CN116058284A

CN116058284A - Non-symbiotic germination propagation method for paphiopedilum seed

Info

Publication number: CN116058284A
Application number: CN202310105239.4A
Authority: CN
Inventors: 付传明; 黄宁珍; 冼康华; 苏江; 刘宝骏; 何金祥
Original assignee: Guangxi Institute of Botany of CAS
Current assignee: Guangxi Institute of Botany of CAS
Priority date: 2023-02-13
Filing date: 2023-02-13
Publication date: 2023-05-05

Abstract

The invention discloses a non-symbiotic germination propagation method for paphiopedilum flutriantis seeds, and belongs to the technical field of plant tissue culture. The method of the invention comprises the following steps: after the surface disinfection of the collected paphiopedilum capsules, the seeds are sequentially subjected to germination culture, differentiation culture, subculture multiplication culture and strong seedling rooting culture, and the illumination conditions of all culture stages are controlled. The invention takes the seed with specific maturity of the paphiopedilum as an explant, and obtains the complete plant of the paphiopedilum through seeding under specific culture medium and light environment conditions, germination, differentiation, secondary proliferation and rooting culture of strong seedlings. The method has high germination rate, quick emergence and good seedling quality, and can produce a large amount of high-quality paphiopedilum seedlings in a short time.

Description

Non-symbiotic germination propagation method for paphiopedilum seed

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a non-symbiotic germination propagation method for paphiopedilum pubescens seeds.

Background

The paphiopedilum [ Paphiodilumbarishii (Rchb.F.) Stein ] is a paphiopedilum perennial plant of Orchidaceae, which is only distributed in Geng Maxian and Menyan county in southwest of Yunnan, grows on the trunk or rock under shrubs in evergreen broadleaf forests or on the shading rock wall in limestone areas, and has very narrow distribution region and special habitat. In recent years, due to the damage of habitat and artificial excessive digging, the wild resources of the paphiopedilum in the bandeau are extremely rare, and a plurality of distribution points originally recorded are difficult to find out the trail. At present, the paphiopedilum is a first-class important protection plant in China, is listed in annex I of International trade Commission on Aristolochia (CITES) of endangered wild animal and plant species, and is also listed as a serious danger species (CR) in the red directory of the world natural protection consortium (IUCN) to be protected urgently.

The flower shape of the paphiopedilum is unique, the sepal is large and striking, the lip is in the shape of a helmet, the petals are bright in color and long and drooping like the ribbon, the flower shape is more unique than other paphiopedilum, and the flower shape is taken as the exquisite paphiopedilum and has extremely high ornamental value. Meanwhile, the ribbon pocket is one of the 2-3 kinds of flower pocket which are only available in China, is a difficultly obtained material for developing high-grade flowers and cultivating new types of flowers, and has very important scientific research value and economic value.

The resource protection, development and utilization of the paphiopedilum are carried out, and the propagation of enough seedlings is key. The traditional propagation method of the paphiopedilum is usually divided into plant propagation (asexual propagation), namely, clustered sprouts are divided for independent cultivation, 1-2 new sprouts are induced in the process of growing, aging and withering of the sprouts, 2-3 years are usually required for completing the process, the average propagation coefficient is lower than 2, the propagation speed is extremely slow, the large-scale propagation cannot be achieved due to the limitation of materials, and the requirements of species protection and commodity production are difficult to meet; in sexual reproduction, the paphiopedilum can pollinate to obtain fructification, and the number of seeds in single-grain capsules is huge, but the seeds are tiny like dust, endosperm is not contained, cotyledons and primordium are incompletely differentiated, the nutrition is insufficient, and specific fungi are required to symbiosis under natural conditions to sporadically germinate, so that the germination rate is extremely low.

Therefore, there is a need to provide a rapid propagation method for non-symbiotic germination of paphiopedilum flutriantis seeds, which has high germination rate, rapid emergence and good seedling quality, and the breeding research of paphiopedilum flutriantis has not been reported at present.

Disclosure of Invention

Therefore, the invention aims to provide a rapid propagation method for non-symbiotic germination of paphiopedilum flutriantis seeds. The method provided by the invention has the advantages of high seed germination rate, fast seedling emergence and good seedling quality, can be used for large-scale seedling propagation of the paphiopedilum, and promotes resource protection and sustainable utilization.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a non-symbiotic germination propagation method for paphiopedilum seed, which comprises the following steps:

after harvesting the paphiopedilum capsules and carrying out surface disinfection, sequentially carrying out germination culture, differentiation culture, subculture multiplication culture and strong seedling rooting culture on seeds;

the germination culture medium is 1/2-1/8 MS, 30-50 g/L of banana, 1.0-2.0 g/L of active carbon, 20-30 g/L of sucrose and 3.0-4.0 g/L of agar, and the pH value is 5.4-6.2;

the differentiation medium is as follows: 1/2-1/4 MS+100-200 mL/L of coconut juice+6-benzyladenine 2.0-4.0 mg/L+0.1-0.5 mg/L of naphthylacetic acid+1.0-2.0 g/L of activated carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, and the pH value is 5.4-6.2;

the secondary proliferation culture medium is as follows: 1/2-1/4 MS+50-100 g/L of banana+6-benzyladenine 1.0-3.0 mg/L+0.1-0.5 mg/L of naphthylacetic acid+1.0-2.0 g/L of activated carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, and the pH value is 5.4-6.2;

the seedling strengthening and rooting culture medium is as follows: 1/2-1/4 MS+50-100 g/L of banana+0.5-1.0 mg/L of silver nitrate+0.5-1.0 mg/L of naphthylacetic acid+1.0-2.0 g/L of active carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, and the pH value is 5.4-6.2;

shading culture is carried out during germination culture, and shading rate is 80-100%; the illumination intensity of the differentiation culture is 500-1000 lx; the illumination intensity of the secondary proliferation culture and the strong seedling rooting culture is independently 1500-3000 lx.

As one implementation mode, artificial pollination is carried out at 8-10 am to obtain capsules 3-10 d after the flowers of the paphiopedilum in the streamer are opened.

Preferably, watering the paphiopedilum in the bandeau within 3-5 days after artificial pollination.

As one embodiment, the maturity of the paphiopedilum capsules is 120-210 DAP, and the appearance is green.

As one embodiment, the illumination time of the differentiation culture, the secondary proliferation culture and the strong seedling rooting culture is independently 10-16 h/d.

As an implementation mode, the temperatures of germination culture, differentiation culture, secondary proliferation culture and strong seedling rooting culture are 23-27 ℃ and the environmental humidity is 60-70%.

As one implementation mode, the germination culture is carried out for 40-90 d to obtain protocorms, the differentiation culture is carried out for 60-120 d to obtain buds, the secondary proliferation culture is carried out for 60-120 d to obtain cluster buds, and the strong seedling rooting culture is carried out for 60-90 d to obtain complete plants.

As one implementation mode, the invention further comprises the seedling hardening and transplanting steps, wherein the seedling hardening and transplanting season is 3-5 months or 10-11 months each year.

Preferably, the seedling hardening is performed under natural light temperature for 10-20 d, the transplanted cultivation matrix is planted Jin Danhe pine bark, and the volume ratio is 1:1 to 3.

Compared with the prior art, the invention has the following technical effects:

the invention takes the seed with a certain maturity of the paphiopedilum as an explant, and obtains the complete plant of the paphiopedilum after germination, differentiation, secondary proliferation and strong seedling rooting culture under specific culture medium and light environment conditions by sowing. Experimental results show that the artificial pollination and fructification of the paphiopedilum in the method provided by the invention reach 88.5%, the germination rate of the seed germination culture is 36.5%, the bud seedlings with 1-3 leaves can be obtained after differentiation culture for 60d, cluster buds can be formed after subculture for 90d, the proliferation coefficient is 2.8, complete plants are formed after rooting culture of strong seedlings, the rooting rate reaches 100%, and the survival rate reaches 91% after seedling hardening and bottle discharging transplantation.

The invention improves the germination and growth rate of seeds, overcomes the problem of brown death of protocorms, has the advantages of high propagation speed, large emergence quantity, good seedling quality and the like, and can produce a large amount of high-quality paphiopedilum seedlings in a short time. The bred paphiopedilum seedling can be applied to the field protection and natural regression, can also be directly used as a high-grade orchid and used for the supply market of the genetic improvement cultivation of new species of flowers, and is beneficial to the resource protection and sustainable utilization of the paphiopedilum.

Drawings

FIG. 1 is a photograph of a banded paphiopedilum of the present invention flowering and pollinating fruit set during the protection of the locus;

FIG. 2 is a photograph of germination of paphiopedilum seed according to the present invention: the left is white protocorm formed by seed germination, and the right is the brown and dead protocorm;

FIG. 3 is a photograph showing the differentiation of the protocorm of the paphiopedilum to a young seedling in the present invention;

FIG. 4 is a photograph of a cluster bud formed by the secondary proliferation of the paphiopedilum in accordance with the present invention;

FIG. 5 is a photograph showing the strong seedling and rooting culture of the paphiopedilum in the present invention;

FIG. 6 is a photograph of a transplant of a banded paphiopedilum out of a bottle in the present invention;

FIG. 7 is a photograph of the growth of a banded paphiopedilum of the present invention after transplanting.

Detailed Description

the germination culture medium is 1/2-1/8 MS+30-50 g/L of banana+1.0-2.0 g/L of active carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, and the pH value is 5.4-6.2;

in conventional tissue culture of paphiopedilum, the illumination conditions used in the stages of seed germination, differentiation culture, secondary proliferation, rooting culture and the like are usually the same illumination conditions: the illumination intensity is 1500-3000 lx, and the illumination time is 10-16 h/d. According to different illumination conditions used in the stages of seed germination, differentiation culture, secondary proliferation, rooting culture and the like of paphiopedilum: shading culture is carried out during germination culture, and shading rate is 80-100%; the illumination intensity of the differentiation culture is 500-1000 lx; the illumination intensity of the secondary proliferation culture and the strong seedling rooting culture is independently 1500-3000 lx.

According to the invention, artificial pollination is carried out at 8-10 am in 3-10 d of the opening of the flower of the paphiopedilum in the ribbon, so as to obtain capsules. In the present invention, the pollination time is preferably 4 to 8d, specifically 8 to 10 am, more preferably 5 to 7d, specifically 8 to 9 am, when the flowers are fully opened. In the invention, the artificial pollination is preferably to take different flowering plants or different flowers of the same plant as parent parents. The pollination mode is achieved by a conventional pollination method in the field. As an embodiment, the process of cutting the pericardium of the labial lobe of the female parent flower with scissors is performed first, and then the pollen sac of the male parent is smeared on the surface of the female parent column.

In the invention, capsule management and maintenance are also needed after pollination, specifically, watering is stopped within 3-5 d after pollination, more preferably, watering is stopped within 4d after pollination, and then management is carried out according to normal water and fertilizer. Experiments prove that watering is carried out in 3d after artificial pollination of the paphiopedilum, the flowers of the plants wilt 5-10 d after pollination, and the pedilum also gradually becomes wilting and cannot successfully bear fruits. The artificial pollination method adopted by the invention has the setting percentage as high as 88.5%, improves the plumpness and the seed quality of the capsules, and further improves the germination rate of the paphiopedilum seed culture.

The invention preferably collects capsules with the maturity of 120-210 DAP and green surface, inoculates seeds on a germination culture medium after surface sterilization, induces the seeds to germinate in a germination light environment, and cultures 40-90 d to obtain protocorms. Experiments prove that the capsule with the maturity lower than 120DAP has green epidermis, white block shape of internal seeds, strong viscosity and difficult dispersion, can only germinate sporadically after sowing, and has extremely low germination rate; the capsules with the maturity of 120-210 DAP also have differences in the appearance color and the growth state of internal seeds due to the differences in the environmental conditions and the physiological states of the plants. The appearance of a part of capsules is still green, the internal seeds are in powder form, the color is changed from white to black brown, and the seeding and germination effects are optimal. The outer surface part of the other capsule is changed into yellow, the inner seeds are yellow or black brown, and germination is difficult after sowing; the capsule with the maturity higher than 210DAP has all turned yellow in appearance, the internal seeds are black brown, and the germination rate after sowing is 0.

In the present invention, the maturity is calculated from the number of days grown from pollination to harvest (days after pollinatiaon, DAP). In the invention, the maturity of the paphiopedilum capsules is 120-210 DAP, preferably 125-180 DAP, more preferably 130-150 DAP, and the appearance color is green.

In the present invention, the sterilization treatment preferably includes: brushing off dust and sundries on the surface of the capsule under tap water by using a soft brush, soaking the capsule in alcohol with the volume fraction of 70-80% for 60-120 s, and rinsing the capsule with sterile water for 1-2 times; then sterilizing for 10-15 min by using 0.1-0.15% of mercuric chloride solution by mass fraction, and rinsing for 4-5 times by using sterile water; more preferably, the method comprises: brushing dust and sundries on the surface of the capsule under tap water by using a soft brush, soaking the capsule in alcohol with the volume fraction of 75% for 90s, rinsing the capsule with sterile water for 1 time, disinfecting the capsule with mercuric chloride solution with the mass fraction of 0.1% for 12min, and rinsing the capsule with sterile water for 5 times. In the invention, the disinfection treatment can kill microorganisms such as bacteria on the surface of capsules and promote non-symbiotic germination of later-stage seeds.

In the present invention, the germination medium is: 1/2-1/8 MS+30-50 g/L of banana+1.0-2.0 g/L of active carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, and the pH value is 5.4-6.2; preferably 1/2-1/4 MS+35-45 g/L of banana+1.0-1.5 g/L of active carbon+20-25 g/L of sucrose+3.2-3.8 g/L of agar, and the pH value is 5.8-6.2; more preferably 1/4 MS+40 g/L of banana+1.0 g/L of activated carbon+20 g/L of sucrose+3.5 g/L of agar, and the pH value is 5.8.

In the present invention, the germination is performed in a light-shielding culture, and the light-shielding rate is 80 to 100%, more preferably 90 to 95%. An alternative embodiment is to perform shading culture with black shading cloth. Under the light environment of germination culture, the germination speed and germination rate of paphiopedilum seed can be improved.

The invention transfers the protocorm obtained by germination culture to a differentiation culture medium, induces the protocorm to differentiate in a differentiation light environment, and cultures for 60-120 days to obtain the bud seedlings.

In the invention, the differentiation culture medium is 1/2-1/4 MS+100-200 mL/L of coconut juice+6-benzyl adenine 2.0-4.0 mg/L+0.1-0.5 mg/L of naphthylacetic acid+1.0-2.0 g/L of active carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, and the pH value is 5.4-6.2; preferably, the composition is 1/2 MS+120-180 mL/L of coconut juice+6-benzyladenine 2.5-3.5 mg/L+naphthalene acetic acid 0.2-0.4 mg/L+activated carbon 1.0-1.5 g/L+sucrose 20-25 g/L+agar 3.5-3.8 g/L, and the pH value is 5.8-6.0; more preferably, the pH value is 5.8, and the total concentration is 1/2 MS+150 mL/L of coconut juice+6-benzyladenine 3.0 mg/L+naphthalene acetic acid 0.3 mg/L+activated carbon 1.0 g/L+sucrose 20 g/L+agar 3.5 g/L; in the present invention, the coconut juice is juice from the interior of fresh coconut.

In the invention, the light environment of the differentiation culture is preferably weak scattering illumination culture, the illumination intensity is 500-1000 lx, and the illumination time is 10-16 h/d; preferably, the illumination intensity is 600-800 lx, and the illumination time is 12-14 h/d. The culture temperature of the differentiation culture is 23-27 ℃ and the humidity is 60-70%. In the invention, the protocorm is transferred onto the differentiation culture in time, and the culture in the light environment of the differentiation culture can overcome the problem that the protocorm is easy to brown and die, and promote the differentiation of the protocorm into buds.

The obtained sprouts are transferred to a secondary proliferation culture medium, and are subjected to secondary proliferation culture for 60-120 days to obtain clustered sprouts.

In the invention, the secondary proliferation culture medium is a culture medium of 1/2-1/4 MS+50-100 g/L of banana+6-benzyladenine 1.0-3.0 mg/L+naphthalene acetic acid 0.1-0.5 mg/L+activated carbon 1.0-2.0 g/L+sucrose 20-30 g/L+agar 3.0-4.0 g/L, and the pH value is 5.4-6.2; preferably 1/2-1/4 MS+60-90 g/L of banana+6-benzyl adenine 1.5-2.5 mg/L+0.15-0.35 mg/L of naphthylacetic acid+1.0-2.0 g/L of active carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, and the pH value is 5.6-6.0; more preferably, the pH value of the culture medium is 5.8, wherein the culture medium is 1/2MS, 80g/L of banana, 2.0mg/L of 6-benzyladenine, 0.2mg/L of naphthylacetic acid, 1.0g/L of activated carbon, 20g/L of sucrose and 3.5-3.8 g/L of agar; the bananas in the culture medium are prepared by removing the skin of mature bananas and then putting the bananas into a juicer and adding water to uniformly stir.

The illumination intensity of the relay multiplication culture is 1500-3000 lx, and the illumination time is 10-16 h/d; preferably, the illumination intensity is 2000-2500 lx, and the illumination time is 12-14 h/d; the temperature of the culture is preferably 23-27 ℃ and the ambient humidity is 60-70%.

The invention divides the cluster buds into single seedlings and inoculates the single seedlings to a strong seedling rooting culture medium, and the whole plants are obtained after culturing for 60-90 days.

In the invention, the strong seedling rooting culture medium is 1/2-1/4 MS+50-100 g/L of banana+0.5-1.0 mg/L of silver nitrate+0.5-1.0 mg/L of naphthylacetic acid+1.0-2.0 g/L of active carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, and the pH value is 5.4-6.2; preferably 1/2-1/4 MS+70-90 g/L of banana+0.7-0.9 mg/L of silver nitrate+0.6-0.9 mg/L of naphthylacetic acid+1.0-2.0 g/L of active carbon+20-30 g/L of sucrose+3.2-3.8 g/L of agar, and the pH value is 5.6-6.0; more preferably 1/2 MS+80 g/L of banana+0.8 mg/L of silver nitrate+0.8 mg/L of naphthylacetic acid+1.0 g/L of active carbon+20 g/L of sucrose+3.5 g/L of agar, and the pH value is 5.8;

the illumination intensity of the strong seedling rooting culture is 1500-3000 lx, and the illumination time is 10-16 h/d; preferably, the illumination intensity is 2000-2500 lx, and the illumination time is 12-14 h/d; the temperature of the culture is preferably 23-27 ℃ and the ambient humidity is 60-70%.

The invention exercises seedlings of the complete plants, and the seedlings of the paphiopedilum are obtained by bottle-out transplanting.

In the invention, the plant preferably has 2-4 leaves and 2-4 roots, and seedling hardening and bottle discharging transplanting are carried out when the plant height reaches more than 2.5 cm.

In the present invention, the season of hardening off and transplanting is preferably 3 to 5 months or 10 to 11 months each year.

In the invention, the seedling hardening is preferably performed in a natural light temperature environment in a facility greenhouse. The seedling hardening time is preferably 10 to 20 days, more preferably 15 to 18 days.

In the invention, the bottle-out transplanting is preferably to take out plants by opening a bottle cap, clean the culture medium attached to the root, soak the plants in the culture medium for 2 to 3 minutes by diluting 1000 times of carbendazim, air the plants in a shade place for 6 to 12 hours, and collect 1 to 3 single plants into a cluster after the root system is slightly whitened, and plant the plants into a seedling cup.

In the present invention, the transplanted cultivation substrate preferably has a volume ratio of 1: 1-3, more preferably 1: 1-2 of gold-planted stone and pine bark. The sources of the gold-planted stone and pine bark are not particularly limited in the present invention, and preferably Japanese imported gold-planted stone having a particle size of 3 to 6mm and New Zealand imported Ornithine defatted pine bark having a particle size of 6 to 9mm. As one implementation mode, pine bark and the plant gold stone are soaked in clear water and then mixed uniformly.

In the invention, the humidity and the temperature are kept after the transplanting, and after the transplanting for 90 days, the plant can generate a new root system, and the transplanting survival rate reaches more than 90%.

The technical scheme of the present invention is described in further detail below with reference to specific embodiments, and the technical scheme of the present invention includes but is not limited to the following embodiments.

Example 1

(1) Artificial pollination and fructification of paphiopedilum

In the orchid locus protection garden of Guangxi Guilin, in the flower period of the paphiopedilum, the healthy plants are selected to carry out artificial pollination in the 3 rd d of the complete flower opening. During pollination, firstly, the bag of the female parent labial lobe is sheared off by scissors, then the pollen bag of the male parent is smeared on the head surface of the female parent column by the toothpick, the bag and the label are sleeved, the pollination date and the parental nature are recorded, the female parent plant is stopped watering within 3d after pollination, and then normal light, temperature, water, fertilizer and other artificial cultivation management is carried out. The result shows that flowers which are successfully pollinated wilt 15-20 days after pollination, and the wilt flowers are timely picked off at the moment, because the wilt flowers remain on the ovary, the flowers are extremely easy to wet in rainy seasons and grow a large amount of mould to spread and damage the ovary. The pedicel of the plant successfully pollinated always keeps fresh green until capsules are ripe, and the statistical fruiting rate is 85% after 120d pollination. As shown in FIG. 1, the left side is flowering in the protected field and the right side is pollination of a firm capsule.

(2) Harvesting, sterilizing and non-symbiotic germination culture of capsules

Due to the differences in environmental conditions and physiological states of plants, the appearance color of capsules with different maturity and the growth state of internal seeds of the paphiopedilum can also be different. The capsule skin with the maturity lower than 120DAP is green, the internal seeds are white lumps, the viscosity is strong, and the capsules are not easy to disperse; the capsule with the maturity of 120-210 DAP, a part of the capsule is still green in appearance, the internal seeds are in powder form, and the color is being changed from white to black brown; the other part of the capsule has been changed to yellow in appearance, and the inner seed is yellow or black brown; the capsule with maturity higher than 210DAP had all turned yellow in appearance and the internal seeds were black brown. The germination effects after surface sterilization of the harvesting of the different maturity banded paphiopedilum capsules are shown in table 1 below, and the surface sterilization is specifically as follows: firstly brushing dust and sundries on the surface of capsules under tap water by using a soft brush, soaking the capsules in alcohol with the volume fraction of 70% for 60s, rinsing the capsules with sterile water for 1 time, disinfecting the capsules with mercuric chloride solution with the mass fraction of 0.1% for 10min, and rinsing the capsules with sterile water for 5 times. Wiping the surface water of the sterilized capsule with sterile filter paper, longitudinally cutting the capsule with a cutter sterilized at high temperature, and inoculating the internal seeds onto the surface of a germination culture medium, wherein the germination culture medium comprises the following formula: 1/2 MS+30 g/L of banana+1.0 g/L of activated carbon+20 g/L of sucrose+3.0 g/L of agar, and the pH value is 5.4. Inducing seed germination in a germination light environment, wherein the germination light environment is as follows: the black shading cloth was used for shading culture, and the shading rate was 80%. The results showed that the germination was evident at 40d, based on the macroscopic protrusion of the seed becoming white and gradually expanding to form white granular protocorms, with a germination rate of 34.5% at 120 d.

TABLE 1 germination and growth of capsules of different maturity

(3) Differentiation culture of protocorms

After the paphiopedilum seed germinates, the formed protocorm particles are transferred to a differentiation medium in time for culture, and the differentiation medium is as follows: 1/2 MS+100 mL/L of coconut juice+6-benzyladenine 2.0 mg/L+naphthalene acetic acid 0.1 mg/L+active carbon 1.0 g/L+sucrose 20 g/L+agar 3.0-4.0 g/L, and the pH value is 5.4; inducing the protocorm to differentiate into bud seedlings in a differentiation light environment, wherein the differentiation light environment is as follows: the illumination intensity is 500lx, the illumination time is 10h/d, the temperature is 23-27 ℃, the humidity is 60-70%, and the bud with 1-3 leaves is obtained by culturing for 90d, as shown in figure 3.

(4) Subculture multiplication of sprouts

Cutting off the buds, and transferring the buds to a secondary proliferation culture medium, wherein the secondary proliferation culture medium is as follows: 1/2MS, 50g/L of banana, 0.0 mg/L of 6-benzyladenine, 0.1mg/L of naphthylacetic acid, 1.0g/L of active carbon, 20g/L of sucrose, 3.0g/L of agar and pH value of 5.4 are subjected to multiplication culture in a bud propagation mode, and 6 strains are inoculated in each bottle by adopting a orchid bottle with the capacity of 650mL, wherein the culture illumination intensity is 1500lx and the illumination time is 10h/d; the temperature is 23-27 ℃ and the humidity is 60-70%. Cluster buds formed when cultured for 90d, and the proliferation factor was 2.6, as shown in fig. 4.

(5) Culturing strong seedling and rooting

Selecting buds with the height of more than 1.5cm, dividing the buds into single plants, and inoculating the single plants onto a strong seedling rooting culture medium, wherein the formula of the strong seedling rooting culture medium is as follows: 1/2MS, 50g/L of banana, 0.5mg/L of silver nitrate, 0.5mg/L of naphthylacetic acid, 1.0g/L of active carbon, 20g/L of sucrose and 3.5g/L of agar, and the pH value is 5.8. The illumination intensity of the culture is 1500lx, and the illumination time is 10h/d; the temperature is 23-27 ℃ and the humidity is 60-70%. The result shows that after 90d culture, the average leaf number is 2.6, the average root number is 2.5, the average root length is 1.3cm, the rooting rate reaches 100%, and as shown in figure 5, the left side is a photograph of the front side of the bottle seedling, and the right side is a photograph of the root system at the bottom of the bottle seedling.

(6) Seedling hardening and transplanting

Transferring the strong seedlings with developed root systems from the culture room to a greenhouse for hardening seedlings for 10d, opening the bottle cap to take out plants, cleaning the culture medium attached to the root, soaking the plants in 1000-time diluted thiophanate methyl for 2min, taking out the plants, placing the plants in a shade place for 6h, and transplanting the plants, as shown in figure 6. The transplanting matrix is made of gold-planted stone (imported from Japan, particle size is 3-6 mm) and pine bark (imported from New Zealand, particle size is 6-9 mm), and the materials are fully soaked in clear water and then uniformly mixed according to the volume ratio of 1:1. 1-3 single plants are gathered into a cluster and transplanted into a plastic seedling raising cup with the height of 7.0cm and the caliber of 8.5cm, and the humidity and the temperature are kept after the transplanting. The result shows that the plant can generate a new root system after 90 days, and the transplanting survival rate reaches 85 percent, as shown in figure 7.

Example 2

(1) Artificial pollination and fructification of paphiopedilum

In the orchid locus protection garden located in Guangxi Guilin, in the paphiopedilum flowering period, the healthy plants are selected to carry out artificial pollination at the 7 th d of complete flower opening, the female parent plants are stopped watering within 4d after pollination, and other cultivation management and protection after pollination are the same as in example 1. The results showed 88.5% statistical seed setting rate at 120d post pollination.

(2) Harvesting, sterilizing and non-symbiotic germination culture of capsules

The surface disinfection is carried out on the green capsules with the harvest maturity of 150DAP, then the internal seeds are inoculated on 1/4MS of germination culture medium, 40g/L of banana, 1.5g/L of active carbon, 25g/L of sucrose and 3.5g/L of agar, the pH value is 5.8, and the seeds are placed in a light environment which is shaded by black shading cloth for 90 percent for cultivation. The results showed that the germination signs were seen at the time of sowing 40d and gradually enlarged to form white granular protocorms, and the germination rate at 120d was 36.5%.

(3) Differentiation culture of protocorms

After the paphiopedilum seed germinates, the paphiopedilum seed is timely transferred to a differentiation medium of 1/2MS, 150mL/L of coconut juice, 3.0mg/L of 6-benzyl adenine, 0.3mg/L of naphthylacetic acid, 1.0g/L of activated carbon, 20g/L of sucrose and 3.5g/L of agar, and the pH value is 5.8; placing the seedlings in a condition that the illumination intensity is 800lx, the illumination time is 12h/d, the temperature is 23-27 ℃, the humidity is 60-70%, and culturing is 60d to obtain the sprouts with 1-3 leaves.

(4) Subculture multiplication of sprouts

Cutting the bud seedlings, transferring the bud seedlings to a secondary proliferation culture medium 1/2MS, banana 80g/L, 6-benzyl adenine 2.0mg/L, naphthylacetic acid 0.2mg/L, active carbon 1.0g/L, sucrose 20g/L and agar 3.0g/L for culture, wherein the pH value is 5.8, and placing the bud seedlings on the secondary proliferation culture medium with the illumination intensity of 2000lx and the illumination time of 12h/d; culturing at 23-27 deg.c and 60-70% humidity. The result showed that cluster buds were formed by culturing for 90d, and the proliferation factor was 2.8.

(5) Culturing strong seedling and rooting

The bud seedlings with the height of more than 1.5cm are selected and separated into single plants, and inoculated onto a strong seedling rooting culture medium of 1/2MS, 80g/L of banana, 0.8mg/L of silver nitrate, 0.8mg/L of naphthylacetic acid, 1.0g/L of active carbon, 20g/L of sucrose and 3.5g/L of agar, wherein the pH value is 5.8. The light intensity is 2000lx, and the light time is 12h/d; culturing at 23-27 deg.c and 60-70% humidity. The result shows that after 90d culture, the average leaf number is 3.0, the average root number is 2.7, the average root length is 1.6cm, and the rooting rate reaches 100%.

(6) Seedling hardening and transplanting

Transferring the strong seedlings with developed root systems from the culture room to a greenhouse for hardening 15d, opening a bottle cap to take out plants, cleaning a culture medium attached to the root, soaking the plants in 1000-fold diluted thiophanate methyl for 3min, taking out the plants, and placing the plants in a shade place for 8h for transplanting. The transplanting matrix is prepared by uniformly mixing gold-planting stones (imported from Japan, particle size of 3-6 mm) and pine bark (imported from New Zealand, particle size of 6-9 mm) according to a volume ratio of 1:2, gathering 1-3 single plants into a cluster, transplanting the cluster into a plastic seedling cup with a height of 7.0cm and a caliber of 8.5cm, and keeping humidity and temperature after transplanting. The result shows that the plant can generate a new root system after 90 days, and the transplanting survival rate reaches 91%.

Example 3

(1) Artificial pollination and fructification of paphiopedilum

In the orchid field protection garden located in Guangxi Guilin, in the paphiopedilum flowering period, the healthy plants are selected to carry out artificial pollination at the 10 th d of complete flower opening, the female parent plants are stopped watering within 5d after pollination, and other cultivation management and protection after pollination are the same as in example 1. The results showed that the statistical seed setting rate at 120d after pollination was 80%.

(2) Harvesting, sterilizing and non-symbiotic germination culture of capsules

The surface disinfection is carried out on the green capsules with the harvest maturity of 240DAP, then the internal seeds are inoculated on 1/8MS of germination culture medium, 50g/L of banana, 2.0g/L of active carbon, 30g/L of sucrose and 3.5g/L of agar, the pH value is 6.2, and the seeds are placed in a light environment which is shaded by black shading cloth for 100 percent for cultivation. The results showed that the germination signs were seen at the time of sowing 40d and gradually enlarged to form white granular protocorms, and the germination rate at 120d was 10.5%.

(3) Differentiation culture of protocorms

After the paphiopedilum seed germinates, the paphiopedilum seed is timely transferred to a differentiation medium of 1/4MS, 200mL/L of coconut juice, 4.0mg/L of 6-benzyl adenine, 0.5mg/L of naphthylacetic acid, 2.0g/L of activated carbon, 30g/L of sucrose and 3.5g/L of agar, and the pH value is 5.8; culturing under the conditions that the illumination intensity is 1000lx, the illumination time is 16h/d, the temperature is 23-27 ℃ and the humidity is 60-70%. As a result, it was found that the seedlings having 1 to 3 leaves could be obtained by culturing 70 d.

(4) Subculture multiplication of sprouts

Cutting the bud seedlings, transferring the bud seedlings to a secondary proliferation culture medium 1/4MS, 100g/L of banana, 3.0mg/L of 6-benzyl adenine, 0.5mg/L of naphthylacetic acid, 1.0g/L of activated carbon, 20g/L of sucrose and 3.0g/L of agar for culture, wherein the pH value is 5.8, and placing the bud seedlings on the secondary proliferation culture medium with illumination intensity of 3000lx and illumination time of 16 hours/d; culturing at 23-27 deg.c and 60-70% humidity. The result showed that cluster buds were formed by culturing for 90d, and the proliferation factor was 2.5.

(5) Culturing strong seedling and rooting

The bud seedlings with the height of more than 1.5cm are selected and are divided into single plants to be inoculated onto 1/4MS of strong seedling rooting culture medium, 100g/L of banana, 1.0mg/L of silver nitrate, 1.0mg/L of naphthylacetic acid, 1.0g/L of active carbon, 20g/L of sucrose and 3.5g/L of agar, and the pH value is 5.8. Placing the glass at the illumination intensity of 3000lx and the illumination time of 16h/d; culturing at 23-27 deg.c and 60-70% humidity. The result shows that after 90d culture, the average leaf number is 2.5, the average root length is 1.3cm, and the rooting rate reaches 100%.

(6) Seedling hardening and transplanting

Transferring the strong seedlings with developed root systems from the culture room to a greenhouse for hardening seedlings for 20d, opening a bottle cap to take out plants, cleaning a culture medium attached to the root, soaking the plants in 1000-fold diluted thiophanate methyl for 3min, taking out the plants, and placing the plants in a shade place for 12h for transplanting. The transplanting matrix is made of diamond (imported from Japan, particle size is 3-6 mm) and pine bark (imported from New Zealand, particle size is 6-9 mm) according to the volume ratio of 1:3, uniformly mixing, gathering 1-3 single plants into a cluster, transplanting the cluster into a plastic seedling cup with the height of 7.0cm and the caliber of 8.5cm, and keeping the humidity and the temperature after transplanting. The result shows that the plant can generate a new root system after 90 days, and the transplanting survival rate reaches 89.5%.

Example 4

Influence of germination environment on germination rate of paphiopedilum seed

Sowing the paphiopedilum seed by adopting the method in the step (2) in the embodiment 1, and culturing in different germination light environments, wherein the germination light environments are specifically as follows: fluorescent lamp with light source of composite wavelength (European T) ₅ A light source), the distance between the light source and the culture flask is about 25-35 cm, the illuminance is 1500-3000 lx, and different shading rates are set by covering the upper part of the culture flask with shading cloth (table 2).

The results show that the seeds of treatment number 1 (without shading) did not see germination; although the treatment numbers 2 to 4 (the shading rate is 20 to 60 percent) can also germinate, the germination starts after 50 to 60 days of sowing, and the germination rate is less than 5 percent at 120 days; the germination signs can be seen when seeds with treatment numbers 5-6 (shading rate 80-100%) are sown for 40d, the germination rate reaches 34.5% when the seeds are sown for 120d, and the germination effect is optimal. Therefore, the paphiopedilum seed is very sensitive to the light environment, and can be cultured in a proper germination light environment, so that the germination speed and germination rate of the paphiopedilum seed can be improved.

TABLE 2 growth of seeds in different germination light environments

Example 5

Effect of differentiation Medium and light Environment on the differential culture of paphiopedilum

In the non-symbiotic germination and propagation of paphiopedilum seeds, differentiated buds can be obtained without transferring continuous culture after the seeds germinate. In the comparison experiment, the protocorm is completely brown and dies after 120d without transferring and continuing to culture after the paphiopedilum flutriantis seeds germinate, and can not differentiate into buds.

The protocorm of the paphiopedilum seed after germination is transferred to a conventional paphiopedilum seed differentiation culture medium and cultured in a differentiation light environment. The conventional paphiopedilum seed differentiation culture medium comprises the following components: 1/2MS+6-benzyladenine 1.0 mg/L+naphthalene acetic acid 0.1 mg/L+sucrose 20 g/L+agar 3.0g/L, light environment condition is 1500-3000 lx, illumination time is 10-16 h/d, protocorms are also gradually brown and dead, and are difficult to differentiate into buds. As shown in FIG. 2, the left is a white protocorm formed by seed germination, and the right is a brown and dead protocorm.

By adopting the method of the invention, the formula of the culture medium is as follows: 1/2-1/4 MS+100-200 g/L of coconut juice+6-benzyl adenine 2.0-4.0 mg/L+0.1-0.5 mg/L of naphthylacetic acid+1.0-2.0 g/L of active carbon+20-30 g/L of sucrose+3.0-4.0 g/L of agar, the light environment condition is 500-1000 lx, the illumination time is 10-16 h/d, and the method can induce the differentiation of protocorms into buds (figure 3).

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims

1. The non-symbiotic germination propagation method for the paphiopedilum seed is characterized by comprising the following steps of:

2. The method of claim 1, wherein the capsules are obtained by artificial pollination at 8-10 am, 3-10 d of the opening of the flowers of the paphiopedilum.

3. The method of claim 2 wherein watering of the banderolum is stopped within 3 to 5 days after the artificial pollination.

4. The method of claim 1, wherein the ribbon has a maturity of 120 to 210DAP and a green appearance.

5. The method according to claim 1, wherein the illumination time of the differentiation culture, the secondary proliferation culture and the rooting culture of the strong seedlings is independently 10-16 h/d.

6. The method according to claim 1, wherein the temperatures of germination culture, differentiation culture, secondary proliferation culture and rooting culture are 23-27 ℃ and the environmental humidity is 60-70%.

7. The method according to claim 1, wherein the germination is cultured for 40-90 d to obtain protocorms, the differentiation is cultured for 60-120 d to obtain buds, the secondary proliferation is cultured for 60-120 d to obtain cluster buds, and the strong seedling is rooting is cultured for 60-90 d to obtain complete plants.

8. The method according to claim 1, further comprising the steps of hardening off and transplanting, wherein the season of hardening off and transplanting is 3-5 months or 10-11 months each year.

9. The method of claim 8, wherein the hardening off is hardening off under natural light temperature for 10-20 d.

10. The method according to claim 8, wherein the transplanted cultivation substrate is plant Jin Danhe pine bark with a volume ratio of 1:1 to 3.