CN113317200B - Tissue culture medium for male populus diversifolia plants and application of tissue culture medium - Google Patents
Tissue culture medium for male populus diversifolia plants and application of tissue culture medium Download PDFInfo
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- CN113317200B CN113317200B CN202110709558.7A CN202110709558A CN113317200B CN 113317200 B CN113317200 B CN 113317200B CN 202110709558 A CN202110709558 A CN 202110709558A CN 113317200 B CN113317200 B CN 113317200B
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Abstract
The invention discloses a tissue culture medium for male poplar plants and application thereof, and a method for establishing a regeneration system of the male poplar plants. The tissue culture medium for the male poplar plant comprises a callus induction medium for inducing an explant to form callus, a callus differentiation medium for inducing the callus to grow adventitious buds and a rooting medium for inducing the adventitious buds to root. The tissue culture medium for the male poplar plant can be used for quickly and efficiently obtaining the male poplar plant and in-vitro regenerated seedlings.
Description
Technical Field
The invention relates to the field of plant culture, in particular to a tissue culture medium for male populus diversifolia plants and application thereof.
Background
The poplar, a generic name of Populus (Populus) plants in Salicaceae, has the characteristics of strong adaptability, wide distribution, strong regeneration capacity, straight trunk, fast lumber formation, relatively small genome and the like, so that the Populus is planted in a large quantity in the global range and is always used as a mode plant for forest molecular research after the first sequencing of Populus trichocarpa genome is completed. The genus Populus is further classified into five groups of Populus tremula (Tacamahaca), Populus alba (Leuce), Populus nigra (Aigeiros), Populus euphratica (Turanga), and Populus megalophylla (Leucoses) on the classification system. Populus japonica (Populus cathayana) is one of the poplar group members, favors wet or dry cold climate, has straight wood texture, fine structure and easy processing, and is commonly used as artificial forest and street tree planting in various parts of China.
The plant tissue culture technology is a biological technology for culturing plant tissues in vitro and obtaining complete plants, which is proposed in the 19 th century based on the cell totipotency hypothesis of Schwann. The entire plant tissue culture process generally comprises five steps: carrying out detoxification treatment on the explant; inducing the explant to dedifferentiate to obtain callus; inducing the callus to redifferentiate to obtain adventitious buds; inducing adventitious buds to root; hardening and transplanting the seedlings. Tissue culture technology is one of important technical means for rapid propagation of plants which are difficult to propagate conventionally, and has important application value in genetic breeding due to low influence of environmental factors, rapid propagation and large quantity, and capability of preserving excellent characters of parents. In addition, with the rapid development of transgenic technology, the use of tissue culture technology as a means to obtain a large number of stably inherited transgenic plants is also an important means to transgenically transform many plants with longer normal growth cycles.
The origin of poplar tissue culture is earlier, and scholars continuously use various poplars such as black populus tremuloides and populus deltoides as explant materials to perform in vitro regeneration culture in the early development stage of plant tissue culture technology, but the poplar tissue culture technology is rapidly developed until the first complete poplar regeneration plant is obtained in the 70 th 20 th century. To date, scholars at home and abroad have great breakthrough in the research of regeneration systems of all poplars in the major market, including poplars of the populus tremuloides group, populus 84K, populus Sinkiangensis and populus deltoides, populus tremuloides of the populus tremuloides group, populus tremuloides of the populus tremuloides group, populus tremula plants with mature regeneration systems, mostly unisexual clonal plants, and few reports on the rapid and effective regeneration systems established for male populus tremula.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a tissue culture medium for male populus diversifolia plants.
The invention also aims to provide the application of the tissue culture medium for the male poplar plant in establishing a regeneration system of the male poplar plant.
The invention also aims to provide a method for establishing a poplar male plant regeneration system.
One of the purposes of the invention is realized by adopting the following technical scheme: a tissue culture medium for male poplar plants comprises a callus induction medium for inducing explants to form callus, a callus differentiation medium for inducing the callus to grow adventitious buds and a rooting medium for inducing the adventitious buds to root;
the callus induction culture medium comprises 2.7g/L of WPM, 30g/L of sucrose, 6g/L, TDZ 0.1.1-0.3 mg/L, IBA 0.3.3-0.5 mg/L of agar, or the callus induction culture medium comprises 2.7g/L of WPM, 30g/L of sucrose, 6g/L, TDZ 0.1.1-0.3 mg/L, NAA 0.3.3-0.5 mg/L of agar, or the callus induction culture medium comprises 2.7g/L of WPM, 30g/L of sucrose, 6g/L of agar, 1.0mg/L of 6-BA, and 0.5-1.0 mg/L of 2,4-D, or the callus induction culture medium comprises 2.7g/L of WPM, 30g/L of sucrose, 6g/L of agar, 0g/L of agar, 0.3mg/L of 0.5-0 mg/L of agar, or the callus induction culture medium comprises 2.7g/L of WPM, 30g/L of sucrose, 6g/L of agar, ZT 2.0mg/L, NAA 1.0.0 mg/L, or the callus induction culture medium comprises WPM 2.7g/L, sucrose 30g/L, agar 6g/L, 6-BA 0.5 mg/L-1.5 mg/L, NAA 0.8 mg/L-1.5 mg/L;
the callus differentiation culture medium comprises 4.74g/L MS, 30g/L sucrose, 7g/L agar and 1.0-1.5 mg/L, NAA 0.1 mg/0.1-1.0 mg/L6-BA;
the rooting culture medium comprises 2.6g/L WPM, 20g/L sucrose and 7g/L, IBA 0.1.1-0.2 mg/L, NAA 0.02.02-0.2 mg/L agar.
As an embodiment, the callus induction medium comprises WPM 2.7g/L, sucrose 30g/L, agar 6g/L, TDZ 0.1.1 mg/L, NAA 0.3.3 mg/L; and/or the presence of a gas in the atmosphere,
the callus differentiation culture medium comprises 4.74g/L MS, 30g/L sucrose, 7g/L agar and 1.0mg/L, NAA 0.1.1 mg/L6-BA; and/or the presence of a gas in the atmosphere,
the rooting medium comprises 2.6g/L WPM, 20g/L sucrose and 7g/L, IBA 0.2.2 mg/L, NAA 0.02.02 mg/L agar.
In one embodiment, the callus induction medium, the callus differentiation medium and the rooting medium each have a pH of 5.8.
The second purpose of the invention is realized by adopting the following technical scheme: the tissue culture medium for the male poplar plant is applied to the establishment of a regeneration system of the male poplar plant.
The third purpose of the invention is realized by adopting the following technical scheme: a method for establishing a poplar male plant regeneration system comprises the following steps:
1) selection and sterilization of explants: selecting leaves and top stem segments of male populus diversifolia plants as explants, placing the explants in an aseptic bottle in an aseptic environment after washing with tap water, sterilizing the explants with ethanol and aseptic water, then sterilizing the explants with NaClO and washing the explants with aseptic water, then sucking water on the surfaces of the explant materials with aseptic filter paper, cutting the leaf explants into small blocks, cutting the stem segment explants into small segments with an aseptic scalpel, and cutting a plurality of wounds on the back surfaces of the block explants and the segment explants;
2) callus induction: inoculating the block explant obtained in the step 1) into a solid callus induction culture medium, and culturing for 20-30 days in a dark environment to induce callus; wherein the callus induction culture medium comprises WPM 2.7g/L, sucrose 30g/L, agar 6g/L, TDZ 0.1-0.3 mg/L, IBA 0.3-0.5 mg/L, or the callus induction culture medium comprises WPM 2.7g/L, sucrose 30g/L, agar 6g/L, TDZ 0.1-0.3 mg/L, NAA 0.3.3-0.5 mg/L, or the callus induction culture medium comprises WPM 2.7g/L, sucrose 30g/L, agar 6g/L, 6-BA 1.0mg/L, 2, 4-D0.5 mg/L-1.0 mg/L, or the callus induction culture medium comprises WPM 2.7g/L, sucrose 30g/L, agar 6-BA 1.0mg/L, 2, 4-D0.5 mg/L-1.0 mg/L, 6g/L, ZT 2.0.0 mg/L, NAA 1.0.0 mg/L agar, or 2.7g/L WPM, 30g/L sucrose, 6g/L agar, 0.5 mg/L-1.5 mg/L, NAA 0.8 of 6-BA and 0.8 mg/L-1.5 mg/L callus induction culture medium; when the small leaf is inoculated on the callus induction culture medium, the paraxial surface is upwards placed on the callus induction culture medium, and when the small stem is inoculated on the callus induction culture medium, the morphological upper end is upwards inserted into the callus induction culture medium and the half of the morphological upper end is exposed outside;
3) differentiation culture: inoculating the callus formed in the step 2) to a callus differentiation culture medium, culturing for 25-35 days, and culturing to obtain adventitious buds of male populus diversifolia plants; wherein the callus differentiation culture medium comprises 4.74g/L MS, 30g/L sucrose, 7g/L agar and 1.0 mg/L-1.5 mg/L, NAA 0.1 mg/L-1.0 mg/L6-BA;
4) rooting culture: taking out the adventitious bud of the male poplar plant formed in the step 3), cutting off a bud of 2-3 cm, transferring the bud into a rooting culture medium, and culturing for 25-35 days to obtain a rooted male poplar plant sterile tissue culture seedling; wherein the rooting culture medium comprises 2.6g/L of WPM, 20g/L of cane sugar and 7g/L, IBA 0.1.1-0.2 mg/L, NAA 0.02.02-0.2 mg/L of agar;
5) hardening and transplanting tissue culture seedlings: transplanting the tissue culture seedlings of the male populus diversifolia plants with the seedling height of 4.5-5.5 cm into sterile soil, covering a preservative film for moisturizing, gradually and partially opening the preservative film after 7 days until the leaves of the seedlings are completely exposed in the air and cannot be dehydrated, and finishing the establishment of the regeneration system of the male populus diversifolia plants.
As an implementation mode, 2-3 young leaves at the top end of a 1-month-old cutting seedling and a stem section with the top end of 4-6 cm are selected as explants in the step 1); and/or the presence of a gas in the atmosphere,
the step 1) is to sterilize the explant with 75% ethanol for 30s, and/or,
in the step 1), 1% of NaClO is adopted to disinfect the leaf explant for 2min, 1% of NaClO is adopted to disinfect the stem explant for 5min, and/or,
in the step 1), the leaf explant is cut into small blocks with the size of 1 multiplied by 1cm by using a sterile scalpel, and the stem explant is cut into small sections with the size of 1 cm-2 cm.
As an embodiment, the callus induction medium employed in the step 2) comprises WPM 2.7g/L, sucrose 30g/L, agar 6g/L, TDZ 0.1.1 mg/L, NAA 0.3.3 mg/L; and/or the presence of a gas in the atmosphere,
the pH value of the callus induction culture medium adopted in the step 2) is 5.8; and/or the presence of a gas in the atmosphere,
the temperature in the dark environment in the step 2) is 25 ℃, and the humidity is 70%.
As one embodiment, the callus differentiation medium used in the step 3) comprises MS 4.74g/L, sucrose 30g/L, agar 7g/L, 6-BA 1.0mg/L, NAA 0.1.1 mg/L; and/or the presence of a gas in the atmosphere,
the pH value of the callus differentiation culture medium adopted in the step 3) is 5.8; and/or the presence of a gas in the atmosphere,
in the step 3), the callus inoculated on the callus differentiation culture medium is cultured with adventitious buds under the conditions of 25 ℃ of temperature, 70% of humidity, 1000lux of illumination intensity and 14 hours/day of illumination time.
As an embodiment, the rooting medium adopted in the step 4) comprises 2.6g/L of WPM, 20g/L of cane sugar, 7g/L, IBA 0.2.2 mg/L, NAA 0.02.02 mg/L of agar; and/or the presence of a gas in the atmosphere,
the pH values of the rooting culture media adopted in the step 4) are all 5.8; and/or the presence of a gas in the atmosphere,
and in the step 4), the adventitious bud inoculated on the rooting culture medium is used for culturing the rooted tissue culture seedling under the conditions of 25 ℃ of temperature, 70% of humidity, 1000lux of illumination intensity and 14 hours/day of illumination time.
As an embodiment, the sterile soil in the step 5) is prepared by mixing the following components in a volume ratio of 1: 1 and vermiculite.
Compared with the prior art, the invention has the beneficial effects that: the method for establishing the poplar male plant regeneration system can quickly and efficiently obtain the poplar male plant and the in-vitro regenerated seedling, can obtain a large number of regenerated plants, and has the advantages of high repeatability, short culture period and robust regenerated seedling. According to the characteristics of the male poplar plant, the method for establishing the regeneration system of the male poplar plant selects leaves and stem sections as explants, sets different culture mediums and hormone combinations at different culture stages, improves the dedifferentiation and redifferentiation efficiency of the male poplar plant, obtains a large number of regenerated plants through three-step culture, has low cost and high efficiency, and can quickly expand and propagate through subculture.
Drawings
FIG. 1A, FIG. 1B and FIG. 1C are graphs showing callus results obtained by induction culture using male stem segments of populus diversifolia as explants;
FIGS. 1D and 1E are graphs showing the results of differentiating and culturing adventitious buds using male stem segments of populus diversifolia as explants;
FIG. 1F is a diagram showing the results of regenerated seedlings obtained by rooting culture using stem segments of male populus diversifolia as explants;
FIG. 1G is a result diagram showing the growth of adventitious bud roots in rooting culture using male stem segments of populus diversifolia as explants;
FIG. 2A is a diagram showing callus results obtained by induction culture using male leaves of populus diversifolia as explants; wherein the hormone composition in the callus induction culture medium is 6-BA + NAA;
FIG. 2B is a diagram showing callus results obtained by induction culture using male leaves of populus diversifolia as explants; wherein the hormone combination in the callus induction culture medium is ZT + NAA;
FIG. 2C is a diagram showing callus results obtained by induction culture using male leaves of populus diversifolia as explants; wherein the hormone composition in the callus induction culture medium is TDZ + IBA;
FIG. 2D is a diagram showing callus results obtained by induction culture using male leaves of populus diversifolia as explants; wherein the hormone composition in the callus induction culture medium is TDZ + NAA.
Detailed Description
The present invention will be further described with reference to the accompanying drawings and the detailed description, and it should be noted that any combination of the embodiments or technical features described below can be used to form a new embodiment without conflict.
The tissue culture medium for the male poplar plants provided by the embodiment of the invention comprises a callus induction medium for inducing explants to form callus, a callus differentiation medium for inducing the callus to grow adventitious buds and a rooting medium for inducing the adventitious buds to root.
The callus induction culture medium takes a WPM culture medium as a basic culture medium, the callus induction culture medium comprises 2.7g/L of WPM, 30g/L of sucrose, 6g/L of agar, 0.1-0.3 mg/L of thiadiazolyl urea (TDZ) and 0.3-0.5 mg/L of indolebutyric acid (IBA), or the callus induction culture medium comprises 2.7g/L of WPM, 30g/L of sucrose, 6g/L, TDZ 0.1 of agar, 0.1-0.3 mg/L of agar and 0.3-0.5 mg/L of naphthylacetic acid (NAA), or the callus induction culture medium comprises 2.7g/L of WPM, 30g/L of sucrose, 6g/L of agar, 1.0mg/L of 6-benzylaminopurine (6-BA), 2, 4-dichlorophenoxyacetic acid (2,4-D)0.5 mg/L-1.0 mg/L, or the callus induction culture medium comprises WPM 2.7g/L, sucrose 30g/L, agar 6g/L, Zeatin (ZT)2.0mg/L, NAA 1.0.0 mg/L, or the callus induction culture medium comprises WPM 2.7g/L, sucrose 30g/L, agar 6g/L, 6-BA 0.5 mg/L-1.5 mg/L, NAA 0.8 mg/L-1.5 mg/L. The callus differentiation culture medium takes an MS culture medium as a basic culture medium and comprises 4.74g/L of MS, 30g/L of cane sugar, 7g/L of agar and 1.0-1.5 mg/L, NAA 0.1.1-1.0 mg/L of 6-BA. The rooting culture medium takes a WPM culture medium as a basic culture medium and comprises 2.6g/L of WPM, 20g/L of cane sugar and 7g/L, IBA 0.1.1-0.2 mg/L, NAA 0.02.02-0.2 mg/L of agar. The induction rate of the male poplar plants is improved by the matching and the using amount of the components in the callus induction culture medium, the callus differentiation culture medium and the rooting culture medium provided by the embodiment of the invention. The starting materials used in the examples of the present invention are all those conventionally purchased by those skilled in the art, unless otherwise specifically required.
The method for establishing the poplar male plant regeneration system provided by the embodiment of the invention comprises the following operation steps:
s1: selecting suitable explants for washing and sterilization to obtain sterile explants;
s2: inoculating the sterile explant to a callus induction culture medium to culture and induce callus;
s3: inoculating the obtained callus to a callus differentiation culture medium for culture, and differentiating adventitious buds;
s4: inoculating the adventitious bud to a rooting culture medium for culturing, and inducing to root to obtain a rooted seedling;
s5: hardening and transplanting the seedlings to obtain complete plants.
Wherein, the populus is a tree species of a populus group of populus of salicaceae.
The specific operation method comprises the following steps:
(1) appropriate explant selection and Disinfection
Selecting 2-3 young leaves at the top end of a 1-month-old cutting seedling and a stem section with the top end of about 5cm as explants, and washing for 30min by using tap water; then transferring the leaves into a sterile bottle in an ultraclean workbench, sterilizing the leaves with 75% ethanol for 30s, cleaning the leaves with sterile water for 2-3 times, sterilizing the leaf explants with 1% NaClO (effective chlorine content) for 2min, sterilizing the stem explants with 1% NaClO (effective chlorine content) for 5min, cleaning the leaf explants and the stem explants with sterile water for 5-6 times, and then sucking the water on the surfaces of the leaves and the stem surface with filter paper; finally, the leaves are cut into small pieces with the size of about 1 multiplied by 1cm by a sterile scalpel, a plurality of tiny wounds are cut on the back, and the stem segments are cut into small segments with the size of 1 cm-2 cm and a plurality of wounds are cut for standby.
(2) Callus induction
Inoculating the sterilized male populus diversifolia plant explant to a sterile solid callus induction culture medium, wherein the paraxial surface is upwards placed on the callus induction culture medium during leaf inoculation, the morphological upper end is upwards inserted into the callus induction culture medium and is exposed to the outside half, and then the callus is cultured and induced in a climatic chamber for 30 days in dark at the temperature of 25 ℃ and the humidity of 70%. FIG. 1A, FIG. 1B and FIG. 1C are graphs showing the callus results obtained by induction culture using male stem segments of populus diversifolia as explants, and it can be seen that the callus grows well. FIGS. 2A, 2B, 2C and 2D are graphs showing callus results obtained by induction culture using leaves of male poplar as explants, wherein the hormone combination in the callus induction medium of FIG. 2A is 6-BA + NAA; FIG. 2B callus induction medium hormone combination ZT + NAA; FIG. 2C shows the callus induction medium with TDZ + IBA as the hormone combination; the hormone combination in the callus induction medium of FIG. 2D was TDZ + NAA. As can be seen from the figure, the callus obtained with the stem segment as explant was better in quality than the callus obtained with the leaf segment as explant.
The callus induction culture medium takes a WPM culture medium as a basic culture medium, the callus induction culture medium comprises WPM (2.7g/L) + sucrose (30g/L) + agar (6g/L) + TDZ (0.1 mg/L-0.3 mg/L) + IBA (0.3 mg/L-0.5 mg/L), or the callus induction culture medium comprises WPM (2.7g/L) + sucrose (30g/L) + agar (6g/L) + TDZ (0.1 mg/L-0.3 mg/L) + NAA (0.3 mg/L-0.5 mg/L), or the callus induction culture medium comprises WPM (2.7g/L) + sucrose (30g/L) + agar (6g/L) +6-BA (1.0mg/L) +2,4-D (0.5 mg/L-1.0 mg/L), or the callus induction culture medium comprises WPM (2.7g/L) + sucrose (30g/L) + agar (6g/L) + ZT (2.0mg/L) + NAA (1.0mg/L), or the callus induction culture medium comprises WPM (2.7g/L) + sucrose (30g/L) + agar (6g/L) +6-BA (0.5 mg/L-1.5 mg/L) + NAA (0.8 mg/L-1.5 mg/L). Table 1 shows the effect of the WPM medium and MS medium, respectively, in combination with different concentrations of hormones on callus induction of male poplar plants. As can be seen from Table 1, a large amount of callus can be rapidly obtained from young stem segments of male populus diversifolia plants under various culture conditions, and the effect of obtaining the callus from the stem segment explants is better compared with leaf explants. And when the WPM culture medium is used as a basic culture medium and the TDZ concentration is 0.1mg/L, NAA concentration is 0.3mg/L, the stem section is used as an explant, a large amount of compact green callus can be obtained, the quality of the formed callus is good, and the induction effect is good. Therefore, the TDZ concentration is preferably 0.1mg/L, and the NAA concentration is preferably 0.3 mg/L. As a preferred embodiment, the callus induction medium comprises WPM (2.7g/L) + sucrose (30g/L) + agar (6g/L) + TDZ (0.1mg/L) + NAA (0.3 mg/L). Wherein the callus induction medium has a pH of 5.8.
TABLE 1
(3) Differential culture
Taking out the obtained callus from the culture bottle, removing the redundant callus induction culture medium, cutting off the browned part, transferring to a fresh callus differentiation culture medium, and culturing in an artificial climate box. The culture conditions were: the temperature is 25 ℃, the humidity is 70 percent, and the illumination is carried out for 14 hours/day by 1000 lux. After 30 days or so of differentiation culture, the adventitious bud of the male poplar plant is cultured. FIGS. 1D and 1E are graphs showing the results of differentiating and culturing adventitious buds with male stem segments of populus diversifolia as explants, which are abundant and robust.
The callus differentiation culture medium takes an MS culture medium as a basic culture medium and comprises MS (4.74g/L), sucrose (30g/L), agar (7g/L), 6-BA (1.0 mg/L-1.5 mg/L) and NAA (0.1 mg/L-1.0 mg/L). The callus differentiation culture medium provided by the embodiment of the invention takes the MS culture medium as the basal medium, and is more beneficial to callus differentiation adventitious buds because the WPM culture medium is used as the basal medium in the differentiation culture stage of the male poplar and the callus is easy to brown. Table 2 shows the effect of MS medium in combination with different concentrations of hormones on callus differentiation of male populus diversifolia.
Although the 6-BA can promote the adventitious bud differentiation of the male poplar plant in a sufficient amount, when the concentration of the 6-BA reaches 1.5mg/L, the adventitious bud elongation is inhibited, and the NAA has little influence on the growth of the adventitious bud of the male poplar plant when the concentration of the NAA is 0.1 mg/L-1.0 mg/L. As can be seen from Table 2, at a concentration of 1.0mg/L, NAA for 6-BA, 0.1mg/L or 0.3mg/L or 0.8mg/L, the number of shoots on average was about 7 and the shoots were robust. In view of cost saving, the concentration of 6-BA is preferably 1.0mg/L, NAA and the concentration is preferably 0.1 mg/L. As a preferred embodiment, the callus differentiation medium comprises MS (4.74g/L) + sucrose (30g/L) + agar (7g/L) +6-BA (1.0mg/L) + NAA (0.1 mg/L).
TABLE 2
(4) Rooting culture
Taking out the clustered adventitious buds of the male poplar plant, cutting off buds about 2-3 cm, transferring the buds into a rooting culture medium for inducing rooting, wherein the culture conditions are the same as those in the differentiation culture stage, and after rooting culture is carried out for about 30 days, obtaining the rooted male poplar plant sterile tissue culture seedling. FIG. 1F is a diagram showing the results of the regenerated seedlings obtained by rooting culture using the stem segment of male populus tomentosa as an explant, and FIG. 1G is a diagram showing the results of the growth of the adventitious bud root by rooting culture using the stem segment of male populus tomentosa as an explant, which shows that the regenerated seedlings have a large and slender rooting amount.
The rooting culture medium takes a WPM culture medium as a basic culture medium and comprises WPM (2.6g/L), sucrose (20g/L), agar (7g/L), IBA (0.1 mg/L-0.2 mg/L) and NAA (0.02 mg/L-0.2 mg/L). Wherein the rooting medium has a pH of 5.8. Table 3 shows the effect of the WPM culture medium and the hormone combinations with different concentrations on the adventitious bud rooting of the male poplar plant, and it can be seen that the rooting culture medium provided by the embodiment of the invention can improve the rooting rate of the adventitious bud of the male poplar plant. IBA with proper concentration can promote the growth of lateral roots of male populus diversifolia plants, NAA with proper concentration can promote the growth of main roots, but the male populus diversifolia plants are more sensitive to NAA, and as can be seen from the table 3, NAA with the concentration of 0.02mg/L can promote the rooting of the male populus diversifolia plants, and NAA with the concentration of more than 0.1mg/L can obviously inhibit the growth of the male populus diversifolia roots. When the concentration of NAA is 0.02mg/L, the rooting rate is 100%, the roots are long and thin, and the growth is fast, so the concentration of NAA is preferably 0.02 mg/L. As a preferred embodiment, the rooting medium comprises WPM (2.6g/L) + sucrose (20g/L) + agar (7g/L) + IBA (0.2mg/L) + NAA (0.02mg/L), and the rooting medium provided by the embodiment can rapidly obtain a large number of regenerated seedlings with strong roots.
TABLE 3
(5) Transplanting and hardening seedlings
Taking out the regenerated seedlings with the height of about 5cm from the culture bottle, cleaning the rooting culture medium remained at the root, transplanting the regenerated seedlings into sterile soil (wherein the volume ratio of nutrient soil to vermiculite is 1: 1), covering a preservative film for preserving moisture, gradually and partially opening the preservative film after 7 days until the leaves of the seedlings are completely exposed in the air and can not be dehydrated, and normally transplanting the seedlings under the required conditions for culture.
In addition, the composition of the MS medium and the composition of the WPM medium are shown in Table 4 and Table 5, respectively.
TABLE 4
TABLE 5
The method for establishing the poplar male plant regeneration system provided by the embodiment of the invention can quickly and efficiently obtain the poplar male plant and the in-vitro regenerated seedling, can obtain a large number of regenerated plants, and has the advantages of high repeatability, short culture period and robust regenerated seedling. According to the characteristics of the male poplar plant, the method for establishing the male poplar plant regeneration system provided by the embodiment of the invention selects leaves and stem sections as explants, sets different culture mediums and hormone combinations at different culture stages, improves dedifferentiation and redifferentiation efficiency of the male poplar plant, obtains a large amount of regenerated plants through three-step culture, has low cost and high efficiency, and can rapidly propagate through subculture. The invention provides an ideal experimental system for the research of cell engineering, genetic engineering and genetic improvement of poplar, in particular to the research of poplar sex determination.
The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.
Claims (10)
1. A tissue culture medium for male poplar plants is characterized by consisting of a callus induction medium for inducing stem explant to form callus, a callus differentiation medium for inducing the callus to grow adventitious buds and a rooting medium for inducing the adventitious buds to root;
the callus induction culture medium consists of the following substances: WPM 2.7g/L + sucrose 30g/L + agar 6g/L + TDZ 0.1mg/L + NAA 0.3 mg/L-0.5 mg/L, or the callus induction medium is composed of the following substances: WPM 2.7g/L + sucrose 30g/L + agar 6g/L +6-BA 1.0mg/L +2, 4-D0.5 mg/L-1.0 mg/L, or the callus induction medium consists of the following substances: WPM 2.7g/L + sucrose 30g/L + agar 6g/L + ZT 2.0mg/L + NAA 1.0 mg/L; the callus differentiation culture medium consists of the following substances: MS 4.74g/L, sucrose 30g/L, agar 7g/L, 6-BA 1.0 mg/L-1.5 mg/L and NAA 0.1 mg/L-1.0 mg/L;
the rooting medium consists of the following substances: WPM 2.6g/L, sucrose 20g/L, agar 7g/L, IBA 0.1-0.2 mg/L, NAA 0.02-0.2 mg/L.
2. Tissue culture medium for male populus diversifolia strains according to claim 1, characterized in that the callus induction medium consists of: WPM 2.7g/L + sucrose 30g/L + agar 6g/L + TDZ 0.1mg/L + NAA 0.3 mg/L; and/or the presence of a gas in the atmosphere,
the callus differentiation culture medium consists of the following substances: MS 4.74g/L, sucrose 30g/L, agar 7g/L, 6-BA 1.0mg/L and NAA 0.1 mg/L; and/or the presence of a gas in the atmosphere,
the rooting medium consists of the following substances: WPM 2.6g/L + sucrose 20g/L + agar 7g/L + IBA 0.2mg/L + NAA 0.02 mg/L.
3. The tissue culture medium for male populus diversifolia plants according to claim 1 or 2, wherein the callus induction medium, the callus differentiation medium and the rooting medium have a pH value of 5.8.
4. Use of the tissue culture medium for male populus diversifolia plants as claimed in any one of claims 1 to 3 for establishing a regeneration system of male populus diversifolia plants.
5. A method for establishing a poplar male plant regeneration system is characterized by comprising the following steps:
1) selection and sterilization of explants: selecting a stem section at the top end of a male populus diversifolia plant as an explant, placing the stem section in an aseptic bottle in an aseptic environment after washing with tap water, sterilizing with ethanol and aseptic water, cleaning with NaClO and aseptic water, sucking water on the surface of the explant material with aseptic filter paper, cutting the stem section explant into small sections with an aseptic scalpel, and cutting a plurality of wounds on the back of the segmented explant;
2) callus induction: inoculating the segmented explant obtained in the step 1) into a solid callus induction culture medium, and culturing for 20-30 days in a dark environment to induce callus; the callus induction culture medium consists of the following substances: WPM 2.7g/L + sucrose 30g/L + agar 6g/L + TDZ 0.1mg/L + NAA 0.3 mg/L-0.5 mg/L, or the callus induction medium is composed of the following substances: WPM 2.7g/L + sucrose 30g/L + agar 6g/L +6-BA 1.0mg/L +2, 4-D0.5 mg/L-1.0 mg/L, or the callus induction medium consists of the following substances: WPM 2.7g/L + sucrose 30g/L + agar 6g/L + ZT 2.0mg/L + NAA 1.0 mg/L; when the stem segments of the small segments are inoculated on the callus induction culture medium, the morphological upper ends of the small segments are upwards and positively inserted into the callus induction culture medium, and half of the small segments are exposed outside;
3) differentiation culture: inoculating the callus formed in the step 2) to a callus differentiation culture medium, culturing for 25-35 days, and culturing to obtain adventitious buds of male populus diversifolia plants; wherein the callus differentiation medium consists of the following substances: MS 4.74g/L, sucrose 30g/L, agar 7g/L, 6-BA 1.0 mg/L-1.5 mg/L and NAA 0.1 mg/L-1.0 mg/L;
4) rooting culture: taking out the adventitious bud of the male poplar plant formed in the step 3), cutting off a bud of 2-3 cm, transferring the bud into a rooting culture medium, and culturing for 25-35 days to obtain a rooted male poplar plant sterile tissue culture seedling; wherein, the rooting culture medium consists of the following substances: WPM 2.6g/L, sucrose 20g/L, agar 7g/L, IBA 0.1-0.2 mg/L, NAA 0.02-0.2 mg/L;
5) hardening and transplanting tissue culture seedlings: transplanting the tissue culture seedlings of the male populus diversifolia plants with the seedling height of 4.5-5.5 cm into sterile soil, covering a preservative film for moisturizing, gradually and partially opening the preservative film after 7 days until the leaves of the seedlings are completely exposed in the air and cannot be dehydrated, and finishing the establishment of the regeneration system of the male populus diversifolia plants.
6. The method for establishing the poplar male plant regeneration system according to claim 5, wherein a stem section with the top end of 4 cm-6 cm of a 1-month-old cutting seedling is selected as an explant in the step 1); and/or the presence of a gas in the atmosphere,
the step 1) is to sterilize the explant with 75% ethanol for 30s, and/or,
the explant of the stem section is disinfected by 1 percent of NaClO for 5min in the step 1), and/or,
in the step 1), cutting the stem explant into small sections with the size of 1-2 cm by using a sterile scalpel.
7. The method for establishing the male populus diversifolia plant regeneration system according to claim 5, wherein the callus induction medium adopted in the step 2) consists of the following substances: WPM 2.7g/L + sucrose 30g/L + agar 6g/L + TDZ 0.1mg/L + NAA 0.3 mg/L; and/or the presence of a gas in the atmosphere,
the pH value of the callus induction culture medium adopted in the step 2) is 5.8; and/or the presence of a gas in the atmosphere,
the temperature in the dark environment in the step 2) is 25 ℃, and the humidity is 70%.
8. The method for establishing the male populus diversifolia plant regeneration system according to claim 5, wherein the callus differentiation medium adopted in the step 3) consists of the following substances: MS 4.74g/L, sucrose 30g/L, agar 7g/L, 6-BA 1.0mg/L and NAA 0.1 mg/L; and/or the presence of a gas in the atmosphere,
the pH value of the callus differentiation culture medium adopted in the step 3) is 5.8; and/or the presence of a gas in the atmosphere,
in the step 3), the callus inoculated on the callus differentiation culture medium is cultured with adventitious buds under the conditions of 25 ℃ of temperature, 70% of humidity, 1000lux of illumination intensity and 14 hours/day of illumination time.
9. The method for establishing the poplar male plant regeneration system as claimed in claim 5, wherein the rooting medium adopted in the step 4) consists of the following substances: WPM 2.6g/L + sucrose 20g/L + agar 7g/L + IBA 0.2mg/L + NAA 0.02 mg/L; and/or the presence of a gas in the atmosphere,
the pH value of the rooting medium adopted in the step 4) is 5.8; and/or the presence of a gas in the atmosphere,
and in the step 4), the adventitious bud inoculated on the rooting culture medium is used for culturing the rooted tissue culture seedling under the conditions of 25 ℃ of temperature, 70% of humidity, 1000lux of illumination intensity and 14 hours/day of illumination time.
10. The method for establishing the male populus diversifolia plant regeneration system according to claim 5, wherein the sterile soil in the step 5) is prepared from the following raw materials in a volume ratio of 1: 1 and vermiculite.
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