CN109744146B - Method for rooting Chinese fir tissue culture seedlings in bottles - Google Patents
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Abstract
The invention provides a method for rooting Chinese fir tissue culture seedlings in bottles, which comprises the following steps: alternately treating by high-concentration and low-concentration hormone culture media in a proliferation stage to ensure that cluster buds grow robustly and the proliferation coefficient is stable; selecting cluster buds which grow robustly and are 3.0-4.0cm from the cluster buds of the fir as tissue culture inoculated bud seedlings of the fir; inoculating the selected tissue culture spruce bud seedlings into a root primordium induction culture medium, and performing dark culture for 3-5 days; after dark culture, moving to an LED red light and blue light composite light source for culture for 14 hours every day, and culturing for 15-20 days; and (4) transplanting the Chinese fir tissue culture seedlings treated by the LED composite light source into a greenhouse, and hardening the seedlings at room temperature for 15-25 days to finish rooting in the Chinese fir tissue culture seedling bottle. The method ensures that the rooting rate of the Chinese fir tissue culture bottle seedlings before transplantation averagely reaches 91.4 percent, the rooting time is 40-45 days, and the roots of the Chinese fir tissue culture seedlings have no wound healing and are convenient to transplant and survive.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a method for rooting Chinese fir tissue culture seedlings in bottles.
Technical Field
Cunninghamia lanceolata (Cunninghamia lanifolia) is a plant of Cunninghamia of the family Cunninghamia (Taxodiaceae). Because the fir is a cross-pollinated tree species, the individual differentiation is large when the fir is propagated by seeds, and the characters of the fir female parent are difficult to maintain. By adopting the tissue culture method, the excellent properties of the parent can be maintained, and the mass production can be realized.
At present, two methods are mainly adopted for rooting in a fir tissue culture seedling bottle: firstly, adjusting a basic culture medium and a growth regulator, and inducing a fir tissue culture seedling to root; secondly, the LED is used as a new light source to promote the rooting of the tissue culture seedlings of the Chinese fir.
Chinese patent CN101480166A discloses a rooting method for tissue culture of Chinese fir, which comprises cutting Chinese fir, proliferating and subculturing plantlets, inoculating to rooting culture medium, and performing rooting culture, wherein the rooting culture medium is improved MS culture medium, and is added with hormone components such as 0.4-0.8mg/LABT, 0.1-0.5mg/L IBA, 2-6mg/L root solar diluent, etc. After 22 days of culture, the highest rooting rate of the tissue culture seedlings of the Chinese fir reaches 96 percent. But the average rooting rate is not published, and partial seedlings have the phenomena of seedling burning and seedling whitening, so that the method is difficult to apply to industrial production.
Chinese patent CN103229723A discloses a rooting induction method for tissue culture seedlings of Chinese fir, which comprises cutting and proliferating subculture seedlings, inoculating the seedlings in a pre-rooting culture medium for culture, performing pre-rooting culture for 15-25 days, and performing rooting culture. The rooting rate of the tissue culture seedlings of the Chinese fir by the method can reach 96 percent at most. But the pre-rooting culture increases the production process and the rooting time is longer.
Chinese patent CN102726299A discloses a two-step tissue culture and rapid propagation method of Chinese fir based on LED light source, which adopts red light and blue light LED combined light source with adjustable light intensity to perform step-by-step treatment in stages, namely, monochromatic red light is used as a subculture light source of Chinese fir, and then monochromatic blue light is used as a rooting culture light source. The method can improve the growth speed and the seedling robustness of the Chinese fir seedlings to a certain extent, and shorten the growth cycle and the rooting time. However, the method does not disclose specific rooting data such as rooting time, average rooting rate and the like.
Disclosure of Invention
Aiming at the defects of the technology, the invention provides a method for cultivating the seedling by three steps of root primordium induction culture medium (used for forming root primordia), LED composite light source (used for extending root tips) and room temperature seedling hardening (the growth and seedling hardening of roots are carried out at the same stage). The method for rooting in the tissue culture seedling bottle of the Chinese fir step by step fundamentally solves the problem of difficult rooting in the tissue culture process of the Chinese fir, reduces the production cost and can realize the industrialized seedling culture of the tissue culture seedling of the Chinese fir.
The invention specifically comprises the following steps:
(1) pretreating tissue culture seedlings of China fir: alternately treating with high concentration hormone culture medium and low concentration hormone culture medium for 30 days in proliferation stage, i.e. high concentration hormone for amplification at the T th time, and low concentration hormone for amplification at the T +1 th time, to obtain strong sterile fir clump bud;
(2) selecting seedlings: selecting cluster buds which are strong in growth and 3.0-4.0cm in length from the sterile fir cluster buds subjected to multiplication culture as fir tissue culture buds, and cutting off 2-3 leaves at the base of the tissue culture seedlings;
(3) formation of root primordia: inoculating the robust tissue culture seedling of the fir to a root primordium induction culture medium, and performing dark culture at 23-26 ℃ for 3-5 days to induce the formation of the root primordium of the tissue culture seedling of the fir;
(4) elongation of root tip: after dark culture, moving to an LED composite light source to irradiate for 14 hours every day; the composite light source irradiates for 14 hours every day and then continues dark culture for 10 hours; the temperature of the warm culture in the stage is 23-26 ℃, and the culture time is 15-25 days;
(5) root growth and hardening seedling: and (4) transplanting the tissue culture seedlings of the Chinese fir after the root tip elongation stage treatment into a greenhouse, and hardening the seedlings at room temperature for 10-25 days to finish rooting of the tissue culture seedlings of the Chinese fir in bottles.
The high-concentration hormone culture medium in the proliferation stage in the step (1) of the invention is as follows: MS minimal medium, 2.0-4.0mg/LKT, 1.0-2.0 mg/L6-BA, 0.5-1.0mg/L IBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the medium is adjusted to be 6.0.
The low-concentration hormone culture medium in the proliferation stage in the step (1) of the invention is as follows: MS minimal medium +1.0-2.0mg/LKT +0.2-0.5 mg/L6-BA +0.2-0.5mg/L IBA +3.0% sucrose +0.65% agar, and adjusting the pH of the medium to 6.0.
The illumination used in the proliferation culture stage in step (1) of the invention is a fluorescent lamp, and the illumination intensity is 1800 plus 2000 lux.
The root primordium induction culture medium in the step (3) of the invention is as follows: 1/4MS minimal medium, 0.01-0.1mg/LIBA, 0.01-0.1mg/L ABT1 rooting powder, 1.5% sucrose and 0.65% agar, and adjusting the pH value of the medium to 6.0.
The LED light source adopted in the root tip elongation stage in the step (4) of the invention is as follows: the ratio of red light to blue light is 1-6: 1; the red light wave peak is 660 +/-20 nm, and the blue light wave peak is 450 +/-20 nm.
Compared with the prior art, the invention has the outstanding advantages that:
1. in the proliferation stage, high-concentration and low-concentration hormones are alternately treated, so that the Chinese fir tissue culture seedling has moderate proliferation multiple, stable proliferation rate and strong seedling growth, and can create good conditions for rooting in the bottle of the later-stage Chinese fir tissue culture seedling. Because the Chinese fir tissue culture seedlings are easy to inhibit the growth of plants after being proliferated under high-concentration hormone for a long time, even cause the distortion of the plants; and the growth is slow under the low-concentration hormone, the proliferation rate is not high, and the production cost is overhigh.
2. Before the tissue culture seedlings of the Chinese fir are treated by the LED light, the tissue culture seedlings of the Chinese fir are firstly inoculated to a root primordium induction culture medium to induce the formation of the root primordium of the tissue culture seedlings of the Chinese fir. The culture medium is added with IBA of 0.01-0.1mg/L and ABT1 rooting powder of 0.01-0.1mg/L, which is only one tenth of the hormone dosage of the common rooting culture medium of tissue culture seedlings of China fir. The purpose is to promote the formation of root primordium, but not to complete the rooting of the whole fir tissue culture seedling. The culture medium can effectively promote the formation of the root primordium of the tissue culture seedlings of the fir, and can not form callus on the basal parts of the tissue culture seedlings to cause the falling of the roots in the later period.
3. And (3) in the root tip elongation stage, transferring the dark cultured tissue culture seedlings to an LED red light and blue light composite light source for culture. The aim is to use an LED composite light source to promote the extension of the root tip of the tissue culture seedling of the fir. According to the photosynthetic absorption spectral characteristics of the plants, the plants are most sensitive to the reactions of blue light with the wavelength of 400-510nm and red light with the wavelength of 610-720 nm; in addition, the red light is the essential light quality for the normal growth of crops, and plays an important role in regulating and controlling photomorphogenesis through the photosensitive pigment; and the blue light is the necessary supplementary light quality for the growth of crops, and the photosynthetic rate of the leaves can be effectively improved under the condition of red and blue compound light. Therefore, the red blue light proportioning under the wavelength can effectively promote the extension of the root tip of the fir tissue culture seedling.
4. In the root growth and hardening stage, the fir tissue culture seedlings processed by the LED composite light source are moved into a greenhouse, and the root growth and hardening process is completed at the same time. Because the extension of the root tip of the tissue culture seedling of the cedarwood is promoted by the LED composite light source, the tissue culture seedling can be directly moved into a greenhouse, so that the aim of root growth and seedling hardening can be fulfilled. The method shortens rooting time of tissue culture seedling of Cunninghamiae Lanceolatae, and reduces production cost.
Drawings
FIG. 1 shows a tissue culture seedling of Cunninghamiae Lanceolatae which has been inoculated onto a root stock induction medium;
FIG. 2 shows tissue culture seedlings of Cunninghamiae Lanceolatae after 5 days of dark culture;
FIG. 3 shows the bottle seedlings after 17 days of cultivation in an LED composite light source (R: B ratio of red light to blue light is 2: 1);
FIG. 4 shows the bottle seedlings after 20 days of seedling hardening in the greenhouse.
Detailed Description
The technical solution of the present invention is further illustrated by the following examples.
Example 1: the method is carried out in a certain tissue culture factory in Kyssal in 2016, and comprises the following specific steps:
(1) pretreating tissue culture seedlings of China fir: alternately treating with high concentration hormone and low concentration hormone in proliferation stage; the subculture seedlings are cultured in a high-concentration hormone culture medium in a proliferation stage for 30 days and then are cultured in a low-concentration hormone culture medium in a proliferation stage for 30 days, so that the clustered shoots can grow robustly and the proliferation coefficient is stabilized at 3.5 times. Wherein, the high-concentration hormone culture medium in the proliferation stage is as follows: MS basic culture medium, 4.0mg/L KT, 2.0 mg/L6-BA, 1.0mg/L IBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the culture medium is adjusted to 6.0; the low-concentration hormone culture medium in the proliferation stage comprises: MS basic culture medium, 1.0mg/L KT, 0.2 mg/L6-BA, 0.5mg/LIBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the culture medium is adjusted to be 6.0.
(2) A fir tissue culture seedling root primordium induction culture medium screening experiment comprises the following steps: inoculating the healthy and strong tissue culture spruce seedlings into root primordium induction culture media containing hormones with different concentrations, wherein the dark culture days are 5 days; after dark culture, moving the culture medium to an LED composite light source, and irradiating for 14 hours every day; after the composite light source irradiates for 14 hours every day, the dark culture is continued for 10 hours; the culture temperature at this stage is 23-26 deg.C, and the culture time is 17 days; then the fir tissue culture seedlings processed by the LED composite light source are moved into a greenhouse and hardened for 20 days at room temperature. Wherein, the root primordium induction culture medium is 1/4MS minimal medium, and IBA and ABT1 rooting powder with different concentrations are added in the culture medium; 1.5 percent of sucrose and 0.55 percent of agar, and adjusting the pH value of the culture medium to 6.0; the red and blue light mixing ratio R to B in the LED composite light source adopted in the root tip extension stage is 2 to 1.
The experiment is repeated for 5 times, 100 plants are cultured each time, and the rooting rate after 20 days of seedling hardening at room temperature is counted and used as the basis for screening the optimal root primordium induction culture medium.
The results in Table 1 show that the addition of IBA 0.05mg/L and ABT1 # rooting powder 0.05mg/L to the root stock induction culture medium is optimal, and the rooting rate can reach 91.4%.
Table 1: statistical result of root primordium induction screening experiment of Chinese fir tissue culture seedling
Example 2: the method is carried out in a certain tissue culture factory in Kyssal in 2017, and comprises the following specific steps:
(1) pretreating tissue culture seedlings of China fir: alternately treating with high concentration hormone and low concentration hormone in proliferation stage; the subculture seedlings are cultured in a high-concentration hormone culture medium in a proliferation stage for 30 days and then are cultured in a low-concentration hormone culture medium in a proliferation stage for 30 days, so that the clustered shoots can grow robustly and the proliferation coefficient is stabilized at 3.5 times. Wherein, the high-concentration hormone culture medium in the proliferation stage is as follows: MS basic culture medium, 4.0mg/L KT, 2.0 mg/L6-BA, 1.0mg/L IBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the culture medium is adjusted to 6.0; the low-concentration hormone culture medium in the proliferation stage comprises: MS basic culture medium, 1.0mg/L KT, 0.2 mg/L6-BA, 0.5mg/LIBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the culture medium is adjusted to be 6.0.
(2) LED composite light source screening experiment: inoculating the robust tissue culture spruce bud seedlings into a root primordium induction culture medium, and performing dark culture for 5d to induce the formation of the root primordium of the tissue culture spruce seedlings; after dark culture, moving the culture medium to a composite light source with different proportions of red light and blue light of an LED for 14 hours, and continuously performing dark culture for 10 hours after the composite light source irradiates for 14 hours every day, wherein the culture temperature is 23-26 ℃. Wherein, the root primordium induction culture medium is as follows: 1/4MS minimal medium, 0.05mg/L IBA, 0.05mg/L ABT1 rooting powder, 1.5% sucrose and 0.55% agar, and adjusting the pH value of the medium to 6.0.
The experiment is repeated for 5 times, 100 strains are obtained each time, and the shortest days for extending the longest root tip to 1cm and the obtained number of the root tips are used as the basis for screening the optimal LED composite light source.
Table 2: statistical result of LED composite light source screening experiment
By the method, the screened optimal LED composite light source has the red-blue light ratio R: B of 2:1, the average required days for the longest root tip to extend to 1cm is the shortest, and the average required days are 16.8 days; and the number of the root tips obtained by each fir tissue culture seedling is the maximum, and reaches 5.4 root tips.
Example 3: the method is carried out in a certain tissue culture factory in Kyssal in 2017, and comprises the following specific steps:
(1) pretreating tissue culture seedlings of China fir: alternately treating with high concentration hormone and low concentration hormone in proliferation stage; the subculture seedlings are cultured in a high-concentration hormone culture medium in a proliferation stage for 30 days and then are cultured in a low-concentration hormone culture medium in a proliferation stage for 30 days, so that the clustered shoots can grow robustly and the proliferation coefficient is stabilized at 3.5 times. Wherein, the high-concentration hormone culture medium in the proliferation stage is as follows: MS basic culture medium, 4.0mg/L KT, 2.0 mg/L6-BA, 1.0mg/L IBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the culture medium is adjusted to 6.0; the low-concentration hormone culture medium in the proliferation stage comprises: MS basic culture medium, 1.0mg/L KT, 0.2 mg/L6-BA, 0.5mg/LIBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the culture medium is adjusted to be 6.0.
(2) Selecting seedlings: selecting cluster buds which are strong in growth and 3.0-4.0cm in length from the sterile fir cluster buds subjected to multiplication culture as fir tissue culture buds, and cutting off 2-3 leaves at the base of the tissue culture seedlings;
(3) formation of root primordia: inoculating the robust tissue culture seedling of Cunninghamiae Lanceolatae into root primordium induction culture medium (shown in figure 1), and dark culturing at 23-26 deg.C for 5 days to induce formation of root primordium of the tissue culture seedling of Cunninghamiae Lanceolatae; the root primordium induction culture medium comprises: 1/4MS minimal medium, 0.05mg/L IBA, 0.05mg/L ABT1 rooting powder, 1.5% sucrose and 0.65% agar, and adjusting the pH value of the minimal medium to 6.0; the effect after 5 days of dark culture is shown in FIG. 2;
(4) elongation of root tip: after dark culture, moving to an LED composite light source to irradiate for 14 hours every day; the composite light source irradiates for 14 hours every day and then continues dark culture for 10 hours; the temperature of the warm culture at the stage is 23-26 ℃, and the culture time is 17 days; the LED light source used in the root tip elongation stage is: the ratio of red light to blue light R to B is 2 to 1; the red light wave peak is 660 +/-20 nm, and the blue light wave peak is 450 +/-20 nm; the effect at this stage after 17 days of culture is shown in FIG. 3;
(5) root growth and hardening seedling: and (4) transferring the fir tissue culture seedlings after the root tip elongation stage treatment into a greenhouse, and hardening the seedlings at room temperature for 20 days. The effect 20 days after acclimatization is shown in figure 4.
Claims (2)
1. A method for rooting in a fir tissue culture seedling bottle is characterized by comprising the following steps:
(1) pretreating tissue culture seedlings of China fir: alternately treating with high concentration hormone culture medium and low concentration hormone culture medium for 30 days at proliferation stage, i.e. high concentration hormone for amplification at the T th time, and low concentration hormone for amplification at the T +1 th time, to obtain strong sterile fir clump bud; the high-concentration hormone culture medium in the proliferation stage comprises: MS basic culture medium, 4.0mg/L KT, 2.0 mg/L6-BA, 1.0mg/L IBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the culture medium is adjusted to 6.0; the low-concentration hormone culture medium in the proliferation stage comprises: MS basic culture medium, 1.0mg/L KT, 0.2 mg/L6-BA, 0.5mg/LIBA, 3.0 percent of cane sugar and 0.65 percent of agar, and the pH value of the culture medium is adjusted to be 6.0;
(2) selecting seedlings: selecting cluster buds which are strong in growth and 3.0-4.0cm from the sterile fir cluster buds subjected to multiplication culture as fir tissue culture buds, and cutting off 2-3 leaves at the base of the tissue culture seedlings;
(3) formation of root primordia: inoculating the robust tissue culture seedling of the fir to a root primordium induction culture medium, and performing dark culture at 23-26 ℃ for 3-5 days to induce the formation of the root primordium of the tissue culture seedling of the fir; the root primordium induction culture medium is 1/4MS minimal medium, 0.05mg/L IBA, 0.05mg/L ABT1 rooting powder, 1.5 percent of sucrose and 0.65 percent of agar, and the pH value of the culture medium is adjusted to 6.0;
(4) elongation of root tip: after dark culture, moving to an LED composite light source to irradiate for 14 hours every day; continuously culturing in dark for 10 hours after the LED composite light source irradiates for 14 hours every day, wherein the proportion of red light to blue light is R: B =2: 1; the red light wave peak is 660 +/-20 nm, and the blue light wave peak is 450 +/-20 nm; the culture temperature at this stage is 23-26 deg.C, and the culture time is 15-20 days;
(5) root growth and hardening seedling: and (4) transplanting the tissue culture seedlings of the Chinese fir after the root tip elongation stage treatment into a greenhouse, and hardening the seedlings at room temperature for 15-25 days to finish rooting of the tissue culture seedlings of the Chinese fir in bottles.
2. The method for rooting in tissue culture fir seedlings in bottles as claimed in claim 1, wherein the illumination used in the propagation culture stage in step (1) is fluorescent lamp, and the illumination intensity is 1800 and 20001 ux.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104429956A (en) * | 2014-11-24 | 2015-03-25 | 广西大学 | Method for test-tube rooting of cunninghamia lanceolata tissue culture seedlings by using compound LED light source |
| CN106922534A (en) * | 2017-03-27 | 2017-07-07 | 河池乐康生态农业科技有限公司 | A kind of method that tissue cultures quickly breed China fir |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104429956A (en) * | 2014-11-24 | 2015-03-25 | 广西大学 | Method for test-tube rooting of cunninghamia lanceolata tissue culture seedlings by using compound LED light source |
| CN106922534A (en) * | 2017-03-27 | 2017-07-07 | 河池乐康生态农业科技有限公司 | A kind of method that tissue cultures quickly breed China fir |
Non-Patent Citations (1)
| Title |
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| 杉木再生系统的比较研究;朱木兰等;《分子细胞生物学报》;20070831;第40卷(第4期);第240页左栏第3段和表3 * |
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