阅读说明：本技术 一种蝴蝶兰花梗茎段快速繁殖方法 (Rapid propagation method for stem segments of flower stalks of phalaenopsis ) 是由 吴勇 于 2019-11-22 设计创作，主要内容包括：本发明公开了一种蝴蝶兰花梗茎段快速繁殖方法,包括如下步骤：(1)培养基的配制；(2)外植体的制备；(3)接种；(4)接种后管理。本发明提供了一种蝴蝶兰花梗茎段快速繁殖方法,不仅有效的防止了愈伤组织褐化变质,还大大的降低了蝴蝶兰组织培养的染菌率,提高了愈伤组织分裂、分化、增殖的能力,进而提高了蝴蝶兰组织培养的繁殖率,极具市场推广价值。(The invention discloses a method for rapidly propagating stems of phalaenopsis amabilis, which comprises the following steps: (1) preparing a culture medium; (2) preparing an explant; (3) inoculating; (4) and (5) managing after inoculation. The invention provides a rapid propagation method for stem segments of butterfly orchid stalks, which not only effectively prevents browning and deterioration of callus, but also greatly reduces the strain contamination rate of butterfly orchid tissue culture, improves the capability of division, differentiation and proliferation of the callus, further improves the propagation rate of butterfly orchid tissue culture, and has great market popularization value.)

1. A method for rapidly propagating stem segments of flower stalks of phalaenopsis is characterized by comprising the following steps:

(1) preparation of a culture medium:

a. firstly weighing 13.5-14.5 parts of wax gourd peel, 8.5-9.5 parts of watermelon peel, 5.5-6.5 parts of amaranth stem, 11-12 parts of cucumber peel and 13-14 parts of pumpkin vine together, placing the materials on a wood sieve, then carrying out steam fumigation below the wood sieve, and taking out the mixed raw materials for later use after 32-38 min of fumigation;

b. b, putting the mixed raw material subjected to fumigation treatment in the operation a into a cryogenic grinder for grinding treatment, and sieving the mixed raw material with a 50-70-mesh sieve for later use after grinding treatment is carried out for 24-28 min;

c. putting the powder sieved in the operation b and sterile water into a micro-jet high-pressure homogenizer together according to the weight-volume ratio of 1g to 4.2-4.8 mL, then carrying out homogenization treatment, and filtering by using 3-5 layers of gauze after 11-13 min of homogenization treatment to obtain filtrate and filter cakes for later use;

d. weighing 210-290 parts of filtrate obtained in the operation c, 2.2-2.8 parts of citric acid, 1.2-1.8 parts of trace elements, 0.65-0.75 part of 6-benzylaminopurine, 0.32-0.38 part of naphthylacetic acid, 3.2-3.8 parts of powder additives, 7.5-8.5 parts of agar powder and 510-590 parts of purified water in corresponding parts by weight for later use;

e. firstly putting all the raw materials weighed in the operation d into a stirring tank together, raising the temperature in the stirring tank to 98-100 ℃ at a speed of 42-48 ℃/min, stirring at a rotating speed of 620-680 rpm in the process of raising the temperature until agar powder in the raw materials is completely melted, then stopping stirring, raising the temperature in the stirring tank to 122-128 ℃, raising the pressure to 3.2-3.8 MPa, maintaining the temperature and the pressure for 21-23 min, then cooling and reducing the pressure, and taking out the product for subpackaging when the pressure is reduced to normal pressure and the temperature is reduced to 72-78 ℃ to obtain a culture medium for later use;

(2) preparing an explant:

a. firstly, selecting a strong butterfly orchid pedicel which is withered and has a past flowering phase, and then cutting the selected pedicel into 4-6 cm stem sections;

b. firstly, putting the stem section of the stem of the phalaenopsis obtained in the operation a into sterilizing liquid for soaking, stirring while soaking, washing the stem section of the phalaenopsis with sterile water until the sterilizing liquid on the surface is washed clean after soaking for 15-19 min, and then sucking the sterile water on the surface of the stem section of the obtained stem section with sterile water absorption paper for later use;

c. cutting off two ends and the surface of the sterile stem section obtained in the operation b, and then cutting the rest stem section into small sections of 0.7-1 cm to obtain explants for later use;

(3) inoculation:

inoculating the explants obtained in the step (2) into the culture medium obtained in the step (1), wherein 4-6 explants are inoculated into each 50mL of culture medium;

(4) and (3) management after inoculation:

and (4) placing the culture medium inoculated in the step (3) into an illumination incubator for culture, keeping the relative humidity of air in the incubator at 50-60% and the temperature at 25-27 ℃ in the culture process, and controlling the illumination condition by using an LED light source capable of adjusting the spectrum in the period.

2. The method for rapidly propagating stem segments of phalaenopsis amabilis is characterized in that the Chinese waxgourd peel, the watermelon peel, the amaranth stem, the cucumber peel and the pumpkin vine in the operation a in the step (1) are all freshly picked.

3. The method for rapidly propagating stem segments of phalaenopsis amabilis according to claim 1, wherein the method for preparing steam in the operation a of the step (1) is as follows: weighing 5.5-6.5 parts of Hangzhou chrysanthemum, 2.2-2.8 parts of honeysuckle, 5.2-5.8 parts of hypericum, 2.2-2.8 parts of wormwood and 1.2-1.8 parts of tripterygium wilfordii according to the corresponding weight parts, putting into 320-380 parts of deionized water, boiling with strong fire, heating with medium fire, slowly dripping absolute ethyl alcohol at a constant speed while heating, and taking steam emitted after 32-38 min as steam for fumigation treatment in the operation a in the step (1), wherein the dripping amount of the absolute ethyl alcohol is 10-20% of the deionized water.

4. The method for rapidly propagating the stalk segment of the phalaenopsis amabilis according to claim 1, wherein the temperature in the pulverizer is controlled to be-30 to-20 ℃ during the pulverizing treatment in the operation b of the step (1), and the rotating speed of the pulverizer is 1250 to 1350 rpm.

5. The method for rapidly propagating stem segments of phalaenopsis amabilis according to claim 1, wherein the working pressure of the microjet high-pressure homogenizer is 145-155 MPa during homogenizing treatment in the operation c of the step (1).

6. The method for rapidly propagating stem segments of phalaenopsis amabilis according to claim 1, wherein the trace elements in the operation d of the step (1) comprise the following components in parts by weight: 0.0022 to 0.0028 part of boric acid, 0.00012 to 0.00018 part of manganese sulfate, 0.0032 to 0.0038 part of zinc sulfate, 0.00022 to 0.00028 part of sodium molybdate, 0.000012 to 0.000018 parts of copper sulfate and 0.000023 to 0.000025 part of cobalt chloride.

7. The method for rapidly propagating stem segments of phalaenopsis amabilis according to claim 1, wherein the preparation method of the powder additive in the operation d of the step (1) is that the filter cake obtained in the step (3) and activated carbon are put into a bead mill together according to the weight ratio of 4.2-4.8: 1.2-1.8, liquid nitrogen is added into the bead mill, the grinding is carried out at the rotating speed of 420-480 rpm, and after the grinding is carried out for 11-13 min, the mixed powder is taken out and sieved by a sieve of 80 meshes.

8. The method for rapidly propagating stem segments of butterfly orchid stalks according to claim 1, wherein the sterilization liquid in the operation b of the step (2) comprises the following components in parts by weight: 3-5 parts of fulvic acid, 45-55 parts of 75% ethanol and 1-2 parts of sodium hypochlorite.

9. The method for rapidly propagating stem segments of butterfly orchid stems as claimed in claim 1, wherein the whole operations of the step (2) and the step (3) are carried out in a clean bench.

10. The method for rapidly propagating stem segments of butterfly orchid stalks according to claim 1, wherein the control of the illumination conditions in the step (4) is specifically as follows: culturing in dark conditions within 8 days after inoculation, culturing for 16-18 h in light every day after 8 days, and culturing for 6-8 h in dark, wherein the light culture is carried out by adopting a red-blue light ratio of 1: 2-3, and the light intensity is 1300-1500 Lux.

Technical Field

The invention belongs to the technical field of flower planting, and particularly relates to a method for quickly propagating stems of butterfly orchid stalks.

Background

Butterfly orchid (Phalaenopsis amabilis) Also called butterfly orchid, belongs to tropical aerial orchid and is a perennial herb. Phalaenopsis is a famous cut flower species and is one of the four most commercially valuable tropical orchids internationally. The butterfly orchid is a beautiful name of 'orchid queen', the production prospect of the butterfly orchid is better and better along with the increase of the domestic market demand, and the butterfly orchid has extremely high economic value. Because the phalaenopsis belongs to the uniaxial aerial orchid, the seeds are small, and the embryos, the endosperm and the like are not completely developed, the phalaenopsis is not suitable for sowing by using the traditional method. The mass propagation of phalaenopsis seedlings by tissue culture is an effective way which is rapid, convenient and free from time constraint. Although there are many studies on the tissue culture of phalaenopsis nowadays, many problems of phalaenopsis are still not well improved in the tissue culture process, explants are easy to brown and deteriorate and then die in the tissue culture process, and the prevention of bacterial contamination in the explant induction process is a common problemThe method has the advantages of improving the propagation rate of butterfly orchid tissue culture and improving the quality of tissue culture seedlings.

Disclosure of Invention

The invention aims to solve the existing problems and provides a rapid propagation method for a stem segment of a butterfly orchid stalk, which not only effectively prevents browning and deterioration of callus, but also greatly reduces the contamination rate of butterfly orchid tissue culture, improves the capabilities of division, differentiation and proliferation of the callus, further improves the propagation rate of the butterfly orchid tissue culture and has great market popularization value.

The invention is realized by the following technical scheme:

a method for rapidly propagating stems of phalaenopsis includes the following steps:

(1) preparation of a culture medium:

a. firstly weighing 13.5-14.5 parts of wax gourd peel, 8.5-9.5 parts of watermelon peel, 5.5-6.5 parts of amaranth stem, 11-12 parts of cucumber peel and 13-14 parts of pumpkin vine together, placing the materials on a wood sieve, then carrying out steam fumigation below the wood sieve, and taking out the mixed raw materials for later use after 32-38 min of fumigation;

b. b, putting the mixed raw material subjected to fumigation treatment in the operation a into a cryogenic grinder for grinding treatment, and sieving the mixed raw material with a 50-70-mesh sieve for later use after grinding treatment is carried out for 24-28 min;

c. putting the powder sieved in the operation b and sterile water into a micro-jet high-pressure homogenizer together according to the weight-volume ratio of 1g to 4.2-4.8 mL, then carrying out homogenization treatment, and filtering by using 3-5 layers of gauze after 11-13 min of homogenization treatment to obtain filtrate and filter cakes for later use;

d. weighing 210-290 parts of filtrate obtained in the operation c, 2.2-2.8 parts of citric acid, 1.2-1.8 parts of trace elements, 0.65-0.75 part of 6-benzylaminopurine, 0.32-0.38 part of naphthylacetic acid, 3.2-3.8 parts of powder additives, 7.5-8.5 parts of agar powder and 510-590 parts of purified water in corresponding parts by weight for later use;

e. firstly putting all the raw materials weighed in the operation d into a stirring tank together, raising the temperature in the stirring tank to 98-100 ℃ at a speed of 42-48 ℃/min, stirring at a rotating speed of 620-680 rpm in the process of raising the temperature until agar powder in the raw materials is completely melted, then stopping stirring, raising the temperature in the stirring tank to 122-128 ℃, raising the pressure to 3.2-3.8 MPa, maintaining the temperature and the pressure for 21-23 min, then cooling and reducing the pressure, and taking out the product for subpackaging when the pressure is reduced to normal pressure and the temperature is reduced to 72-78 ℃ to obtain a culture medium for later use;

(2) preparing an explant:

a. firstly, selecting a strong butterfly orchid pedicel which is withered and has a past flowering phase, and then cutting the selected pedicel into 4-6 cm stem sections;

b. firstly, putting the stem section of the stem of the phalaenopsis obtained in the operation a into sterilizing liquid for soaking, stirring while soaking, washing the stem section of the phalaenopsis with sterile water until the sterilizing liquid on the surface is washed clean after soaking for 15-19 min, and then sucking the sterile water on the surface of the stem section of the obtained stem section with sterile water absorption paper for later use;

c. cutting off two ends and the surface of the sterile stem section obtained in the operation b, and then cutting the rest stem section into small sections of 0.7-1 cm to obtain explants for later use;

(3) inoculation:

inoculating the explants obtained in the step (2) into the culture medium obtained in the step (1), wherein 4-6 explants are inoculated into each 50mL of culture medium;

(4) and (3) management after inoculation:

and (4) placing the culture medium inoculated in the step (3) into an illumination incubator for culture, keeping the relative humidity of air in the incubator at 50-60% and the temperature at 25-27 ℃ in the culture process, and controlling the illumination condition by using an LED light source capable of adjusting the spectrum in the period.

Furthermore, the Chinese waxgourd peel, the watermelon peel, the amaranth stem, the cucumber peel and the pumpkin vine in the operation a in the step (1) are all picked fresh.

Further, the preparation method of the steam in the operation a of the step (1) is as follows: weighing 5.5-6.5 parts of Hangzhou chrysanthemum, 2.2-2.8 parts of honeysuckle, 5.2-5.8 parts of hypericum, 2.2-2.8 parts of wormwood and 1.2-1.8 parts of tripterygium wilfordii according to the corresponding weight parts, putting into 320-380 parts of deionized water, boiling with strong fire, heating with medium fire, slowly dripping absolute ethyl alcohol at a constant speed while heating, and taking steam emitted after 32-38 min as steam for fumigation treatment in the operation a in the step (1), wherein the dripping amount of the absolute ethyl alcohol is 10-20% of the deionized water.

Further, the temperature in the pulverizer is controlled to be-30 to-20 ℃ during the pulverizing treatment in the step (1) and the rotating speed of the pulverizer is 1250 to 1350 rpm.

Further, when homogenizing in the operation c in the step (1), the working pressure of the micro-jet high-pressure homogenizer is 145-155 MPa.

Further, the trace elements in the step (1) and the operation d comprise the following components in parts by weight: 0.0022 to 0.0028 part of boric acid, 0.00012 to 0.00018 part of manganese sulfate, 0.0032 to 0.0038 part of zinc sulfate, 0.00022 to 0.00028 part of sodium molybdate, 0.000012 to 0.000018 parts of copper sulfate and 0.000023 to 0.000025 part of cobalt chloride.

Further, the preparation method of the powder additive in the operation d in the step (1) comprises the steps of putting the filter cake obtained in the step (3) and the activated carbon into a bead mill together according to the weight ratio of 4.2-4.8: 1.2-1.8, adding liquid nitrogen into the bead mill, grinding at the rotating speed of 420-480 rpm, taking out the mixed powder after grinding for 11-13 min, and sieving with a 80-mesh sieve.

Further, the sterilization liquid in the step (2) and the operation b comprises the following components in parts by weight: 3-5 parts of fulvic acid, 45-55 parts of 75% ethanol and 1-2 parts of sodium hypochlorite.

Further, the whole operation of the step (2) and the step (3) is carried out in an ultra-clean bench.

Further, the controlling of the illumination condition in the step (4) specifically comprises: culturing in dark conditions within 8 days after inoculation, culturing for 16-18 h in light every day after 8 days, and culturing for 6-8 h in dark, wherein the light culture is carried out by adopting a red-blue light ratio of 1: 2-3, and the light intensity is 1300-1500 Lux.

The invention comprehensively considers the problems existing in the rapid propagation process of phalaenopsis, and provides a rapid propagation method of the stem section of the phalaenopsis, which mainly aims at preventing the browning of an explant, reducing the bacterial contamination rate and improving the propagation rate of the phalaenopsis. During the preparation of the culture medium, firstly, steam fumigation is carried out on fresh Chinese waxgourd peels, watermelon peels and the like, the steam contains a large amount of volatile alcohol and other organic substances, the expansion of raw material cells can be promoted, worm eggs and various bacteria existing in the raw materials can be fully killed by the hot steam, and in addition, partial nutrient components in the raw materials can be promoted to decompose and are converted into an existing form which is more favorable for the absorption of explants by the hot steam. And the raw materials are crushed quickly and effectively under the freezing condition, so that the foundation is laid for subsequent operation, pathogenic bacteria and worm eggs possibly left in the raw materials can be further killed under the freezing condition, and the quality of the culture medium is further ensured. Then carrying out micro-jet high-pressure homogenization treatment on the obtained powder and sterile water together, and carrying out high-speed collision, high-frequency oscillation, instantaneous pressure drop, strong shearing, cavitation and other actions on the material in an oscillation reaction cavity, thereby realizing the crushing of material cells and promoting the outflow of cell contents, and under the condition of micro-jet high pressure, the polysaccharide molecular chain is broken and can be degraded, the composition, the granularity and the molecular aggregation state of monosaccharide are changed, and the monosaccharide is converted into a structure which is easier to digest and absorb by an explant, and the obtained filtrate can provide sufficient nutrient components for the division, differentiation and proliferation of cells of the phalaenopsis explant. The filtrate obtained by homogenizing treatment and other raw material components are put into a stirring tank together to be stirred and mixed uniformly, and the sterilization effect is achieved through the control of high-temperature and high-pressure conditions. The filter liquor obtained by homogenization treatment is added into the raw materials, citric acid, trace elements, 6-benzyl aminopurine, naphthylacetic acid, powder additives and the like are also added, the trace elements are mainly added to make up the problem of insufficient content of the trace elements in the filter liquor, the 6-benzyl aminopurine and the naphthylacetic acid are used as plant growth regulators and can play an auxiliary role to accelerate the division and differentiation of cells, the powder additives are mainly prepared by taking homogenized filter cakes as base materials and then carrying out bead milling treatment together with active carbon, the active carbon is adsorbed into fiber pores of the filter cakes and is added into the raw materials together with the citric acid, the oxidation and browning of explants can be effectively prevented, and in the process of tissue culture, the explant metabolic products can be absorbed to prevent the toxic action of the metabolic products on the explants, callus tissues, differentiated seedlings and the like, further improving the quality of the culture medium. In the preparation of the explant, the stalk stem of the phalaenopsis is selected as the explant material, the stalk stem of the phalaenopsis is convenient to obtain, rich in raw materials and strong in cell differentiation capacity, and can be used as the first choice material for rapid propagation of phalaenopsis, after the stalk is subjected to sterilization treatment by a bactericide, surface tissues are damaged, the surface and two ends are cut off, sterile stalk segments with vigorous vitality are exposed, and are cut into small segments with proper sizes, so that the small segments are easy to die and can not sufficiently absorb nutrition, then the small segments are inoculated into the prepared culture medium, dark condition culture is adopted in 8 days after inoculation, browning of the explant can be effectively relieved, the synergistic effect of citric acid and a powder additive in the culture medium is added, the leaching of secretion is inhibited to a certain degree, the browning phenomenon is relieved, then the light condition is controlled by using an LED light source capable of adjusting spectrum, enhance the activity of various enzymes in the explant, accelerate the respiration rate of cells and promote the formation of callus.

Compared with the prior art, the invention has the following advantages:

the invention provides a rapid propagation method for stem segments of butterfly orchid stalks, which not only effectively prevents browning and deterioration of callus, but also greatly reduces the strain contamination rate of butterfly orchid tissue culture, improves the capability of division, differentiation and proliferation of the callus, further improves the propagation rate of butterfly orchid tissue culture, and has great market popularization value.

Detailed Description