/cm2Sterilizing at high pressure and high temperature of 120-.
The following data are combined to show the optimal standardized production process of acacia melanoxylon tissue culture seedlings:
1) acacia melanoxylon inoculation cycle
Selecting 60 bottles of acacia melanoxylon tissue culture seedlings with consistent growth, wherein the propagation culture period is respectively 15 days, 20 days, 25 days and 30 days, counting after 60 days of culture, namely the culture period is 15 days for total transfer 4 times, namely the culture period is 20 days for total transfer 3 times, namely the culture period is 25 days and 30 days for total transfer 2 times, and the result is that the propagation rate is the highest in the 15 days of transfer month and the propagation rate is the next in the 20 days of culture. Therefore, the propagation culture period of the acacia melanoxylon tissue culture seedling is preferably 15 to 20 days.
TABLE 1 Effect of the propagation seedling switching cycle on the growth of the seedlings
2) Inoculation density of acacia melanoxylon proliferated seedlings
Selecting acacia melanoxylon tissue culture proliferated seedlings with consistent growth, dividing the proliferated seedlings into 4-5 plants/clumps and 5-6 plants/clumps, respectively inoculating 8, 9, 10, 11 and 12 clumps into each bottle of culture medium, continuously inoculating 20 bottles for 1000 clumps, and finally, inoculating 9 clumps in each bottle with the highest proliferation rate and the largest number of seedlings larger than 2.0 cm; the proliferation rate of 8 seedlings inoculated in each bottle and the number of seedlings larger than 2.0cm are set as 2 (table 2), and the results of dividing the proliferated seedlings into 4-5 seedlings/seedling and 5-6 seedlings/seedling are the same. Therefore, the proliferated seedlings are divided into 4-6 strains/cluster, and 8-9 clusters are inoculated in each bottle, so that the production efficiency is highest.
TABLE 2 Effect of inoculation Density on Acacia melanoxylon tissue culture growth
3) Transfer cycle of production phase
Selecting 60 bottles of acacia melanoxylon tissue culture proliferated seedlings with consistent growth, respectively culturing in a culture room for 25, 30, 35 and 40 days after inoculation to generate rooted seedlings, obtaining the most rooted seedlings after 40 days of culture as a result, culturing for 35 days, continuously performing the proliferated culture on the residual seedlings after cutting the rooted seedlings, and continuously generating the rooted seedlings in the next period. Therefore, the best switching period in the production stage is 35-40 days.
TABLE 3 Effect of the transfer cycle of rooted shoots on yield
4) Inoculation density of rooted seedlings
The cut rooted seedlings are respectively inoculated into 10, 15, 20 and 25 strains to be cultured in a rooting culture medium for 20 days, so that the highest rooting rate is obtained by inoculating 20 strains, and the seedling height and the single plant biomass are the highest by inoculating 10 strains/bottle.
Therefore, in consideration of the production cost and the seedling rooting rate, 20 plants are preferably inoculated to each bottle of rooted seedlings.
TABLE 4 Effect of inoculation Density of rooted seedlings on growth of seedlings
5) Hardening time of rooted seedlings
The inoculated rooting seedlings are uniformly cultured for 30 days, respectively placed in a culture room and a sunlight greenhouse for different times, placed in the culture room for 4, 8, 12, 16, 20 and 24 days, then correspondingly moved into the sunlight greenhouse for hardening seedling and culturing for 26, 22, 18, 14, 10 and 6 days, and the rooting rate and the rooting number are better in such a way that the rooting rate and the rooting number are placed in the culture room for 4 to 8 days firstly and then are placed in the sunlight greenhouse for 22 to 26 days.
TABLE 5 influence of different time rooting and seedling hardening culture on seedlings
The above description is only a part of the embodiments of the present invention, but the design concept of the present invention is not limited thereto, and any insubstantial modifications made by the design concept should fall within the scope of infringing the present invention. However, any simple modification, equivalent change and modification of the above embodiments according to the technical essence of the present invention will still fall within the protection scope of the technical solution of the present invention without departing from the content of the technical solution of the present invention.
Claims (1)
1. A standardized production process of acacia melanoxylon tissue culture seedlings is characterized by comprising the following steps: a) after obtaining the acacia melanoxylon tissue culture seedling, carrying out proliferation culture for 15-20 days to obtain a proliferation seedling; b) transferring the proliferated seedlings for inoculation; c) culturing the inoculated proliferation seedlings for 35-40 days; d) cutting single buds in the proliferated seedlings to serve as rooted seedlings, wherein the number of the cut rooted seedlings is 25-50; e) inoculating a rooting seedling; f) culturing the rooted seedlings for 4-8 days at 23-27 ℃ and adding 1500-2000Lx artificial illumination for 10-11h in the daytime; g) hardening seedlings for 22-26 days at 25-35 ℃ under 7000-10000Lx illumination;
in the step b), the inoculation density of the proliferated seedlings is 8-9 bundles of seedlings inoculated in each bottle, and 4-6 seedlings are inoculated in each bundle;
the culture medium for the propagation seedling culture in the step c) takes MS as a basic culture medium, 0.4-0.6mg of 6BA, 25-30g of white sugar and 5-6g of agar are added into each liter of the basic culture medium, the pH value is adjusted to 5-6, and the culture medium is pre-packaged at 1.0-1.2kg/cm2Sterilizing under high pressure and high temperature;
the inoculation density of the rooted seedlings in the step e) is 10-20 seedlings inoculated in each bottle;
the culture medium for culturing the rooting seedling in the step f) takes 1/2MS as a basic culture medium, 1.0-1.5mg of IBA, 25-30g of white sugar and 5-6g of agar are added into each liter of the basic culture medium, the pH value is adjusted to 5-6, and the culture medium is pre-packaged at 1.0-1.2kg/cm2Sterilizing under high pressure and high temperature.
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