CN110896856A - Method for inducing complete inflorescence in vitro by axillary bud of inflorescence shaft of phalaenopsis - Google Patents
Method for inducing complete inflorescence in vitro by axillary bud of inflorescence shaft of phalaenopsis Download PDFInfo
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- CN110896856A CN110896856A CN201911081189.0A CN201911081189A CN110896856A CN 110896856 A CN110896856 A CN 110896856A CN 201911081189 A CN201911081189 A CN 201911081189A CN 110896856 A CN110896856 A CN 110896856A
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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Abstract
The invention belongs to the technical field of phalaenopsis culture, and particularly relates to a method for in vitro induction of complete inflorescences by axillary buds of inflorescences of phalaenopsis. The method successfully induces and cultivates a complete phalaenopsis inflorescence by treating the axil axillary bud of the phalaenopsis and placing the base part of the axil axillary bud on an induction culture medium in combination with a temperature and illumination interaction mode, is not limited by seasonal conditions, can realize long-term induction cultivation, saves test materials for researching the flowering mechanism of the phalaenopsis, can eliminate the interference of a plurality of unknown factors by completing the inflorescence under the in vitro condition, is a material for researching the flowering mechanism of the phalaenopsis, and lays a foundation for the reproductive induction cultivation of the phalaenopsis.
Description
Technical Field
The invention belongs to the technical field of phalaenopsis culture, and particularly relates to a method for in vitro induction of complete inflorescences by axillary buds of inflorescences of phalaenopsis.
Background
The phalaenopsis amabilis is a plant of the phalaenopsis of the orchidaceae, is favored by people due to beautiful flower color, various varieties, beautiful flower appearance and long flowering phase, and is extremely popular in the flower market in recent years, but the natural flowering phase of the phalaenopsis amabilis is relatively late, generally in the months of 4 to 5 every year, and cannot meet the demand of people on the phalaenopsis amabilis all the year round, so that the phalaenopsis amabilis can be flowered as required by manual regulation and control in the cultivation of the phalaenopsis amabilis.
At present, the research on the tissue culture of the phalaenopsis is very deep, and the leaves, the root tips, the stem tips and the inflorescence axes of the phalaenopsis can be used for inducing complete plants, but no effective method is available for inducing the phalaenopsis to complete the inflorescence under the in vitro condition. The butterfly orchid is a flower of an annual night, the natural flowering phase of the butterfly orchid is about 3 months every year, and the key link of butterfly orchid production is that the butterfly orchid flowers according to needs by artificially regulating and controlling the flowering phase. In production, the temperature is controlled by means of air conditioning cooling or high mountain flower forcing and the like to meet the flowering requirement of the butterfly orchid in the night year, but the production cost of the butterfly orchid is greatly increased, and a large number of mature seedlings are needed to perform related tests every year for exploring the response mechanism of butterfly orchid flowering.
Taking the inflorescence axis of the phalaenopsis as an explant, inducing axillary buds of the inflorescence axis into a complete plant according to the requirement of the existing method, and then culturing the plant into the complete inflorescence, wherein the process needs a long time and is difficult to meet the market requirement; however, there is no effective method for directly inducing the axile axillary bud of the inflorescence to form a complete inflorescence at present, so that a method for inducing the complete inflorescence in vitro by the axile axillary bud of the phalaenopsis is needed to shorten the cultivation period of the phalaenopsis.
Disclosure of Invention
Aiming at the defect and problem that no axillary bud of an inflorescence shaft of the phalaenopsis can be induced into a complete inflorescence in vitro at present, the invention provides a method for inducing the complete inflorescence in vitro by the axillary bud of the inflorescence shaft of the phalaenopsis.
The technical scheme adopted by the invention for solving the technical problems is as follows: a method for inducing complete inflorescences of axillary buds of inflorescences of phalaenopsis comprises the steps of shearing the inflorescences of phalaenopsis, sterilizing the inflorescences of phalaenopsis, inoculating the inflorescences on an induction culture medium for induction culture, wherein the culture temperature is as follows: the temperature ranges of day and night are 22-26 ℃ and 15-20 ℃, respectively, and the culture illumination is as follows: light intensity of 150--2·s-1And culturing for 25-30 days at the light-dark ratio of 12h/12h, and counting the number of complete inflorescences of the phalaenopsis.
The day temperature and the night temperature are respectively 24 ℃ and 18 ℃, and the light intensity is 200 mu mol.m-2·s-1。
The method for inducing the complete inflorescence in vitro of the axillary bud of the inflorescence shaft of the phalaenopsis comprises the step of adding 0.1-0.2mg/LTDZ, 0.8-1.2mg/L6-BA and 0.1-0.2mg/LNAA into an MS culture medium.
The method for inducing the complete inflorescence in vitro of the axillary bud of the inflorescence shaft of the phalaenopsis comprises the following steps of adding 20% of coconut juice, 7.5g/L of agar, 20g/L of sucrose and 1g/L of activated carbon into an induction medium.
Before the induction culture, the method also comprises the step of placing the base part of the inflorescence shaft into a 2,4-D solution of 0.1-0.2mg/L for soaking for 10-30s after beveling the two ends of the inflorescence shaft.
In the method for inducing complete inflorescences in vitro by using axillary buds of inflorescences of phalaenopsis, the light for culturing illumination is scattered light.
The invention has the beneficial effects that: the invention adopts the inflorescence shaft of the phalaenopsis as an explant, the base part of the inflorescence shaft of the phalaenopsis is placed in a 2,4-D solution for soaking treatment, then inoculating the axillary buds of the inflorescence axis in a special culture medium, performing regeneration induction on the axillary buds of the inflorescence axis through the interaction of temperature and illumination, successfully induces and cultures the complete inflorescence of the phalaenopsis by the synergistic action of nutrient components, temperature and illumination of the culture medium, reveals the optimal development environment for developing the complete inflorescence, can obviously improve the induction rate of the in vitro induced complete inflorescence of the axillary bud of the inflorescence shaft of the phalaenopsis amabilis, shortens the induction culture time, is not limited by seasonal conditions, can realize long-term induced cultivation, saves test materials for researching the flowering mechanism of the phalaenopsis, and, the technology is used for finishing inflorescence under the condition of in vitro induction, can eliminate the interference of a plurality of unknown factors, and lays a foundation for the reproductive induction culture of phalaenopsis.
Drawings
FIG. 1 is a photograph of an intact inflorescence induced by axillary bud of inflorescence shaft of phalaenopsis of the present invention in vitro; the length of the axillary bud of the inflorescence axis from left to right in the figure is 0.5cm, 1cm, 1.5cm and 2cm respectively.
FIG. 2 is a photograph of whole plants induced by axillary buds of inflorescence axes of comparative examples of the present invention in vitro.
Detailed Description
The invention is further illustrated below with reference to the accompanying drawings and examples, in order to better illustrate the invention, the invention uses a phalaenopsis 'capsicum annuum' variety as a test variety.
The invention of the technology can save most of test materials for researching the flowering mechanism of the phalaenopsis, and the technology is used for finishing the inflorescence induced under the in vitro condition, can eliminate the interference of a plurality of unknown factors, and is a material for researching the flowering mechanism of the phalaenopsis.
Example 1: the embodiment provides a method for inducing complete inflorescences of inflorescences in vitro by axillary buds of inflorescences of large hot peppers of phalaenopsis, which comprises the following steps:
firstly, cutting a inflorescence shaft of the phalaenopsis large hot pepper by using scissors, taking the first two axillary buds from top to bottom, and cutting the inflorescence shaft into small sections of 3-4cm by taking the axillary buds of the inflorescence shaft as the center;
secondly, removing the scales wrapping the axillary buds, and wiping the scales for 3-5 times by using an alcohol cotton ball;
thirdly, sterilizing the rachis with 0.1 percent mercuric chloride for 10min on a super-clean workbench, and then washing with sterile water for 3-5 times;
fourthly, beveling two ends of the rachis, and soaking the rachis base in 0.1 mg/L2, 4-D solution for 20 s; adding inflorescence shaft baseInoculating into induction culture medium (1/2MS +1mg/L6-BA +0.2mg/L TDZ +0.2mg/L LNAA + 20% coconut juice +7.5g/L agar +20g/L sucrose +1g/L active carbon), inoculating 8 dormant buds of phalaenopsis rachis each bottle, adding 10 bottles into a group, placing into artificial climate incubator for induction culture, setting day and night temperature at 24 deg.C and 18 deg.C, respectively, and setting culture light intensity at 200 μmol/m-2·s-1Culturing for 26d with the light-dark ratio of 12h/12h, and counting the number of the induced complete inflorescences of the large butterfly orchid capsicum.
Example 2: the embodiment provides a method for inducing complete inflorescences of inflorescences in vitro by axillary buds of inflorescences of large hot peppers of phalaenopsis, which comprises the following steps:
firstly, cutting a inflorescence shaft of the phalaenopsis large hot pepper by using scissors, taking the first two axillary buds from top to bottom, and cutting the inflorescence shaft into small sections of 3-4cm by taking the axillary buds of the inflorescence shaft as the center;
secondly, removing the scales wrapping the axillary buds, and wiping the scales for 3-5 times by using an alcohol cotton ball;
thirdly, sterilizing the rachis with 0.1 percent mercuric chloride for 15min on a super-clean workbench, and then washing with sterile water for 3-5 times;
fourthly, beveling two ends of the rachis, and soaking the rachis base in 0.1 mg/L2, 4-D solution for 10 s; aseptically inoculating the basal part of the inflorescence shaft downwards on an induction culture medium (1/2MS +1.2 mg/L6-BA +0.1mg/L TDZ +0.1mg/L LNAA + 20% coconut juice + agar 7.5g/L +20g/L sucrose +1g/L active carbon), inoculating 8 phalaenopsis inflorescence shaft dormant buds in each bottle, combining 10 bottles into a group, putting the group into an artificial climate incubator for induction culture, setting the day and night temperature to be 22 ℃ and 15 ℃ respectively, and setting the culture illumination to be 150 mu mol m light intensity-2·s-1And culturing for 30d at the light-dark ratio of 12h/12h, and calculating the number of the induced complete inflorescences of the large butterfly orchid capsicum.
Example 3: the embodiment provides a method for inducing complete inflorescences of inflorescences in vitro by axillary buds of inflorescences of large hot peppers of phalaenopsis, which comprises the following steps:
firstly, cutting a inflorescence shaft of the phalaenopsis large hot pepper by using scissors, taking the first two axillary buds from top to bottom, and cutting the inflorescence shaft into small sections of 3-4cm by taking the axillary buds of the inflorescence shaft as the center;
secondly, removing the scales wrapping the axillary buds, and wiping the scales for 3-5 times by using an alcohol cotton ball;
thirdly, sterilizing the rachis with 0.1 percent mercuric chloride for 12min on a super-clean workbench, and then washing with sterile water for 3-5 times;
fourthly, beveling two ends of the rachis, and soaking the rachis base in 0.15 mg/L2, 4-D solution for 12 s; inoculating the base part of the inflorescence shaft downwards to an induction culture medium (1/2MS +0.8 mg/L6-BA +0.3mg/L TDZ +0.3mg/L LNAA + 20% coconut juice + agar 7.5g/L +20g/L sucrose +1g/L active carbon), inoculating 8 phalaenopsis inflorescence shaft dormant buds in each bottle, combining 10 bottles into a group, putting the group into an artificial climate incubator for induction culture, setting the day and night temperature to be 26 ℃ and 20 ℃ respectively, and setting the culture illumination to be 250 mu mol.m-2·s-1And culturing for 28 days at the light-dark ratio of 12h/12h, and calculating the number of the induced complete inflorescences of the large butterfly orchid capsicum.
Examples 4 to 6
Examples 4-6 provide methods for inducing intact inflorescences in vitro by axillary buds of inflorescences of Phalaenopsis Capsicum frutescens var. lappa in substantially the same manner as in example 1, except that the induction medium and the culture environment are slightly different, as shown in Table 1.
TABLE 1 culture media and culture environments used in examples 4-6 of the present invention
Comparative example
The method for performing in-vitro induction on the inflorescence shaft of the large butterfly orchid hot pepper provided by the comparative example comprises the following steps:
firstly, cutting a inflorescence shaft of the phalaenopsis large hot pepper by using scissors, taking the first two axillary buds from top to bottom, and cutting the inflorescence shaft into small sections of 3-4cm by taking the axillary buds of the inflorescence shaft as the center;
secondly, removing the scales wrapping the axillary buds, and wiping the scales for 3-5 times by using an alcohol cotton ball;
thirdly, sterilizing the rachis with 0.1 percent mercuric chloride for 10min on a super-clean workbench, and then washing with sterile water for 3-5 times;
fourthly, beveling both ends of the rachis, aseptically inoculating the rachis base downwards on an induction culture medium (1/2MS +10mg/L6-BA + 20% coconut juice +7.5g/L agar +20g/L sucrose +1g/L active carbon), inoculating 8 phalaenopsis rachis dormant buds in each bottle, combining 10 bottles into a group, putting the group into an artificial climate incubator for induction culture, setting the day and night temperature to be 30 ℃ and 28 ℃ respectively, and setting the culture illumination to be 30 mu mo 1m-2.s-1The light-dark ratio is 12h/12h, the culture is carried out for 25d, and the induction result is observed.
By observing the appearance shape of the induction of the inflorescence shaft of the large hot pepper of the comparative phalaenopsis, the result is shown in figure 2, and the fact that the method of the comparative example is adopted to perform in vitro induction on the inflorescence shaft of the phalaenopsis is found, and the inflorescence shaft is induced to develop into a complete plant in the development process and does not develop towards the direction of developing into a complete inflorescence.
Induction results
The induction results of examples 1-6 are detailed in Table 2.
TABLE 2 statistics of in vitro induced intact rachis of the invention
| Group of | Dormant bud (seed) | Inflorescence shaft (root) | Inductivity (%) |
| Example 1 | 80 | 78 | 97.5 |
| Example 2 | 80 | 77 | 96.25 |
| Example 3 | 80 | 75 | 93.75 |
| Example 4 | 80 | 75 | 93.75 |
| Example 5 | 80 | 76 | 95 |
| Example 6 | 80 | 73 | 91.25 |
As can be seen from Table 2, the induction rate of inducing the inflorescence axes of the phalaenopsis by adopting the method disclosed by the invention exceeds 91%, while the induction rate of the inflorescence axes by adopting the method disclosed by the embodiment 1 is as high as 97.5%, which indicates that the phalaenopsis inflorescence axes explants can be efficiently and quickly isolated into complete inflorescence axes by using the culture medium provided by the invention and combining the interaction mode of temperature and illumination.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and scope of the present invention are intended to be covered thereby.
Claims (6)
1. A method for inducing complete inflorescences of axillary buds of inflorescences of phalaenopsis amabilis in vitro is characterized by comprising the following steps: the method comprises the following steps:
firstly, cutting a inflorescence shaft by using scissors, taking the first two axillary buds from top to bottom, and cutting the inflorescence shaft into small sections of 3-4cm by taking the axillary buds of the inflorescence shaft as the center;
secondly, removing the scales wrapping the axillary buds, and wiping the scales for 3-5 times by using an alcohol cotton ball;
thirdly, sterilizing the rachis with 0.1 percent mercuric chloride for 10-15min on a super-clean workbench, and then washing with sterile water for 3-5 times;
and fourthly, beveling two ends of the rachis, inoculating the rachis base part downwards on an induction culture medium in a sterile way for induction culture, wherein the culture temperature is as follows: day and night temperature ranges are 22-26 ℃ and 15-20 ℃, and culture illumination is as follows: light intensity of 150--2·s-1Culturing for 25-30 days at the light-dark ratio of 12h/12h to obtain the complete inflorescence of the phalaenopsis.
2. The method for inducing the complete inflorescence in vitro by the axillary bud of the inflorescence shaft of the phalaenopsis as claimed in claim 1, which is characterized in that: the day and night temperatures are 24 and 18 ℃, respectively, and the light intensity is 200 mu mol.m-2·s-1。
3. The method for inducing the complete inflorescence in vitro by the axillary bud of the inflorescence shaft of the phalaenopsis as claimed in claim 1, which is characterized in that: the induction culture medium is prepared by adding 0.1-0.3mg/L TDZ, 0.8-1.2mg/L6-BA and 0.1-0.3mg/L NAA into MS culture medium.
4. The method for inducing the complete inflorescence in vitro by the axillary bud of the inflorescence shaft of the phalaenopsis as claimed in claim 3, wherein the method comprises the following steps: the induction culture medium also contains 20% of coconut juice, 7.5g/L of agar, 20g/L of sucrose and 1g/L of activated carbon.
5. The method for inducing the complete inflorescence in vitro by the axillary bud of the inflorescence shaft of the phalaenopsis as claimed in claim 1, which is characterized in that: before induction culture, the method also comprises the step of soaking the base part of the rachis in a 2,4-D solution of 0.1-0.2mg/L for 10-30s after beveling the two ends of the rachis.
6. The method for inducing the complete inflorescence in vitro by the axillary bud of the inflorescence shaft of the phalaenopsis as claimed in claim 1, which is characterized in that: the light of the culture illumination is scattered light.
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Cited By (1)
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| CN115251057A (en) * | 2022-08-26 | 2022-11-01 | 郑州师范学院 | Method for inducing germination of phalaenopsis seedlings by utilizing phytohormone composition |
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Application publication date: 20200324 |