CN108719061B - Method for inducing cherokee rose leaf to directly generate adventitious bud and regenerate plant - Google Patents
Method for inducing cherokee rose leaf to directly generate adventitious bud and regenerate plant Download PDFInfo
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- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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Abstract
The invention discloses a method for inducing cherokee rose leaf blades to directly generate adventitious buds and regenerate plants, which comprises the following steps: cutting single leaf with petiole of aseptic seedling of fructus Rosae Laevigatae, inoculating to induction culture medium, dark culturing for 8-10 days, transferring the dark cultured leaf to illumination time of 16h/d, and culturing at 25 + -2 deg.C for 25-40 days to generate adventitious bud at petiole; cutting and separating adventitious buds, transferring to an enrichment medium for continuous culture, and growing for about 20-30 days to obtain a large number of cluster buds; cutting and separating the cluster buds into single buds, transferring the single buds into a strong seedling culture medium for 25-40 days to make the strong seedlings grow into seedlings with the plant height of 3-5cm, transferring the seedlings into a rooting culture medium for inducing rooting, wherein the rooting rate is more than 95% after 20-30 days; transferring the rooted seedlings to a potting medium for culture to obtain complete adult cherokee rose-hip plants. The method is simple and easy to implement, the induction period is short, the regeneration plants are robust, and the production is rapid.
Description
Technical Field
The invention belongs to the technical field of bioengineering, relates to a method for directly inducing adventitious bud differentiation and plant regeneration from leaves by utilizing a tissue culture technology, and particularly relates to a method for inducing cherokee rose leaves to directly generate adventitious buds and plant regeneration.
Background
Rosa laevigata Michx is a plant used as both medicine and food in Rosa of Rosaceae, and its root, stem, flower and fruit can be used as medicine, and are mostly in semi-wild state. With the development of modern biotechnology, more researchers adopt tissue culture method to culture cherokee rose fruit, but utilize the methodThe regeneration of the plant obtained from the isolated leaf of the cherokee rose fruit is difficult, and the axillary bud and the stem tip are mainly used for rapid propagation in China[1-2]However, the whole strain obtained by the isolated leaf of the cherokee rose fruit has not been reported.
Reference documents:
[1] liaoli, Lanzu planting, Lingdian, Cherokee rose tissue culture and plant regeneration research [ J ] modern Chinese medicine research and practice 2010(6) 22-24.
[2] Tissue culture and rapid propagation of Xiaoxian, Wai Tao, Yangling, Cherokee rose fruit [ J ] Jiangxi forestry science and technology, 2011(4):31-32.
Disclosure of Invention
The invention aims to provide a method for directly inducing and regenerating adventitious buds from cherokee rose leaves by utilizing a tissue culture technology so as to regenerate plants. The method is simple and easy to implement, short in induction period, strong in regeneration plants and rapid in production, and lays a foundation for genetic transformation of the cherokee rose fruits.
The purpose of the invention is realized by the following technical scheme:
a method for inducing cherokee rose leaf to directly generate adventitious buds and regenerate plants comprises the following steps:
step (1), adventitious bud induction: the compound leaves of the cherokee rose set cultured seedlings are cut and cut into single leaves with petioles, considering that the leaves comprise more chloroplasts on the front side, the front side faces to a light source and can better perform photosynthesis, the back side contains air holes and can better absorb nutrients of a culture medium, the single leaves with the petioles are directly inoculated to an adventitious bud induction culture medium downwards on the back side for dark culture for 8-10 days, so that the adventitious buds can be obtained, and the browning rate is kept at a lower degree; transferring the dark-cultured leaves to the normal photoperiod condition for continuous culture for 25-40 days, expanding and growing light green bud points on part of leaf stalks, then germinating the bud points, and performing branching and leaf expansion to generate adventitious buds;
step (2), adventitious bud proliferation: cutting and separating adventitious buds generated at the petiole, transferring to a multiplication culture medium for continuous culture, and growing for 20-30 days to generate a large amount of cluster buds with the multiplication coefficient of about 3.5;
step (3), strong seedling culture: cutting and separating the cluster buds into single buds, transferring the single buds into a strong seedling culture medium, culturing the strong seedlings for 25-40 days, and growing the bud seedlings into seedlings with the height of 3-5 cm;
transferring the seedlings to a rooting culture medium to induce rooting, wherein the rooting rate is over 95 percent after 20-30 days of culture, and the rooted seedlings have stronger survival and environmental adaptability;
and (5) selecting seedlings with developed root systems, robust growth and plant height not less than 5cm, cleaning a culture medium with root adhesive tapes, quickly transferring the seedlings into a potting medium, culturing the seedlings indoors for 1 month, and then transferring the seedlings to natural illumination for culturing for 2-5 months to obtain complete adult cherokee rose-hip plants. Before transplanting, the seedlings are not required to be hardened by uncovering, and as long as sufficient moisture is ensured in the early stage, the transplanting survival rate can reach 100 percent;
in the step (1), the cherokee rose fruit group culture seedling is a cherokee rose fruit sterile group culture seedling. The aseptic tissue culture seedling of the cherokee rose takes the single axillary bud stem section of the soil culture seedling of the cherokee rose as an explant, and the aseptic tissue culture seedling is obtained by primary culture, subculture multiplication culture and strong seedling culture in sequence after disinfection; wherein the culture temperature of each stage is 25 +/-2 ℃, the illumination intensity is 5000lx, and the illumination time is 16 h/d. The primary culture medium is MS + NAA0.1mg/L +6-BA0.5mg/L + GA30.4mg/L, 30g/L of cane sugar and 7.5g/L of agar, and the pH value is 5.8-6.0; wherein GA3Filtering and sterilizing. The subculture multiplication medium comprises MS, NAA0.1mg/L, 6-BA1mg/L, sucrose 30g/L and agar 7.5g/L, and the pH value is 5.8-6.0. The strong seedling culture medium is MS, sucrose 30g/L and agar 7.5g/L, and the pH value is 5.8-6.0.
The compound leaves of the culture seedlings of the cherokee rose are cut on a super clean workbench and then cut into single leaves with petioles. The cutting mode is to cut a knife perpendicular to the main vein of the leaf and reserve a petiole with the length of 0.1-1 mm.
The adventitious bud induction culture medium is 1/2MS + NAA (naphthylacetic acid) 0-0.0005mg/L + TDZ (thidiazuron) 1.5mg/L + CH (acid hydrolyzed casein) 100mg/L + AgNO310mg/L of silver nitrate, 30g/L of cane sugar and 7.5g/L of agar, and the pH value is 5.8-6.0; wherein TDZ and AgNO3Suction filtration sterilization is required.
Preferably, the adventitious bud induction culture medium is 1/2MS + NAA0.0005mg/L + TDZ1.5mg/L + CH100mg/L+AgNO310mg/L + sucrose 30g/L + agar 7.5g/L, pH value 5.8-6.0.
The temperature of dark culture is 25 +/-2 ℃; the normal photoperiod culture condition is that the illumination time is 16h/d, the illumination intensity is 5000-.
In the step (2), the proliferation culture medium is MS + NAA0.1mg/L +6-BA (6-benzyladenine) 1mg/L + sucrose 30g/L + agar 7.5g/L, and the pH value is 5.8-6.0.
The proliferation culture conditions comprise 16h/d of illumination time, 5000-10000lx of illumination intensity and 25 +/-2 ℃.
In the step (3), the strong seedling culture medium is not added with any hormone, and is MS + sucrose 30g/L + agar 7.5g/L, and the pH value is 5.8-6.0.
The strong seedling culture conditions comprise the illumination time of 16h/d, the illumination intensity of 5000-.
In the step (4), the rooting medium is MS, NAA0.1mg/L, sucrose 30g/L and agar 7.5g/L, and the pH value is 5.8-6.0.
The rooting culture conditions comprise 16h/d of illumination time, 5000-10000lx of illumination intensity and 25 +/-2 ℃.
In the step (5), the pot culture substrate is prepared from peat soil, vermiculite and perlite in a volume ratio of 1:2:1, and the raw materials are sterilized at 121 ℃ for 30 minutes, cooled, mixed and stirred uniformly.
The indoor culture conditions comprise 16h/d of illumination time, 5000-10000lx of illumination intensity and 25 +/-2 ℃.
The invention has the beneficial effects that:
the method is simple, convenient and easy to implement, short in induction period, strong and strong in regenerated plant and rapid in production, and lays a foundation for genetic transformation of the cherokee rose fruits.
The leaf of the cherokee rose with petioles is used as an explant, the quantity is large, the propagation speed is high, the regeneration capacity is strong, and meanwhile, the adventitious bud induction by using the leaf is convenient to be connected with subsequent genetic transformation. The leaves of the cherokee rose fruit group seedling culture adopted are in an aseptic state, and the operation process is greatly simplified. The leaves in vitro will lose water and will wither, so the cutting of leaves and the inoculation work are performed simultaneously to ensure that the explants in the best state are inoculated on the induction culture medium. In the tissue culture test, the antioxidant silver nitrate is added to reduce the browning phenomenon, in addition, the dark culture of the explant for 8-10 days can slow down the synthesis of phenolic compounds, the quinone oxidation products are correspondingly reduced, and the browning rate of the explant is reduced.
Drawings
FIG. 1 shows the process of adventitious bud induction; wherein, the bud point of FIG. 1A appears, the bud point of FIG. 1B germinates, the adventitious bud of FIG. 1C expands leaves, and the adventitious bud of FIG. 1D shoots.
FIG. 2 growth process from adventitious bud to plant regeneration; wherein FIG. 2A shows adventitious bud proliferation, FIG. 2B shows strong seedling, FIG. 2C shows rooting, and FIG. 2D shows transplanting and potting.
Detailed Description
The technical solution of the present invention will be further described with reference to the following embodiments.
EXAMPLE 1 acquisition of sterile tissue culture seedlings
Selecting branches which grow well in the cherokee rose-hip soil-cultured seedlings and have plump axillary buds but do not germinate, cutting the branches into single-bud stem segments with the length of about 2cm as explants, placing the single-bud stem segments under running water for washing for 12h, washing the single-bud stem segments with washing powder for 10min, and then washing the single-bud stem segments with clear water. Placing on a clean bench, washing with sterile water for 1 time, washing with 75% alcohol for 30s, treating with 5% sodium hypochlorite for 10min, washing with sterile water for 5 times, and drying with sterile filter paper.
Inoculating the sterilized explant to a primary culture medium for culture, after 7d, beginning axillary buds on the stem explant to germinate, beginning bud enlargement, elongation and leaf expansion, cutting the bud from a base part, transferring the bud into a secondary multiplication culture medium, and obtaining a large number of cluster seedlings after 30 d; the seedlings are divided and inoculated in a strong seedling culture medium, and after 30 days, the seedlings grow into rootless seedlings with the plant height of 3-5cm, the leaf length of the seedlings at the stage can reach 0.5-1.5cm generally, and the seedlings have the condition of being used as explants for inducing adventitious buds in the next step. The culture stages are all carried out in a constant-temperature culture room, the temperature of the culture room is 25 +/-2 ℃, the illumination intensity is 5000lx, and the illumination time is 16 h/d.
The primary culture medium is MS + NAA0.1mg/L +6-BA0.5mg/L+GA30.4mg/L, 30g/L of cane sugar and 7.5g/L of agar, and the pH value is 5.8-6.0; wherein GA3Filtering and sterilizing. The subculture multiplication medium comprises MS, NAA0.1mg/L, 6-BA1mg/L, sucrose 30g/L and agar 7.5g/L, and the pH value is 5.8-6.0. The strong seedling culture medium is MS, sucrose 30g/L and agar 7.5g/L, and the pH value is 5.8-6.0.
Example 2 examination of the Effect of plant growth regulators on adventitious bud Induction
Selecting 1/2MS as basic culture medium, adding CH100mg/L + AgNO310mg/L + sucrose 30g/L + agar 7.5g/L, pH 5.8-6.0, and investigating the influence of plant growth regulators NAA and TDZ on adventitious bud induction.
Cutting sterile compound leaves of the cherokee rose fruit group cultured seedlings on a superclean bench, cutting a knife perpendicular to the main vein of the leaves to obtain single leaves with petioles of 0.5-1mm in length, and inoculating the single leaves with the petioles with the back facing downwards on 1/2MS culture media with different hormone ratios to perform adventitious bud induction. Culturing in dark (25 + -2 deg.C) for 8 days, transferring into normal light cycle (illumination time 16h/d, illumination intensity 5000lx, culture temperature 25 + -2 deg.C), culturing, treating 8 culture dishes each, and inoculating 8 explants each. Each treatment was repeated 3 times, and the adventitious bud induction rate was counted after 4 weeks.
TABLE 1 Effect of different plant growth regulators on the induction of adventitious buds of Cherokee Rose
As can be seen from Table 1, the ratio of NAA and TDZ in different concentrations has direct influence on the induction of adventitious buds, and the adventitious buds can be induced only when the concentration of TDZ is 1.5mg/L, while the induction rate is changed along with the change of the concentration of NAA: when the concentration of TDZ is 1.5mg/L and the concentration of NAA is 0.0005, the induction rate of the adventitious bud is the highest and can reach about 10 percent; the concentration of NAA exceeds or is lower than 0.0005, and the induction rate is reduced to different degrees. Therefore, 1/2MS + NAA0-0.0005mg/L + TDZ1.5mg/L + CH100mg/L + AgNO was selected310mg/L + sucrose 30g/L + agar 7.5g/L, pH 5.8-6.0 as adventitious bud induction culture medium, and can avoid adventitious bud induction processThe generation of adventitious roots is avoided, and the adventitious roots are prevented from drawing nutrition and moisture in a culture medium, so that the generation of induced adventitious buds is ensured; in view of the induction rate, 1/2MS + NAA0.0005mg/L + TDZ1.5mg/L + CH100mg/L + AgNO was further selected310mg/L + sucrose 30g/L + agar 7.5g/L, pH 5.8-6.0 is the optimum adventitious bud induction medium.
The adventitious buds induced by the method grow robustly and quickly, and the growth process is approximately as follows: after 10 days of inoculation, the leaves are folded to different degrees, the leaf discs are curled and tilted, the petiole parts are expanded after about 15 days and are in a light green convex shape, and the bulges are bud points; after 25-30 days, the bud points germinate and complex leaves grow, which belongs to direct regeneration (see figure 1).
The inventor adopts the Chinese rose varieties Sichun, lunar powder, green calyx and Samantha which are Rosa of Rosaceae, and inoculates the obtained explants on the optimal adventitious bud induction culture medium according to the method of the embodiment, and no adventitious bud is induced.
Example 3 Effect of dark culture time on adventitious bud Induction
Cutting sterile compound leaf of the Cherokee rose tissue cultured seedling on an ultraclean workbench, cutting a knife perpendicular to the main vein of the leaf to obtain a single leaf with 0.5-1mm leaf stalk, inoculating the single leaf with the stalk with the back face downwards to an induction culture medium (1/2MS + NAA0.0005mg/L + TDZ1.5mg/L + CH100mg/L + AgNO)310mg/L + sucrose 30g/L + agar 7.5g/L, pH 5.8-6.0) were cultured in the dark (culture temperature 25. + -. 2 ℃) for 8 days, 10 days, 12 days, 14 days, respectively, and then transferred to the normal photoperiod (same as example 2) for culture, each treatment of 8 dishes, each dish containing 10 explants. After one week, the explant growth was observed and adventitious bud induction was counted, with the results shown in table 2.
TABLE 2 Effect of different dark culture days on adventitious bud Induction and explant growth
As can be seen from Table 2, the dark culture time has some influence on the browning degree of the explant and the adventitious bud induction rate. With the increase of dark culture days, the differentiation rate of the adventitious bud is increased and then decreased, while the browning degree of the explant is increased all the time. When cultured in the dark for 10 days, the differentiation rate of the adventitious bud reaches the highest. After dark culture for more than 10 days, the differentiation rate of the adventitious bud is reduced, the browning rate of the explant is increased, and the phenomenon of water stain with different degrees is caused. After dark culture is finished, the culture medium is transferred to a normal light cycle for culture, and the water stain phenomenon is relieved.
Example 4 Induction assay of different explants
Cutting compound leaves of the cherokee rose fruit group culture seedlings, cutting a knife perpendicular to the main vein of the leaves, and cutting into single leaves with the length of a leaf stalk of about 0.5-1 mm.
Respectively taking leaf disc, single leaf with leaf stalk length of about 0.5-1mm and young stem segment with length of about 0.5cm of fructus Rosae Laevigatae tissue culture seedling as explants, inoculating to induction culture medium (1/2MS + NAA0.0005mg/L + TDZ1.5mg/L + CH100mg/L + AgNO)310mg/L + sucrose 30g/L + agar 7.5g/L, pH 5.8-6.0), wherein the single leaf with the handle is inoculated with the back side down; dark culture (25 + -2 deg.C) for 10 days, transferring to normal photoperiod condition (same as example 2), treating 8 culture dishes each, inoculating 8 explants each. After 4 weeks, the adventitious bud induction rate was counted. The result shows that the incisions of the leaf disc and the leaf stalk are expanded to different degrees in dark culture for 10 days, the expansion of the leaf stalk part is more obvious, but only the expanded substance of the leaf stalk part can directly differentiate adventitious buds; while the tender stem segment and the leaf disc have no adventitious bud, and the wound of the leaf disc has adventitious root. The results show that the petiole is a key part with extremely strong differentiation capability, and the cutting and placement modes of the petiole have important influence on the induction of adventitious buds. When the leaf blade is cut, the leaf stalk with the length of about 0.5-1mm is reserved, the explant is the best explant with higher adventitious bud induction rate, and the back of the leaf stalk explant with the length faces downwards to be inoculated into an induction culture medium, so that the adventitious bud can be generated.
Example 5
A method for inducing cherokee rose leaf to directly generate adventitious buds and regenerate plants comprises the following steps:
step (1), selecting strong-growing cherokee rose fruit group to cultivate seedlings, and shearing plantsThe compound leaf with larger middle upper part is cut into a single leaf with a petiole with the length of about 0.5-1mm perpendicular to the main vein of the leaf disc as an explant; inoculating single leaf with handle to adventitious bud induction culture medium (1/2MS + NAA0.0005mg/L + TDZ1.5mg/L + CH100mg/L + AgNO)310mg/L, 30g/L of cane sugar and 7.5g/L of agar, the pH value is 5.8-6.0, and dark culture is carried out in a constant-temperature culture chamber (25 +/-2 ℃) for 10 days;
transferring the dark-cultured leaves to normal photoperiod conditions (the photoperiod is 16 hours, the illumination is 8 hours and dark, the illumination intensity is 5000lx, the culture temperature is 25 +/-2 ℃, and the culture conditions are unchanged in the following culture stages), continuously culturing for 4 weeks, expanding part of leaf stalks and growing light green buds, then germinating the buds, and carrying out lopping and leaf expansion;
step (2), cutting and separating the adventitious buds generated at the petioles, transferring the adventitious buds to a multiplication medium for continuous culture for about 2 weeks to generate a large number of cluster buds (figure 2A), wherein the multiplication coefficient is about 3.5; wherein, the enrichment medium MS + NAA0.1mg/L +6-BA1mg/L + sucrose 30g/L + agar 7.5g/L, pH value 5.8-6.0.
Step (3), cutting the adventitious bud cluster into single buds, transferring the single buds to a strong seedling culture medium for strong seedling culture for 3 weeks, and growing the buds into seedlings with the height of 3-5cm (figure 2B); wherein, the strong seedling culture medium MS, sucrose 30g/L and agar 7.5g/L, the pH value is 5.8-6.0;
step (4), transferring the bud seedlings to a rooting culture medium for inducing rooting, wherein the rooting rate reaches more than 95% after 3 weeks (figure 2C); wherein the rooting medium is MS, NAA0.1mg/L, sucrose 30g/L, agar 7.5g/L, pH 5.8-6.0,
selecting seedlings with developed root systems, strong growth and plant height not less than 5cm, washing the culture medium with the root adhesive tapes clean, quickly transferring the seedlings to a potted plant substrate which is thoroughly watered without uncovering and hardening the seedlings, and continuously culturing, wherein the transplanting survival rate can reach 100%; culturing in pot for 2 months to obtain complete fructus Rosae Laevigatae plant (FIG. 2D).
The potting matrix is prepared from peat soil, vermiculite and perlite in a volume ratio of 1:2:1 through sterilizing the raw materials at 121 ℃ for 30min, cooling and uniformly stirring.
Claims (7)
1. A method for inducing cherokee rose leaf to directly generate adventitious buds and regenerate plants is characterized by comprising the following steps:
step (1), adventitious bud induction: cutting sterile compound leaves of the cherokee rose fruit group culture seedling, cutting into single leaves with petioles, inoculating the single leaves with petioles to an adventitious bud induction culture medium with the back facing downwards for dark culture, wherein the adventitious bud induction culture medium is 1/2MS + NAA0-0.0005mg/L + TDZ1.5mg/L + CH (acid hydrolyzed casein) 100mg/L + AgNO310mg/L, 30g/L of cane sugar and 7.5g/L of agar, and the pH value is 5.8-6.0; dark culture for 8-10 days; transferring the dark cultured leaf to normal light cycle condition for 25-40 days to generate adventitious bud at leaf stalk;
step (2), adventitious bud proliferation: cutting and separating adventitious buds, transferring to enrichment medium for continuous culture, and culturing for 20-30 days to generate cluster buds; the proliferation culture medium is MS + NAA0.1mg/L +6-BA1mg/L + sucrose 30g/L + agar 7.5g/L, and the pH value is 5.8-6.0;
step (3), strong seedling culture: cutting and separating the cluster buds into single buds, transferring the single buds into a strong seedling culture medium, culturing strong seedlings for 25-40 days, and growing the buds into seedlings; the strong seedling culture medium is not added with any hormone, and comprises MS, sucrose 30g/L and agar 7.5g/L, and the pH value is 5.8-6.0;
step (4), inducing rooting: transferring the seedlings to a rooting culture medium for inducing rooting, and culturing for 20-30 days to obtain rooted seedlings; the rooting medium is MS, NAA0.1mg/L, sucrose 30g/L and agar 7.5g/L, and the pH value is 5.8-6.0;
and (5) selecting seedlings with developed root systems, robust growth and plant height not less than 5cm, cleaning culture medium with root adhesive tapes, transplanting the seedlings into a potting medium, culturing for 1 month indoors, and then transferring the seedlings to natural light for culturing to obtain complete adult cherokee rose plants.
2. The method for inducing the direct adventitious bud generation and plant regeneration of cherokee rose-hip leaves as claimed in claim 1, wherein the dark culture temperature in step (1) is 25 ± 2 ℃; the normal photoperiod culture condition is that the illumination time is 16h/d, the illumination intensity is 5000-.
3. The method for inducing the direct generation of adventitious buds and plant regeneration of cherokee rose-hip leaves as claimed in claim 1, wherein in step (1), the adventitious bud induction medium is 1/2MS + NAA0.0005mg/L + TDZ1.5mg/L + CH100mg/L + AgNO310mg/L + sucrose 30g/L + agar 7.5g/L, pH value 5.8-6.0.
4. The method for inducing the direct generation of adventitious bud and plant regeneration of cherokee rose-hip leaf as claimed in claim 1, wherein the proliferation culture conditions in step (2) are 16h/d of illumination time, 5000 + 10000lx of illumination intensity and 25 + -2 ℃.
5. The method for inducing the direct generation of adventitious bud and plant regeneration in the leaf of cherokee rose-hip as claimed in claim 1, wherein the strong seedling culture conditions in step (3) are 16h/d of illumination time, 5000-10000lx of illumination intensity and 25 ± 2 ℃.
6. The method for inducing the direct generation of adventitious buds and plant regeneration of cherokee rose-hip leaves as claimed in claim 1, wherein the rooting culture conditions in step (4) are 16h/d of illumination time, 5000-10000lx of illumination intensity and 25 +/-2 ℃.
7. The method for inducing the direct generation of adventitious buds and plant regeneration of cherokee rose-hip leaves according to claim 1, wherein in the step (5), the raw materials of the potting matrix are peat soil, vermiculite and perlite in a volume ratio of 1:2:1, and the raw materials are sterilized at 121 ℃ for 30 minutes, cooled and mixed uniformly;
the indoor culture conditions comprise 16h/d of illumination time, 5000-10000lx of illumination intensity and 25 +/-2 ℃.
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| JPH03103128A (en) * | 1989-09-14 | 1991-04-30 | Kyowa Hakko Kogyo Co Ltd | Adventitious bud formation of plant of genus rose |
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| JPH03103128A (en) * | 1989-09-14 | 1991-04-30 | Kyowa Hakko Kogyo Co Ltd | Adventitious bud formation of plant of genus rose |
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