Pineapple tissue culture method

Abstract

The invention relates to a bromeliad tissue culture method, and aims to solve the problems of long propagation period, low efficiency, poor genetic stability and the like in a traditional bromeliad propagation mode. According to the method disclosed by the invention, the rapid, efficient and stable propagation of the bromeliad is realized through the measures of optimizing a culture medium formula, adjusting the concentration and proportion of growth adjusting substances, adopting specific cells or tissues as explants, optimizing culture conditions and the like. According to the technology, the propagation period of the bromeliad is remarkably shortened, the propagation efficiency is improved, the hereditary character of the stock plant is kept, and the consistency of the plant shape and flower color is ensured. In addition, the operation difficulty and cost of the tissue culture technology are reduced by simplifying the operation process, reducing the operation steps, adopting a novel culture medium and growth regulating substances and the like. The successful implementation of the technology provides powerful support for commercialized production of the bromeliad and promotes rapid development of the bromeliad industry.

Description

Technical Field

[0001] The invention relates to the technical field of pineapple flower cultivation, in particular to a pineapple flower tissue cultivation method. Background Art

[0002] In terms of bromeliad propagation, although traditional seed propagation and division propagation are still in use, tissue culture technology has gradually begun to emerge. Through tissue culture technology, a large number of bromeliad seedlings with consistent genetic traits can be quickly propagated to meet the market demand for high-quality bromeliads.

[0003] Although tissue culture technology has achieved certain results in bromeliad reproduction, there are still some defects and areas for improvement.

[0004] Reproduction efficiency needs to be improved: Although tissue culture technology can shorten the reproduction cycle of pineapple flowers, the current reproduction efficiency still needs to be improved. Especially in large-scale production, how to further improve the reproduction efficiency and reduce production costs is a problem that needs to be solved at present.

[0005] Complex technical operations: Tissue culture technology requires specific culture medium, growth regulators and strict aseptic operating conditions, which leads to certain limitations on the promotion and application of technology.

[0006] Genetic stability issues: Although tissue culture technology can maintain the genetic characteristics of the mother plant to a certain extent, in the actual operation process, genetic variation may still occur due to the influence of culture conditions, cell differentiation and other factors, resulting in different plant morphology and flower color.

[0007] In response to the defects and problems of the existing technology, this patented technology proposes a rapid propagation technology of bromeliads based on tissue culture. Summary of the invention

[0008] In order to solve the problems of the prior art, the present invention provides a pineapple flower tissue culture method.

[0009] In order to solve the above technical problems, the present invention is implemented by the following technical solutions: 1. A pineapple flower tissue culture method, comprising the following steps:

[0010] S1: Explant selection and pretreatment: Select healthy, disease-free and insect-free young leaves, terminal buds or lateral buds of pineapple flowers as explants, and perform surface disinfection to remove surface microorganisms and contaminants;

[0011] S2: Inoculation on high-efficiency induction medium: The pretreated explants were inoculated on a high-efficiency induction medium containing MS basal medium, 6-BA 2.0 mg / L, NAA 0.2 mg / L, sucrose 3%, agar 0.6% and activated carbon 0.5 g / L, and cultured under the predetermined temperature, light and humidity conditions until robust callus tissue was formed;

[0012] S3: Transfer to rapid proliferation medium: transfer the formed callus or clustered buds to a rapid proliferation medium containing MS basal medium, KT 1.5 mg / L, IBA 0.1 mg / L, sucrose 3% and sodium alginate as a suspending agent to rapidly proliferate cells and form a large number of buds;

[0013] S4: Rooting and seedling strengthening treatment: The seedlings that have proliferated to a certain size are transferred to a rooting and seedling strengthening medium containing 1 / 2MS basal medium, 0.5mg / L IBA, 0.1mg / L NAA, 2% sucrose, 0.7% agar and 0.5g / L calcium magnesium phosphate fertilizer for rooting and seedling strengthening treatment to form complete plants;

[0014] S5: Out of bottle acclimatization: The plants after rooting and seedling strengthening treatment are taken out from the culture bottle, cleaned and sterilized, planted in a clean and germ-free cultivation medium, and adapted in the acclimatization room for 30 days;

[0015] S6: After completing domestication, plant the plants in a suitable field environment and strengthen field management to ensure their healthy growth.

[0016] In this method, through tissue culture technology, the same kind of pineapple flower plants can be propagated in large quantities in a short period of time to meet market demand. Tissue culture technology can maintain the excellent genetic characteristics of the mother plant, so that the propagated new plants are consistent with the mother plant in terms of morphology, growth habits, etc. Through specific culture medium formulas and culture conditions, the stress resistance of pineapple flower plants can be improved to a certain extent, so that they can better adapt to adverse environmental conditions.

[0017] Compared with traditional propagation methods, such as division propagation and cutting propagation, tissue culture technology can quickly propagate a large number of new plants in a short period of time. This greatly shortens the propagation cycle and improves production efficiency; tissue culture technology reduces the risk of infection during the propagation process of plants through sterile operation and control of culture conditions, thereby improving the survival rate. In addition, since the culture medium provides sufficient nutrients and growth regulators, it is beneficial to the growth and development of the plants; tissue culture technology can maintain the genetic stability of the mother plant and avoid problems such as genetic variation and trait separation that may occur in traditional propagation methods. This makes the new plants reproduced consistent with the mother plant in terms of morphology, growth habits, etc., which is conducive to maintaining the excellent characteristics of the variety; tissue culture technology can improve the stress resistance of pineapple plants to a certain extent. By adjusting the culture medium formula and culture conditions, different environmental conditions can be simulated so that the plants can gradually adapt to these conditions during the propagation process. This helps to improve the survival rate and growth performance of the plants during the planting process

[0018] In some specific embodiments, the innermost stem top of the pineapple crown bud is preferably used in step S1;

[0019] The disinfection treatment specifically includes immersing the explant in commercially available bleach for 20 minutes, and then rinsing it with sterile water for 3 times, each time for 10 minutes.

[0020] In some specific embodiments, the environmental parameters in step S2, step S3 and step S4 are: temperature is set to 22-25 degrees Celsius, humidity is set to 75%-85%, light is set to 1000-1500 lux (lx), and lighting time is set to 12-16 hours / day.

[0021] In some specific embodiments, the cultivation time of step S2 is set to 2-8 weeks, with the presentation of bright green or light green and compact texture as the reference standard, the cultivation time of step S3 is set to 2-6 weeks, with the presentation of green or light green seedlings as the reference standard, and the cultivation time of step S4 is set to 3-8 weeks, with the presentation of thick root system and strong plants as the reference standard.

[0022] In some specific embodiments, the S4 step further includes stress resistance enhancement culture: when facing adverse environmental conditions, some seedlings or plants can be transferred to a stress resistance enhancement culture medium containing MS basal culture medium, 6-BA 1.0 mg / L, NAA 0.2 mg / L, sucrose 3%, agar 0.7%, paclobutrazol 0.5 mg / L and mannitol 5 g / L for stress resistance enhancement culture to improve the plant's ability to adapt to adversity.

[0023] In some specific embodiments, the disinfection treatment in step S5 adopts the disinfection treatment process of S1.

[0024] In some specific embodiments, the planting conditions of step S6 are set to a temperature of 20-30 degrees Celsius, sufficient light, good ventilation, and fertile, loose, and well-drained soil.

[0025] The beneficial effects of the present invention are:

[0026] 1. The present invention adopts tissue culture technology, which can significantly shorten the reproduction cycle of pineapple flowers from the traditional 12-18 months to only 3-6 months, and can achieve the complete process from explant to transplant. This significant time reduction makes the commercial production of pineapple flowers possible, greatly improves the reproduction efficiency, and meets the market demand for rapid and large-scale reproduction of pineapple flowers;

[0027] 2. Tissue culture technology can be carried out in a strictly controlled laboratory environment, effectively avoiding the intrusion of pests and diseases, thereby significantly improving the survival rate of pineapple seedlings. At the same time, due to the high controllability of growth conditions, the health of the plants is also greatly guaranteed, the leaves are bright in color and beautiful in shape, and the overall growth condition is better than traditional cultivation methods;

[0028] 3. The present invention can reproduce a large number of pineapple seedlings by using a small amount of explants, which greatly optimizes the efficiency of resource utilization. Compared with the traditional cultivation method that requires a large number of seeds or native seedlings and land resources, the present invention is more efficient in resource utilization and has lower long-term costs. In addition, tissue culture technology can also reduce the use of chemical substances such as fertilizers and pesticides, further reducing production costs and improving economic benefits. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Embodiment: A method for bromeliad tissue culture.

[0031] Pineapple flower tissue culture method process

[0032] 1. Explant selection and pretreatment (S1)

[0033] Explant selection:

[0034] Select healthy, disease- and insect-free young leaves, terminal buds or side buds of bromeliads, preferably the top of the stem in the innermost layer of the crown bud.

[0035] Preprocessing:

[0036] For surface disinfection, the explants were immersed in commercially available bleach for 20 minutes.

[0037] After disinfection, rinse with sterile water three times, 10 minutes each time, to remove microorganisms and contaminants on the surface.

[0038] 2. Inoculate in high-efficiency induction medium (S2)

[0039] Culture medium formula:

[0040] MS basal medium

[0041] 6-BA 2.0mg / L

[0042] NAA 0.2mg / L

[0043] Sucrose 3%

[0044] Agar 0.6%

[0045] Activated carbon 0.5g / L

[0046] Culture conditions:

[0047] Temperature: 22-25 degrees Celsius

[0048] Humidity: 75%-85%

[0049] Light: 1000-1500 lux (lx)

[0050] Lighting time: 12-16 hours / day

[0051] Cultivation time:

[0052] 2-8 weeks, with bright green or light green callus and compact texture as the reference standard.

[0053] 3. Transfer to Rapid Proliferation Medium (S3)

[0054] Culture medium formula:

[0055] MS basal medium

[0056] KT 1.5mg / L

[0057] IBA 0.1mg / L

[0058] Sucrose 3%

[0059] Sodium alginate as suspending agent

[0060] Culture conditions:

[0061] Same as step S2.

[0062] Cultivation time:

[0063] 2-6 weeks, with green or light green sprouts as the reference standard.

[0064] 4. Rooting and seedling strengthening treatment (S4)

[0065] Culture medium formula:

[0066] 1 / 2MS basal medium

[0067] IBA 0.5mg / L

[0068] NAA 0.1mg / L

[0069] Sucrose 2%

[0070] Agar 0.7%

[0071] Calcium magnesium phosphate fertilizer 0.5g / L

[0072] Culture conditions:

[0073] Same as steps S2 and S3.

[0074] Cultivation time:

[0075] 3-8 weeks, with strong root system and healthy plants as the reference standard.

[0076] Stress resistance enhancement culture (optional):

[0077] When facing adverse environmental conditions, some seedlings or plants were transferred to a stress-enhanced medium containing MS basal medium, 6-BA 1.0 mg / L, NAA 0.2 mg / L, sucrose 3%, agar 0.7%, paclobutrazol 0.5 mg / L and mannitol 5 g / L for stress-enhanced culture.

[0078] 5. Domestication after bottle removal (S5)

[0079] Processing flow:

[0080] The plants after the rooting and seedling strengthening treatment were taken out from the culture bottle.

[0081] After cleaning and sterilization, it is planted in a clean and germ-free cultivation medium.

[0082] Acclimate in the acclimatization room for 30 days.

[0083] Disinfection:

[0084] Use the disinfection process in step S1.

[0085] VI. Field planting and management (S6)

[0086] Planting conditions:

[0087] Temperature: 20-30 degrees Celsius

[0088] Adequate lighting

[0089] Good ventilation

[0090] The soil is fertile, loose and well-drained

[0091] Field management:

[0092] Strengthen field management, including watering, fertilizing, pest and disease control, to ensure the healthy growth of bromeliad plants.

[0093] The above process describes in detail the various steps of the pineapple flower tissue culture method, including the selection and pretreatment of explants, inoculation in a high-efficiency induction medium, transfer to a rapid proliferation medium, rooting and seedling treatment, bottle acclimatization, and field planting and management. Each step includes specific parameters such as culture medium formula, culture conditions, and culture time to ensure the rapid propagation and healthy growth of pineapple flower plants. At the same time, an optional step of stress resistance enhancement culture is also provided to improve the plant's ability to adapt to adversity.

[0094] In order to compare the difference between this patented technology and traditional cultivation technology in producing pineapple seedlings, we designed a control experiment. The following is the experimental design, including detailed descriptions of the experimental group, control group and blank group:

[0095] Experimental design

[0096] 1. Purpose of the experiment

[0097] Compare the differences between the patented tissue culture method and the traditional cultivation method in terms of pineapple seedling generation speed, survival rate and plant health.

[0098] 2. Experimental Materials

[0099] Experimental group: patented technology was used and tissue culture method was used.

[0100] Control group: Traditional cultivation methods were used, such as seedlings collected directly from the wild and grown naturally for a long time (native seedlings).

[0101] Blank group: No cultivation operation was performed, and the group was only used as an observation object to evaluate the performance of bromeliad flowers under natural growth conditions.

[0102] 3. Experimental Procedure

[0103] Experimental Group

[0104] Sampling: Cut the pineapple crown buds and bring them back to the laboratory. Peel off the outer leaves of the pineapple crown buds to expose the innermost stem top.

[0105] Removal of miscellaneous bacteria: Peel off the outer leaves of the pineapple crown buds and place them in 5 times diluted commercially available bleach for disinfection, then rinse with sterile water.

[0106] Tissue culture: Cut off the top tissue of the pineapple crown bud stem after disinfection and culture it on a culture medium containing plant growth regulators and cytokinins.

[0107] Cluster bud production: Transfer the sterile buds to another culture medium for cultivation to produce cluster buds.

[0108] Branch multiplication: Cut the clustered buds for further multiplication.

[0109] Rooting: Cut the branches and place them on rooting medium until adventitious roots grow at the base of the plant.

[0110] Domestication and transplanting: The cultivated plants are domesticated and then transplanted into a suitable substrate.

[0111] Control group (traditional cultivation method)

[0112] Collect native seedlings: Collect native bromeliad seedlings that have adapted to the natural environment from the wild.

[0113] Transplanting: Transplant the native seedlings into a suitable substrate and carry out routine management.

[0114] Blank group (natural growth)

[0115] Observation object: Select an area with good natural environment as the observation object.

[0116] Record data: Regularly record the growth of bromeliads in the area, including growth rate, survival rate, etc.

[0117] 4. Data Collection and Evaluation

[0118] Growth rate: Record the growth cycle of each group of pineapple seedlings, from the time of sampling or collection to the time of becoming a complete plant.

[0119] Survival rate: After a period of time after transplanting, the survival rate of each group of pineapple seedlings was counted.

[0120] Plant health: Observe and record the leaf color, morphology and other health indicators of each group of pineapple seedlings.

[0121] 5. Experimental results and analysis

[0122] Growth rate: The growth rate of the experimental group may be significantly faster than that of the control group and the blank group because the tissue culture method can accelerate the growth process of plants.

[0123] Survival rate: The survival rates of the experimental group and the control group may be higher, but the experimental group may have higher adaptability due to the domestication process, thus having a higher survival rate. The survival rate of the blank group may be greatly affected by the natural environment and fluctuate greatly.

[0124] Plant health: The plant health indicators of the experimental group and the control group may be similar, but the experimental group may have more controllable growth conditions and more stable plant health. The plant health of the blank group may be greatly affected by the natural environment.

[0125] Precautions

[0126] Experimental conditions: Ensure that the experimental conditions (such as temperature, light, humidity, etc.) of the experimental and control groups are as consistent as possible to eliminate the influence of external factors on the experimental results.

[0127] Data recording: Record all data during the experiment in detail to facilitate accurate analysis and comparison.

[0128] Repeat the experiment: In order to improve the reliability and accuracy of the experiment, it is recommended to conduct multiple repeated experiments and take the average value as the final result.

[0129] Through the above experimental design, we can compare the advantages and disadvantages of the experimental group's tissue culture method and the traditional cultivation method in the production of pineapple seedlings, and provide a scientific basis for actual production.

[0130]

[0131] Remark:

[0132] Growth cycle: The experimental group can significantly shorten the growth cycle of pineapple flowers through tissue culture methods. It usually takes only 3-6 months from explant to transplant. Traditional cultivation technology takes longer, and it may take 12-18 months from seeds or native seedlings to mature plants. The growth cycle of the blank group is greatly affected by the natural environment, and the specific time cannot be determined.

[0133] Survival rate: The survival rate of the experimental group is usually higher because the tissue culture method can be carried out in a controlled environment, reducing the risk of pests and diseases. The survival rate of traditional cultivation technology is affected by factors such as seed quality and environmental adaptability, and is relatively low. The survival rate of the blank group is greatly affected by factors such as the natural environment and natural enemies, and is usually lower.

[0134] Plant health: The health of the plants in the experimental group is better because the growth conditions are controllable and can reduce the impact of adverse factors such as pests and diseases. The health of plants in traditional cultivation techniques may be affected by pests and diseases, environmental changes, etc., and is generally poor. The health of the plants in the blank group is greatly affected by the natural environment and cannot be determined.

[0135] Reproduction efficiency: The experimental group has high reproduction efficiency, which can quickly and massively reproduce pineapple seedlings to meet commercial needs. The reproduction efficiency of traditional cultivation technology is low and it is difficult to meet large-scale needs. The reproduction efficiency of the blank group is limited by the natural environment and is usually low.

[0136] Resource Utilization: The experimental group can efficiently use a small amount of explants to reproduce a large number of plants, which improves resource utilization efficiency. Traditional cultivation technology requires more seeds or native seedlings, and land resources are limited, so resource utilization efficiency is relatively low. The blank group is limited by the natural environment, and the resource utilization efficiency cannot be determined.

[0137] Cost investment: The initial investment in equipment and culture medium for the experimental group is high, but the long-term cost is low because a large number of plants can be reproduced quickly. The cost investment of traditional cultivation technology is relatively low, but the cost may increase due to the use of fertilizers and pesticides. The blank group has no direct cost investment, but may incur indirect costs due to environmental damage.

[0138] Technical difficulty: The experimental group requires professional skills and equipment, and the operation is relatively complicated. Traditional cultivation technology is relatively simple and easy to master. The blank group has no technical requirements, but is limited by the natural environment.

[0139] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A pineapple flower tissue culture method, characterized in that: The following steps are involved: S1: Explant selection and pretreatment: Select healthy, disease-free and insect-free young leaves, terminal buds or lateral buds of pineapple flowers as explants, and perform surface disinfection to remove surface microorganisms and contaminants; S2: Inoculation on high-efficiency induction medium: The pretreated explants were inoculated on a high-efficiency induction medium containing MS basal medium, 6-BA 2.0 mg / L, NAA 0.2 mg / L, sucrose 3%, agar 0.6% and activated carbon 0.5 g / L, and cultured under the predetermined temperature, light and humidity conditions until robust callus tissue was formed; S3: Transfer to rapid proliferation medium: Transfer the formed callus or clustered buds to a rapid proliferation medium containing MS basal medium, KT1.5mg / L, IBA 0.1mg / L, sucrose 3% and sodium alginate as a suspending agent to rapidly proliferate cells and form a large number of buds; S4: Rooting and seedling strengthening treatment: The seedlings that have proliferated to a certain size are transferred to a rooting and seedling strengthening medium containing 1 / 2MS basal medium, 0.5mg / L IBA, 0.1mg / L NAA, 2% sucrose, 0.7% agar and 0.5g / L calcium magnesium phosphate fertilizer for rooting and seedling strengthening treatment to form complete plants; S5: Out of bottle acclimatization: The plants after rooting and seedling strengthening treatment are taken out from the culture bottle, cleaned and sterilized, planted in a clean and germ-free cultivation medium, and adapted in the acclimatization room for 30 days; S6: After completing domestication, plant the plants in a suitable field environment and strengthen field management to ensure their healthy growth.

2. A pineapple flower tissue culture method according to claim 1, characterized in that: In the step S1, the innermost stem top of the pineapple crown bud is preferably selected; The disinfection treatment specifically includes immersing the explant in commercially available bleach for 20 minutes, and then rinsing it with sterile water for 3 times, each time for 10 minutes.

3. A pineapple flower tissue culture method according to claim 1, characterized in that: The environmental parameters in the steps S2, S3 and S4 are: the temperature is set at 22-25 degrees Celsius, the humidity is set at 75%-85%, the light is set at 1000-1500 lux (lx), and the lighting time is set at 12-16 hours / day.

4. A pineapple flower tissue culture method according to claim 1, characterized in that: The cultivation time of step S2 is set to 2-8 weeks, with bright green or light green and compact texture as the reference standard. The cultivation time of step S3 is set to 2-6 weeks, with green or light green sprouts as the reference standard. The cultivation time of step S4 is set to 3-8 weeks, with thick root system and strong plants as the reference standard.

5. A pineapple flower tissue culture method according to claim 1, characterized in that: Further, the step S4 further includes stress resistance enhancement culture: when facing adverse environmental conditions, part of the seedlings or plants can be transferred to a stress resistance enhancement culture medium containing MS basal culture medium, 6-BA 1.0 mg / L, NAA 0.2 mg / L, sucrose 3%, agar 0.7%, paclobutrazol 0.5 mg / L and mannitol 5 g / L, for stress resistance enhancement culture to improve the plant's ability to adapt to adversity.

6. A pineapple flower tissue culture method according to claim 1, characterized in that: The disinfection process in step S5 adopts the disinfection process flow of step S1.

7. A pineapple flower tissue culture method according to claim 1, characterized in that: The planting conditions of step S6 are set as follows: a temperature of 20-30 degrees Celsius, sufficient light, good ventilation, and fertile, loose, and well-drained soil.

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