Tissue culture and rapid propagation method for leaves and flowers of wild primulina
By employing specific treatments and culture medium methods on the leaves and flowers of wild Primrose, the problem of low efficiency in the Primrose regeneration system was solved, achieving efficient tissue culture and rapid propagation, resulting in high survival and rooting rates.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-02-04
- Publication Date
- 2026-04-14
AI Technical Summary
Existing techniques for tissue culture of Primula sylvatica have problems such as long induction time for embryogenic callus, unclear time for callus differentiation into adventitious buds, unclear rooting rate and callus rate, and long regeneration time, resulting in an inefficient regeneration system.
Wild Primrose leaves and flowers were used as explants. Tissue culture was carried out through specific pretreatment, disinfection and addition of exogenous hormones to the culture medium, including running water rinsing, ultraviolet sterilization, alcohol and mercuric chloride disinfection, MS medium with different hormone ratios, and primary, subculture and rooting seedling culture were carried out step by step, and finally seedling hardening and transplanting were carried out.
Rapid propagation of Primrose was achieved. The entire regeneration system has a growth cycle of about 95 days. The survival rate of leaves after disinfection and sterilization is over 82.5%, the survival rate of flowers after disinfection and sterilization is over 92%, the differentiation rate of the first generation culture is 90%, the healing rate of tissue culture seedlings after subculture is 98%, the rooting rate is 95%, and the survival rate of tissue culture seedlings after transplanting is 100%.
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Figure CN121844952A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue culture and rapid propagation technology, and relates to a method for tissue culture and rapid propagation of leaves and flowers of wild Primrose. Background Technology
[0002] Primulina tabacum is a perennial herbaceous plant belonging to the genus Primulina in the family Gesneriaceae. Due to its scattered distribution, harsh living environment, and presence in many limestone caves, as well as the severe human disturbance caused by the development of tourism, its population and distribution area have decreased dramatically.
[0003] Although there have been reports on tissue culture of Primula, the time required to induce embryogenic callus of Primula is long, the time required for callus to differentiate into adventitious buds is not specified, and growth indicators such as rooting rate and callus emergence rate are not specified, resulting in a long time to cultivate regenerated plants.
[0004] Therefore, establishing an efficient regeneration system for Primrose is essential. The establishment of such a system is a prerequisite for improving Primrose resistance and genetic transformation, making it possible to establish an efficient resistance gene transformation system. Summary of the Invention
[0005] This invention provides a method for tissue culture and rapid propagation of leaves and flowers of wild Primrose, comprising the following steps: Step 1: Obtaining explants: Take vigorous young leaves and newly opened flowers from the top of Primrose as explants. Step 2: Pretreatment of explants: After removing the petioles from the tender leaves, cut them into small pieces and rinse them under running water until the surface dust is clean. After rinsing the flowers under running water, place them in a glass bottle and shake them to rinse. Step 3: Disinfection of explants: The pretreated tender leaves are placed in a cleaning bottle in a clean bench, rinsed with distilled water, and drained. The tender leaves in the cleaning bottle are then disinfected by soaking. Place the pre-treated flowers into the cleaning bottle in the laminar flow hood, add distilled water, gently shake and rinse, then drain the water; disinfect the flowers in the cleaning bottle by soaking. Step 4: Initial Culture The disinfected young leaves were inoculated onto the primary leaf culture medium for primary culture; the primary leaf culture medium used MS as the basic medium and was supplemented with exogenous hormones at a ratio of KT 1 mg / L + NAA 0.3 mg / L. The sterilized flowers were inoculated onto the primary flower culture medium for primary culture; the primary flower culture medium used MS as the basic medium, with the addition of exogenous hormones at a ratio of KT 1-2 mg / L + NAA 0.3-0.5 mg / L. Step 5, Subculture: The material for subculture is the tender leaves after primary culture. When the seedlings from primary culture are growing well and reach a height of about 1 cm, take their leaves, cut them into small pieces of 0.5 cm × 0.5 cm, and inoculate them into the subculture medium. After culturing in the dark for 7 days, transfer them to light culture to obtain subcultured seedlings. The subculture medium is MS as the basic medium, with the addition of exogenous plant hormones at a ratio of KT 1 mg / L + NAA 0.4-0.6 mg / L. Step 6: Rooting and strengthening seedling culture: Take healthy seedlings with a height of 0.5-0.6cm, 4 leaves, and no roots, and culture them under light with MS as the basic medium to obtain strong seedlings. Step 7: When the seedlings reach the growth indicators of 1.8-2cm in height, 1.9-2.1cm in root length, and 7-8 leaves, harden them off. After hardening off, transplant them for further cultivation.
[0006] Furthermore, the young leaves should be placed in a glass bottle and rinsed at least three times, with each rinsing session lasting 3-5 minutes.
[0007] Furthermore, the time for rinsing the flowers under running water was set to 3-5 minutes; Rinse the flowers in a glass bottle at least three times, with each rinse lasting 3-5 minutes.
[0008] Furthermore, before placing the pretreated young leaves and flowers into the cleaning bottle of the laminar flow hood, the laminar flow hood is sterilized using ultraviolet light, with the ultraviolet irradiation time set to be no less than 30 minutes.
[0009] Furthermore, the specific process for disinfecting the pretreated young leaves is as follows: Add alcohol to the cleaning bottle and use the soaking method to disinfect the tender leaves in the cleaning bottle for the first time. After the initial disinfection, the alcohol in the cleaning bottle is rinsed with distilled water, the water is drained, and then mercuric chloride is added to the cleaning bottle to disinfect the tender leaves in the cleaning bottle a second time. After secondary disinfection, the tender leaves are rinsed with distilled water. Once there is no mercuric chloride residue on the leaf surface, the disinfected tender leaves are placed on filter paper. Disinfect the tender leaves.
[0010] Furthermore, the specific process for disinfecting the pre-treated flowers is as follows: Add alcohol to the cleaning bottle and use the soaking method to disinfect the flowers in the cleaning bottle for the first time; After the initial disinfection, rinse the alcohol with distilled water, drain the water, and then add mercuric chloride for a second disinfection. After the second disinfection, rinse the flowers with distilled water. Once there is no mercuric chloride residue on the surface of the flowers, place the disinfected flowers on the operating tray and cut off the front end of the disinfected flowers with a blade. Complete the disinfection of the flowers.
[0011] Furthermore, the culture conditions for primary culture, subculture, and rooting and seedling cultivation were all set as follows: First, place the sample in the dark for 7 days under light-protected conditions; After dark culture, the cells were placed under light for 16 hours per day. The temperature of the culture room was (25±2)℃ and the humidity was 70%-85%.
[0012] Furthermore, during the initial culture of the disinfected young leaves, three young leaves were inoculated into each leaf's initial culture medium, and the culture medium was observed daily after inoculation. During the initial culture of the sterilized flowers, three flowers were inoculated into each flower's initial culture medium, and the culture medium was observed daily after inoculation.
[0013] Furthermore, the hardening-off method is as follows: unscrew the cap of the tissue culture bottle to allow air to pass through, with an air passage time of 9 hours per day for 7 days; The volume ratio of coconut fiber bricks, perlite, and vermiculite was set to 2:1:1.
[0014] Furthermore, the steps for preparing 1L MS basal medium are as follows: (1) Add 50mL of prepared macro-element mother liquor, 10mL of micro-element mother liquor, 10mL of iron salt mother liquor, 10mL of organic mother liquor and carrageenan, add 500-600mL of distilled water to dissolve, boil until the solution is translucent, add sucrose, and add distilled water to make up to 1000mL. (2) Adjust the pH to 6.5-7.0; (3) Add various plant growth regulators; (4) Place the culture medium in an autoclave, pressurize it to 1.0 kg / cm2, set the temperature to 121°C, and sterilize it for 20 min.
[0015] Compared with the prior art, the present invention has the following beneficial effects: (1) This invention is the first to successfully cultivate sterile Primrose seedlings from flower organs, with a transplant success rate of over 95%. Using tissue culture of both leaves and flowers, and adjusting sterilization methods, plant growth regulators (types and concentrations), and seedling rooting, wild Primrose is rapidly propagated through tissue culture. The entire regeneration system of the wild Primrose has a growth cycle of approximately 95 days. Tests showed that the survival rate of leaves as explants after sterilization was over 82.5%, and the survival rate of flowers as explants was over 92%. The initial culture differentiation rate was approximately 90%, and the healing rate of the tissue culture seedlings after subculture reached 98%, with a rooting rate of over 95%. The transplant survival rate of the Primrose tissue culture seedlings cultivated through this invention reached 100%.
[0016] (2) In this invention, when young leaves are placed in a primary culture medium, deformation begins to occur at the wound site on the leaf edge after about 20 days, producing yellow-green callus tissue. Roots and buds differentiate after about 30 days. The growth rate of the callus tissue and the differentiation rate of the buds are significantly better than those reported in previous studies.
[0017] In addition to the objectives, features, and advantages described above, the present invention has other objectives, features, and advantages. The invention will now be described in further detail with reference to the figures. Attached Figure Description
[0018] The accompanying drawings, which form part of this application, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an undue limitation of the invention. In the drawings: Figure 1 (A) is a schematic diagram of the leaf growth of wild Primrose leaves 60 days after inoculation under condition A1 in an embodiment of the present invention. Figure 1 (B) is a schematic diagram of the leaf growth of wild Primrose leaves 60 days after inoculation under conditions A2 in an embodiment of the present invention. Figure 1 (C) is a schematic diagram of the leaf growth of wild Primrose leaves 60 days after inoculation under condition B1 in an embodiment of the present invention. Figure 1 (D) is a schematic diagram of the leaf growth of wild Primrose leaves 60 days after inoculation under B2 conditions in an embodiment of the present invention. Figure 2 (A) is a schematic diagram of the treatment of wild Primulina leaves under condition A1 in an embodiment of the present invention; Figure 2 (B) is a schematic diagram of the treatment of wild Primulina leaves under condition A2 in an embodiment of the present invention; Figure 2(C) is a schematic diagram of the treatment of wild Primulina leaves under condition A3 in an embodiment of the present invention; Figure 2 (D) is a schematic diagram of the treatment of wild Primrose leaves under A4 conditions in an embodiment of the present invention; Figure 2 (E) is a schematic diagram of the treatment of wild Primulina leaves under A5 conditions in an embodiment of the present invention; Figure 2 (F) is a schematic diagram of the treatment of wild Primulina leaves under A6 conditions in an embodiment of the present invention; Figure 2 (G) is a schematic diagram of the treatment of wild Primulina leaves under condition B1 in an embodiment of the present invention; Figure 2 (H) is a schematic diagram of the treatment of wild Primrose leaves under condition B2 in an embodiment of the present invention; Figure 2 (I) is a schematic diagram of the treatment of wild Primrose leaves under B3 conditions in an embodiment of the present invention; Figure 2 (J) is a schematic diagram of the treatment of wild Primulina leaves under B4 conditions in an embodiment of the present invention; Figure 2 (K) is a schematic diagram of the treatment of wild Primrose leaves under B5 conditions in an embodiment of the present invention; Figure 2 (L) is a schematic diagram of the treatment of wild Primrose leaves under B6 conditions in an embodiment of the present invention; Figure 2 (M) is a schematic diagram of the treatment of wild Primrose leaves under C1 conditions in an embodiment of the present invention; Figure 2 (N) is a schematic diagram of the treatment of wild Primrose leaves under C2 conditions in an embodiment of the present invention; Figure 2 (O) is a schematic diagram of the treatment of wild Primrose leaves under C3 conditions in an embodiment of the present invention; Figure 2 (P) is a schematic diagram of the treatment of wild Primrose leaves under C4 conditions in an embodiment of the present invention; Figure 2 (Q) is a schematic diagram of the treatment of wild Primrose leaves under C5 conditions in an embodiment of the present invention; Figure 2 (R) is a schematic diagram of the treatment of wild Primulina leaves under C6 conditions in an embodiment of the present invention; Figure 3 (A) is a schematic diagram of the bud growth of wild Primrose leaves under condition A in an embodiment of the present invention; Figure 3 (B) is a schematic diagram of the bud growth of wild Primrose leaves under condition B in an embodiment of the present invention; Figure 3 (C) is a schematic diagram of the bud growth of wild Primrose leaves under condition C in an embodiment of the present invention; Figure 3 (D) is a schematic diagram of the bud growth of wild Primrose leaves under condition D in an embodiment of the present invention; Figure 3 (E) is a schematic diagram of the bud growth of wild Primrose leaves under condition E in an embodiment of the present invention; Figure 3 (F) is a schematic diagram of the bud growth of wild Primrose leaves under condition F in an embodiment of the present invention; Figure 3 (G) is a schematic diagram of the bud growth of wild Primrose leaves under condition G in an embodiment of the present invention; Figure 3 (H) is a schematic diagram of the bud growth of wild Primrose leaves under condition H in an embodiment of the present invention; Figure 3 (I) is a schematic diagram of the bud growth of wild Primrose leaves under condition I in an embodiment of the present invention; Figure 3 (J) is a schematic diagram of the bud growth of wild Primrose leaves under condition J in an embodiment of the present invention; Figure 3 (K) is a schematic diagram of the bud growth of wild Primrose leaves under condition K in an embodiment of the present invention; Figure 3 (L) is a schematic diagram of the bud growth of wild Primrose leaves under L conditions in an embodiment of the present invention; Figure 4 This is a schematic diagram of the seedling transplanting process after tissue culture of wild Primrose leaves in an embodiment of the present invention; Figure 5 (A) is a schematic diagram of the flower growth of wild Primrose in an embodiment of the present invention when the ratio of plant growth regulator is KT 1 mg / L + NAA 0.3 mg / L; Figure 5 (B) is a schematic diagram of the flower growth of wild Primrose in an embodiment of the present invention when the ratio of plant growth regulator is KT 2 mg / L + NAA 0.5 mg / L; Figure 6 (A) is a schematic diagram of the treatment of wild Primrose flowers under condition A1 in an embodiment of the present invention; Figure 6 (B) is a schematic diagram of the treatment of wild Primrose flowers under condition A2 in an embodiment of the present invention; Figure 6 (C) is a schematic diagram of the treatment of wild Primrose flowers under condition A3 in an embodiment of the present invention; Figure 6 (D) is a schematic diagram of the treatment of wild Primrose flowers under conditions A4 in an embodiment of the present invention; Figure 6 (E) is a schematic diagram of the treatment of wild Primulina flowers under conditions A5 in an embodiment of the present invention; Figure 6 (F) is a schematic diagram of the treatment of wild Primrose flowers under A6 conditions in an embodiment of the present invention; Figure 7 (A) is a schematic diagram of the bud growth of wild Primrose flowers under condition A in an embodiment of the present invention; Figure 7 (B) is a schematic diagram of the bud growth of wild Primrose flowers under condition B in an embodiment of the present invention; Figure 7 (C) is a schematic diagram of the bud growth of wild Primrose flowers under condition C in an embodiment of the present invention; Figure 7 (D) is a schematic diagram of the bud growth of wild Primrose flowers under condition D in an embodiment of the present invention; Figure 8 This is a schematic diagram of the seedling transplantation process after tissue culture of wild Primrose flowers in an embodiment of the present invention. Detailed Implementation
[0019] To make the above-mentioned objectives, features, and advantages of the present invention clearer and easier to understand, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings. It should be noted that the accompanying drawings of the present invention are all in a simplified form and use non-precise proportions, and are only used to facilitate and clearly assist in illustrating the implementation of the present invention; the "several" mentioned in the present invention are not limited to the specific number shown in the examples in the accompanying drawings; the orientations or positional relationships indicated by terms such as "front," "middle," "rear," "left," "right," "up," "down," "top," "bottom," and "center" mentioned in the present invention are all based on the orientations or positional relationships shown in the accompanying drawings of the present invention, and do not indicate or imply that the device or component referred to must have a specific orientation, nor should they be construed as limitations on the present invention.
[0020] Example: The present invention provides a method for tissue culture and rapid propagation of leaves and flowers of wild Primrose, comprising the following steps: Step 1: Obtaining explants: Take vigorous young leaves and newly opened flowers from the top of Primrose as explants. Step 2: Pretreatment of explants: After removing the petioles from the tender leaves, cut them into small pieces and rinse them under running water until the surface dust is clean. After rinsing the flowers under running water, place them in a glass bottle and shake them to rinse. Step 3: Disinfection of explants: The pretreated tender leaves are placed in a cleaning bottle in a clean bench, rinsed with distilled water, and drained. The tender leaves in the cleaning bottle are then disinfected by soaking. Place the pre-treated flowers into the cleaning bottle in the laminar flow hood, add distilled water, gently shake and rinse, then drain the water; disinfect the flowers in the cleaning bottle by soaking. Step 4: Initial Culture The disinfected young leaves were inoculated onto the primary leaf culture medium for primary culture; the primary leaf culture medium used MS as the basic medium and was supplemented with exogenous hormones at a ratio of KT 1 mg / L + NAA 0.3 mg / L. The sterilized flowers were inoculated onto the primary flower culture medium for primary culture; the primary flower culture medium used MS as the basic medium, with the addition of exogenous hormones at a ratio of KT 1-2 mg / L + NAA 0.3-0.5 mg / L. Step 5, Subculture: The material for subculture is the tender leaves after primary culture. When the seedlings from primary culture are growing well and reach a height of about 1 cm, take their leaves, cut them into small pieces of 0.5 cm × 0.5 cm, and inoculate them into the subculture medium. After culturing in the dark for 7 days, transfer them to light culture to obtain subcultured seedlings. The subculture medium is MS as the basic medium, with the addition of exogenous plant hormones at a ratio of KT 1 mg / L + NAA 0.4-0.6 mg / L. Step 6: Rooting and strengthening seedling culture: Take healthy seedlings with a height of 0.5-0.6cm, 4 leaves, and no roots, and culture them under light with MS as the basic medium to obtain strong seedlings. Step 7: When the seedlings reach the growth indicators of 1.8-2cm in height, 1.9-2.1cm in root length, and 7-8 leaves, harden them off. After hardening off, transplant them for further cultivation.
[0021] Preferably, in this embodiment, the specific method for pretreating the young leaves is as follows: Remove the petioles from the tender leaves and cut them into small pieces of (0.5cm-1.0cm) × (1.0cm-2.0cm). Rinse them under running water until the surface dust is clean. Place them in a glass bottle and shake them three times, each time for 3 minutes.
[0022] Preferably, in this embodiment, the specific method for pretreating the flowers is as follows: Rinse the flowers under running water for 5 minutes. After rinsing them clean, place them in a glass bottle and shake them three times, each time for 3 minutes.
[0023] Preferably, in this embodiment, before placing the pretreated tender leaves and flowers into the cleaning bottle of the laminar flow hood, the laminar flow hood is sterilized using ultraviolet light, and the ultraviolet irradiation time is set to be no less than 30 minutes.
[0024] Preferably, in this embodiment, the specific process of disinfecting and ultra-cleaning inoculating the pretreated young leaves is as follows: Place the pretreated tender leaves into a washing bottle, rinse three times with distilled water, drain the water, add 75% alcohol for initial disinfection, and soak for 30 seconds. After rinsing the alcohol with distilled water and draining it, add mercuric chloride for secondary disinfection and soak for 6 minutes. After soaking in mercuric chloride, rinse four times with distilled water. Once there is no mercuric chloride residue on the leaf surface, place the disinfected explant on filter paper to complete the disinfection of the tender leaves.
[0025] Furthermore, since the leaves of Primula are relatively delicate and easily damaged, special care should be taken when cleaning them. When shaking the leaves in the cleaning bottle, the movements should be gentle. A brush can be used as an auxiliary tool for cleaning. The method is to gently stir the leaves in the cleaning bottle with the tip of the brush to promote full contact between the explant and the disinfectant.
[0026] Preferably, in this embodiment, the specific process of disinfecting and ultra-cleaning inoculating the pretreated flowers is as follows: Place the pre-treated flowers into a washing bottle, add distilled water, gently shake and rinse, rinse three times with distilled water, and drain. Add 75% alcohol for disinfection and soak for 20 seconds; After rinsing the alcohol off with distilled water, add mercuric chloride and soak for 4 minutes and 30 seconds for disinfection. After disinfection, rinse four times with distilled water to complete the disinfection of the flowers.
[0027] Preferably, the rinsing method involves gently picking up the material with tweezers, placing it into the cleaning bottle, adding the reagent (approximately 2 / 3 of the cleaning bottle's volume), and gently shaking.
[0028] Preferably, in this embodiment, the specific process of primary culture of the disinfected young leaves is as follows: After disinfection, place the tender leaves on the operating tray that has been autoclaved. Use a blade to cut the tender leaves into 2cm×2cm pieces, lay them flat on the surface of the primary culture medium with the surface facing up, and place 3 pieces of the treated leaves evenly on each culture medium. The primary culture medium for leaves was MS as the basal medium, with the addition of exogenous hormones at a ratio of KT 1 mg / L + NAA 0.3 mg / L. Three explants were inoculated into each culture medium. The culture medium was observed daily after inoculation to facilitate timely treatment of bacterial growth and browning, and to avoid cross-infection.
[0029] Preferably, in this embodiment, the specific process of initial culture of the disinfected flowers is as follows: After sterilization, place the flowers on the operating tray, cut off the front end of the sterilized flowers with a blade, and spread them flat on the surface of the culture medium, with two flowers evenly placed on each culture medium. The primary culture medium for flowers was MS as the basal medium, with the addition of exogenous hormones in the ratio of KT 1-2 mg / L + NAA 0.3-0.5 mg / L. Three explants were inoculated into each culture medium. The culture medium was observed daily after inoculation to facilitate timely treatment of mycelial growth and browning, and to avoid cross-infection.
[0030] Preferably, in this embodiment, the seedling hardening process is as follows: the cap of the tissue culture seedling culture bottle is unscrewed to allow ventilation, the ventilation time is 9 hours / day, and the ventilation time is 7 days.
[0031] Preferably, in this embodiment, the volume ratio of coconut brick, perlite and vermiculite is set to 2:1:1, and care should be taken to keep the soil moist after transplanting.
[0032] Preferably, in this embodiment, the steps for preparing 1LMS basal culture medium are as follows: ① Add the prepared macro-element mother liquor (50mL), micro-element mother liquor (10mL), iron salt mother liquor (10mL), organic mother liquor (10mL), and carrageenan. Add 500-600mL of distilled water to dissolve. Boil until the solution becomes translucent. Add sucrose and distilled water to bring the volume to 1000mL.
[0033] ② Adjust the pH to 6.5-7.0.
[0034] ③ Add various plant growth regulators as needed.
[0035] ④ Place the culture medium in an autoclave, set the pressure to 1.0 kg / cm², the temperature to 121°C, and sterilize for 20 minutes.
[0036] Preferably, unless otherwise specified, all chemical reagents used in this application are commonly used reagents in the industry and can be purchased through formal channels.
[0037] As a further aspect of this embodiment, the culture conditions for culturing explants are set as follows: After inoculating the explants, the culture medium was placed in the dark and cultured for 7 days to promote the formation of embryogenic callus and prevent browning. After dark culture, the cells were placed under light for 16 hours per day. The temperature of the culture room was (25±2)℃ and the humidity was 70%-85%.
[0038] As a further embodiment, please refer to Tables 1 to 5 and Figure 1 (A) to Figure 4 As shown, when tissue culture and rapid propagation of wild Primrose leaves, the various parameters obtained by tissue culture and rapid propagation using the method of this application are far superior to those of others.
[0039] Table 1: Effects of different combinations of plant growth regulators on leaf growth of Primula sylvatica Table 2: Effects of different combinations of plant growth regulators on leaf proliferation and differentiation of Primulina purpurea Table 3: Effects of different combinations of plant growth regulators on the leaf growth of Primulina 'Primulina' Table 4: Effects of different combinations of plant growth regulators on vigorous seedling growth of Primula spp. Table 5: Effects of different combinations of plant growth regulators on rooting of Primula sylvatica Note: Data are mean ± standard error. Different letters after the data in the same column indicate significant differences (P < 0.05).
[0040] As a further embodiment, please refer to Tables 6 to 5 and Figure 5 (A) to Figure 8 As shown, in the tissue culture and rapid propagation of wild Primrose flowers, the various parameters obtained by the tissue culture and rapid propagation method of this application are far superior to those of others.
[0041] Table 6: Effects of different combinations of plant growth regulators on the flower growth of Primrose Table 7: Effects of different combinations of plant growth regulators on the proliferation and differentiation of tissue-cultured flowers of Primula sylvatica Table 8: Effects of different combinations of plant growth regulators on the growth of tissue-cultured Primulina flowers Table 9: Effects of different combinations of plant growth regulators on rooting of Primula sylvatica Experimental Example 1: This experimental example specifically employs the following steps for tissue culture and rapid propagation of wild Primrose leaves and flowers: S1. Disinfection and sterilization of explants: The test material was Primula spp., which was collected from the wild. The leaves were the tender new leaves from the top of the plant. 1.1 Blade Pretreatment: Take vigorous young leaves from the top of Primrose as explants. After removing the petioles, cut the leaves into small pieces and rinse them under running water until the surface dust is clean. Place them in a glass bottle and shake them three times, each time for 3 minutes.
[0042] 1.2 Disinfection and Ultra-Clean Inoculation: After UV sterilization, the explants were sterilized on a clean bench. Primrose leaves were placed in a cleaning bottle, rinsed three times with distilled water, drained, and then soaked in 75% alcohol for 30 seconds for initial sterilization. After rinsing off the alcohol with distilled water and draining again, mercuric chloride was added for secondary sterilization, soaking for 6 minutes. After the mercuric chloride soak, the leaves were rinsed four times with distilled water until no mercuric chloride residue remained on the leaf surface. The sterilized explants were then placed on filter paper. Because primrose leaves are delicate and easily damaged, special care is needed during cleaning. The leaves should be gently agitated in the cleaning bottle. A brush can be used as an auxiliary tool; the brush tip should be used to gently stir the leaves in the cleaning bottle to promote full contact between the explants and the disinfectant. After sterilization, the leaves were placed on an autoclaved operating tray and cut into 2cm x 2cm pieces with a blade. The pieces were then laid face up on the culture medium surface, with three pieces evenly placed on each culture medium.
[0043] S2, Initial Cultivation: Take sterilized leaves from S1 and lay them flat in the primary culture medium with the leaf surface facing up and the leaf underside facing down. After inoculation of explants, the culture medium is first placed under dark conditions and cultured for 7 days to promote embryogenic callus formation and prevent browning. After dark culture, it is placed under light for 16 hours / day, with a culture temperature of (25±2)℃ and a humidity of 70%-85%. The composition of the primary culture medium is: MS as the basic medium, with the addition of exogenous hormones in the ratio of KT 1 mg / L + NAA 0.3 mg / L. The pH of the culture medium is adjusted to 6.5-7.0 before sterilization.
[0044] S3, Subculture: Leaves were taken from sterile seedlings of Primula spp., which were cultured in S2 for 25 days. When the seedlings from the primary culture were growing well and reached a height of about 1 cm, their leaves were taken, cut into small pieces of 0.5 cm × 0.5 cm, and inoculated into the subculture medium. After 7 days of dark culture, they were transferred to light culture and cultured at a temperature of (25±2) ℃, a light duration of 16 h / d, and a humidity of 70%-85%. The subculture medium consisted of MS as the basic medium, and exogenous plant hormones were added in the ratio of KT 1 mg / L + NAA 0.4 mg / L, referring to the growth and differentiation of explants in the primary culture in 2). The pH of the medium was adjusted to 6.5-7.0 before sterilization.
[0045] S4. Seedling rooting culture: After 30 days of S3 culture, subculture seedlings with good growth, a height of 0.5-0.6 cm, 4 leaves, and no roots were selected and inoculated onto a strong seedling rooting medium. The seedlings were then placed under light for 16 hours per day, at a temperature of (25±2)℃ and a humidity of 70%-85%. The primary culture medium consisted of MS as the basal medium, supplemented with exogenous hormone IBA at a ratio of 0.7 mg / L. The pH was adjusted to 6.5-7.0 before sterilization.
[0046] S5. Seedling Transplanting: Remove the seedlings from the culture bottle in S4, wash off the culture medium with clean water, and transfer them to 8*8cm nutrient pots. The cultivation substrate in the nutrient pots should be exposed to sunlight for 1 day in advance and thoroughly watered. The mass ratio of the cultivation substrate is: coconut brick, vermiculite and perlite in a mass ratio of 2:1:1.
[0047] The entire regeneration system in this experiment had a growth cycle of 100 days. The survival rate of the explants after sterilization was 82%, the healing rate of the adventitious buds was 97%, the plants grew well and were bright green, the rooting rate of the tissue culture seedlings was 100%, and the final transplant survival rate of the tissue culture seedlings reached 100%.
[0048] Experimental Example 2: (I) Disinfection and sterilization of explants: The test material was Primula spp., collected from the wild. Flowers were taken from the top of the plant when they were just fully open and used as explants. (i) Flower pretreatment: Take the newly opened flowers at the top of Primula as explants. Rinse the flowers under running water for 5 minutes. After rinsing them clean, place them in a glass bottle and shake them three times.
[0049] (ii) Flower disinfection and ultra-clean inoculation: The ultra-clean workbench is sterilized with ultraviolet light for 30 minutes before use. After ultraviolet sterilization, the pre-treated flowers are placed in a washing bottle, distilled water is added, and the mixture is gently shaken and rinsed three times with distilled water, then drained. 75% alcohol is added for disinfection and soaked for 20 seconds. After rinsing off the alcohol with distilled water, mercuric chloride is added and soaked for 4 minutes and 30 seconds. After disinfection, the flowers are rinsed four times with distilled water. The rinsing method involves gently picking up the material with tweezers, placing it in the washing bottle, adding the reagent (approximately 2 / 3 of the bottle's volume), and gently shaking. After disinfection, the flowers are placed on a work tray, the tips are cut off with a blade, and the flowers are laid flat on the surface of the culture medium, with two explants evenly placed on each culture medium.
[0050] (II) Initial Cultivation: Take the sterilized flowers from step (I), cut off the front end of the flower with a blade, and lay it flat on the surface of the culture medium. After inoculating the explants, the culture medium is first placed under dark conditions and cultured for 7 days to promote the formation of embryogenic callus and prevent browning. After the dark culture is completed, it is placed under light for 16 hours / day, with a culture room temperature of (25±2)℃ and a humidity of 70%-85%. The composition of the primary culture medium is: MS as the basic culture medium, with the addition of exogenous hormones in the ratio of KT1-2 mg / L + NAA0.3-0.5 mg / L. The pH of the culture medium is adjusted to 6.5-7.0 before sterilization.
[0051] (III) Successive generations of training: Leaves were taken from sterile seedlings of Primula spp., which were obtained after culturing for 25 days in step (II). When the seedlings in the primary culture were growing well and reached a height of about 1 cm, their leaves were taken, cut into small pieces of 0.5 cm × 0.5 cm, and inoculated into the subculture medium. After culturing in the dark for 7 days, they were transferred to light culture and cultured at a temperature of (25±2) ℃, a light duration of 16 h / d, and a humidity of 70%-85%. The composition of the subculture medium was as follows: MS was used as the basic medium, and exogenous plant hormones were added in the ratio of KT 1 mg / L + NAA 0.4 mg / L, referring to the growth and differentiation of explants in the primary culture in step (II). The pH of the medium was adjusted to 6.5-7.0 before sterilization.
[0052] (IV) Seedling Rooting Culture: After 30 days of cultivation in step (III), select healthy subculture seedlings with a height of 0.5-0.6 cm, 4 leaves, and no roots. Inoculate them onto a strong seedling rooting medium and place them under light for 16 hours / day. The temperature in the culture room is (25±2)℃, and the humidity is 70%-85%. The composition of the primary culture medium is as follows: MS is the basic culture medium, with the addition of exogenous hormone IBA at a ratio of 0.7 mg / L. The pH of the culture medium is adjusted to 6.5-7.0 before sterilization.
[0053] (V) Transplanting of tissue culture seedlings: Remove the seedlings from the culture bottle in step (IV), wash off the culture medium with clean water, and transfer them to 8*8cm nutrient pots. The cultivation substrate in the nutrient pots should be exposed to sunlight for 1 day in advance and thoroughly watered. The mass ratio of the cultivation substrate is: coconut brick, vermiculite and perlite in a mass ratio of 2:1:1.
[0054] The entire regeneration system in this experiment had a growth cycle of 100 days. The survival rate of the explants after sterilization was 95%, the healing rate of the adventitious buds was 98%, the plants grew well and were bright green, the rooting rate of the tissue culture seedlings was 100%, and the final transplant survival rate of the tissue culture seedlings reached 100%.
[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.
Claims
1. A method for tissue culture and rapid propagation of leaves and flowers of wild Primrose, characterized in that, Includes the following steps: Step 1: Obtaining explants: Take vigorous young leaves and newly opened flowers from the top of Primrose as explants. Step 2: Pretreatment of explants: After removing the petioles from the tender leaves, cut them into small pieces and rinse them under running water until the surface dust is clean. After rinsing the flowers under running water, place them in a glass bottle and shake them to rinse. Step 3: Disinfection of explants: The pretreated tender leaves are placed in a cleaning bottle in a clean bench, rinsed with distilled water, and drained. The tender leaves in the cleaning bottle are then disinfected by soaking. Place the pre-treated flowers into the cleaning bottle in the laminar flow hood, add distilled water, gently shake and rinse, then drain the water; disinfect the flowers in the cleaning bottle by soaking. Step 4: Initial Culture The disinfected young leaves were inoculated onto the primary leaf culture medium for primary culture; the primary leaf culture medium used MS as the basic medium and was supplemented with exogenous hormones at a ratio of KT 1 mg / L + NAA 0.3 mg / L. The sterilized flowers were inoculated onto the primary flower culture medium for primary culture; the primary flower culture medium used MS as the basic medium, with the addition of exogenous hormones at a ratio of KT 1-2 mg / L + NAA 0.3-0.5 mg / L. Step 5, Subculture: The material for subculture is the young leaves after the initial culture. When the seedlings from the initial culture are growing well and reach a height of 1 cm, take their leaves, cut them into small pieces of 0.5 cm × 0.5 cm, and inoculate them into the subculture medium. After culturing in the dark for 7 days, transfer them to light culture to obtain subcultured seedlings. The subculture medium is MS as the basic medium, with the addition of exogenous plant hormones at a ratio of KT 1 mg / L + NAA 0.4-0.6 mg / L. Step 6: Rooting and strengthening seedling culture: Take healthy seedlings with a height of 0.5-0.6cm, 4 leaves, and no roots, and culture them under light with MS as the basic medium to obtain strong seedlings. Step 7: When the seedlings reach the growth indicators of 1.8-2cm in height, 1.9-2.1cm in root length, and 7-8 leaves, harden them off. After hardening off, transplant them for further cultivation.
2. The method according to claim 1, characterized in that, The young leaves should be placed in a glass bottle and rinsed at least three times, with each rinsing session lasting 3-5 minutes.
3. The method according to claim 1, characterized in that, Set the time for rinsing the flowers under running water to 3-5 minutes; Rinse the flowers in a glass bottle at least three times, with each rinse lasting 3-5 minutes.
4. The method according to any one of claims 1-3, characterized in that, Before placing the pretreated young leaves and flowers into the cleaning bottle of the laminar flow hood, the laminar flow hood is sterilized with ultraviolet light for at least 30 minutes.
5. The method according to claim 4, characterized in that, The specific process for disinfecting the pretreated young leaves is as follows: Add alcohol to the cleaning bottle and use the soaking method to disinfect the tender leaves in the cleaning bottle for the first time. After the initial disinfection, the alcohol in the cleaning bottle is rinsed with distilled water, the water is drained, and then mercuric chloride is added to the cleaning bottle to disinfect the tender leaves in the cleaning bottle a second time. After secondary disinfection, the tender leaves are rinsed with distilled water. Once there is no mercuric chloride residue on the leaf surface, the disinfected tender leaves are placed on filter paper. Disinfect the tender leaves.
6. The method according to claim 4, characterized in that, The specific process for disinfecting the pre-treated flowers is as follows: Add alcohol to the cleaning bottle and use the soaking method to disinfect the flowers in the cleaning bottle for the first time; After the initial disinfection, rinse the alcohol with distilled water, drain the water, and then add mercuric chloride for a second disinfection. After the second disinfection, rinse the flowers with distilled water. Once there is no mercuric chloride residue on the surface of the flowers, place the disinfected flowers on the operating tray and cut off the front end of the disinfected flowers with a blade. Complete the disinfection of the flowers.
7. The method according to claim 5 or 6, characterized in that, The culture conditions for primary culture, subculture, and rooting and seedling cultivation are all set as follows: First, place the sample in the dark for 7 days under light-protected conditions; After dark culture, the cells were placed under light for 16 hours per day. The temperature of the culture room was (25±2)℃ and the humidity was 70%-85%.
8. The method according to claim 7, characterized in that, During the initial culture of disinfected young leaves, three young leaves were inoculated into the initial culture medium for each leaf, and the culture medium was observed daily after inoculation. During the initial culture of the sterilized flowers, three flowers were inoculated into each flower's initial culture medium, and the culture medium was observed daily after inoculation.
9. The method according to claim 8, characterized in that, The hardening-off method is as follows: unscrew the cap of the tissue culture bottle to allow air to pass through for 9 hours per day for a total of 7 days. The volume ratio of coconut fiber bricks, perlite, and vermiculite was set to 2:1:
1.
10. The method according to claim 9, characterized in that, The steps for preparing 1L MS basal medium are as follows: (1) Add 50mL of prepared macro-element mother liquor, 10mL of micro-element mother liquor, 10mL of iron salt mother liquor, 10mL of organic mother liquor and carrageenan, add 500-600mL of distilled water to dissolve, boil until the solution is translucent, add sucrose, and add distilled water to make up to 1000mL. (2) Adjust the pH to 6.5-7.0; (3) Add various plant growth regulators; (4) Place the culture medium in an autoclave, pressurize it to 1.0 kg / cm2, set the temperature to 121°C, and sterilize it for 20 min.