CN105494103A - Method for tissue culture and rapid propagation of high-quality paphiopedilum maudiae seedlings - Google Patents

Abstract

The invention discloses a rapid propagation method for tissue culture of high-quality Paphiopedilum spp. In the present invention, on the basis of a series of medicament treatments on the mother plant of P. edulis, the new lateral buds are used as explants, and a unique medium is used to induce adventitious buds, proliferate clustered buds, and cultivate rooting to obtain high-quality seedlings quickly. The method of reproduction provides an effective way to meet the needs of the market for the high-quality Paphiopedilum species. The technology is practical and has high application value.

Description

A kind of tissue culture rapid propagation method of high-quality Paphiopedilum morpha species

Technical field:

The invention belongs to the field of plant biotechnology, and in particular relates to a rapid propagation method for tissue culture of high-quality Paphiopedilum species seedlings.

Background technique:

Paphiopedilum Maudiae is a hybrid of P.lawrenceanum and P.callosum. Using it as a parent, many hybrid offspring have been bred. In the market, a class of P. maudiae type with flowers similar to those of P. maudiae type is collectively referred to as "P. maudiaetype". It ranges from purple-black, reddish-brown to green-white, and has abundant spots and lines. It is the main species of Paphiopedilum in the international and domestic markets. Routine propagation of Paphiopedilum genus Modi can be carried out by ramets, but the propagation speed is slow and the reproduction rate is low, which is far from meeting the needs of the commercial market. At present, the Morti class Paphirandos sold in the world market come from aseptic sowing. However, because Paphiopedilum is a hybrid species, the offspring of aseptic sowing have large segregation, and it is impossible to maintain the excellent traits of the parents to obtain seedlings with uniform traits, which seriously restricts its large-scale production. At the same time, due to the difficulty in decontamination of the explants and the slow growth rate of the explants during tissue culture, its tissue culture is extremely difficult. There has not been selected excellent strains from the greenhouse as explants in the world to carry out the cultivation of Paphiopedilum. A report on the rapid propagation of orchid tissue culture.

Invention content:

The purpose of the present invention is to provide a method for tissue culture and rapid propagation of high-quality Paphiopedilum edulis high-quality seedlings, so as to obtain seedlings with uniform properties by asexual cloning of excellent strains of P.

The method for rapid propagation of the high-quality seedlings of Modi class Paphiopedilum by tissue culture of the present invention is characterized in that it comprises the following steps:

a, the induction of adventitious buds and the proliferation of clustered buds: cut the lateral buds of Paphiopedilum explants as explants, after disinfection, inoculate them in the induction medium, induce adventitious buds, and then transfer the induced adventitious buds to Proliferation culture is carried out on the proliferation medium to obtain clustered buds. Both the induction medium and the proliferation medium contain 3.0-10.0 mg of 6-benzylpurine, 0.5-1.0 mg of naphthaleneacetic acid, and 100-200 ml of coconut milk per liter. , 150-200 mg of sodium dihydrogen phosphate, 20-30 grams of sucrose and 6-7 grams of agar, the balance being 1/2 MS medium, pH 5.8-6.0; subculture once every 60 days until the sixth generation , The proliferation coefficient can reach 2.5 to 3.0 times.

B, rooting and strong seedling cultivation: inoculate the clustered buds obtained by the proliferation into the strong seedling medium to strengthen the seedlings, obtain thick clustered shoots, then cut them into single buds and transfer them to the rooting medium for rooting culture, and obtain For a complete plant, each liter of the strong seedling medium contains 1.0-2.0 mg of 6-benzylpurine, 0.5-1.0 mg of naphthaleneacetic acid, 100-200 ml of coconut milk, 150-200 mg of sodium dihydrogen phosphate, and 20-30 mg of sucrose. 6-7 grams of agar, the balance is 1/2 MS medium, pH 5.8-6.0; the rooting medium contains 0.3-0.5 mg of 6-benzylpurine, 0.5-1.0 mg of naphthaleneacetic acid, coconut juice 100-200 ml, sodium dihydrogen phosphate 150-200 mg, banana homogenate 50-150 grams, activated carbon 0.1-1.0 grams, sucrose 20-30 grams and agar 6-7 grams, the balance is 1/2 MS medium, pH5.8～6.0;

c. Transfer the complete plant to natural light for seedling hardening. After hardening, wash the root culture medium and transfer it to the planting stone: bark in a mixed medium with a mass ratio of 1-3:1 to obtain the Paphiopedilum seedlings. Generally, proper ventilation and sufficient humidity are maintained, and the survival rate of transplanting can reach more than 90%.

The step a is inoculated into the induction medium, and transferred to the proliferation medium for proliferation culture, step b is to inoculate the clustered shoots into the strong seedling medium for strong seedlings and cut the clustered shoots into single buds and then transfer Receive into the rooting culture medium and carry out rooting culture, its culture temperature 24～28 ℃, illuminance 1500～2000lx, light 12～16 hours/day.

The lateral buds of the cut Modi class Paphiopedilum are used as explants, and after disinfection, the explants are obtained by the following methods:

Choose P. edulis with strong plant growth potential and round flower shape as the mother plant, and 35 days before cutting the explants for disinfection, water it once with a 500-800-fold diluted aqueous solution of carbendazim and spray the leaves. After 5 days, irrigate once with 500-800 times diluted aqueous solution of 35% gram soil fungus wettable powder and spray the leaves; Spray the leaves, then repeat the above steps once every 5 days, and 5 days after the last irrigation of agricultural streptomycin, use a scalpel sterilized with a volume fraction of 75% alcohol solution to cut off the lateral buds with a length of 1.5 to 2.5 cm as the outer buds. implant.

The disinfection is specifically as follows: soak the excised explants in 75% alcohol aqueous solution for 10-30 seconds on the ultra-clean workbench, and then soak them in hypochlorous acid solution with 1.0% available chlorine for 5-10 minutes. Rinse with sterile water for 4 to 5 times, then sterilize with 0.1% mercury liter solution for 5 to 10 minutes, rinse with sterile water for 4 to 5 times, cut off the leaves, and place them in 1500 to 2000 times diluted medical streptomycin Shake in aqueous solution for 24 hours to obtain sterilized explants. Such treatment can make the disinfection success rate 50-60%, thereby effectively solving the difficult problem of explant decontamination.

MS medium is an international general medium, its composition and configuration method see MurashigeT, SkoogF (1962) (Arevisedmediumforrapidgrowthandbioassaywithtobaccotissuecultures.PhysiolPlant15:473-497). 1/2 MS medium means that the amount of macroelements in MS medium is 1/2 of the original, and the rest of the ingredients remain unchanged.

In the present invention, on the basis of a series of medicament treatments on the mother plant of P. edulis, the new lateral buds are used as explants, and a unique medium is used to induce adventitious buds, proliferate clustered buds, and cultivate rooting to obtain high-quality seedlings quickly. The method of reproduction provides an effective way to meet the needs of the market for the high-quality Paphiopedilum species. The technology is practical and has high application value.

detailed description:

The following examples are to further illustrate the present invention, rather than limit the present invention.

Example 1: Tissue Culture of Red Modi Miracle (Paphiopedilum SCBGMiracle)

(1) Material selection and handling

Paphiopedilum SCBGMiracle, which has strong plant growth vigor and round flower shape, was selected as the mother plant. Before cutting explant (lateral bud) sterilized 35 days, water once and spray blade with 500 times of diluted aqueous solution of carbendazim earlier, water once with 500 times diluted aqueous solution of 35% gram soil bacteria wettable powder after 5 days and Spray the leaves, and after another 5 days, water once and spray the leaves with a 3000-fold diluted aqueous solution of 72% agricultural streptomycin soluble powder. Then every 5 days, the above steps were repeated once, and 5 days after the last irrigation of agricultural streptomycin, the side buds with a length of 2.5 cm were cut with a scalpel sterilized with a volume fraction of 75% alcohol solution as explants.

(2) explant disinfection

On the ultra-clean workbench, the excised explants were first soaked in 75% alcohol aqueous solution for 30 seconds, then soaked in hypochlorous acid solution with 1.0% available chlorine for 10 minutes, rinsed with sterile water for 5 times, and then used Disinfect with 0.1% mercuric chloride aqueous solution for 10 minutes, and rinse with sterile water for 5 times. After obtaining the sterilized explants, cut off the leaves, place the remaining lateral buds in a 2000-fold diluted aqueous solution of medical streptomycin, shake them in a shaker for 24 hours, and then inoculate them into the induction medium at a culture temperature of 28°C. The illuminance is 2000 lx, and the light is 16 hours/day. The induction medium contains 10.0 mg of 6-benzylpurine (6-BA), 1.0 mg of naphthaleneacetic acid (NAA), 200 ml of coconut milk, and 150 mg of sodium dihydrogen phosphate per liter. milligrams, 20 g of sucrose and 6 g of agar, and the balance is 1/2 MS medium, pH 5.8 to 6.0. Its preparation method is to mix the ingredients in the above medium evenly, and then sterilize to obtain the induction medium (the same below). The success rate of disinfection is 60%.

(3) Induction of adventitious buds and multiplication of clustered buds

The explants after inoculation can be seen to form obvious brown secretions in about 10 days, and will be transferred to a new induction medium at 30 days, and then cultivated for about 25 days to form adventitious buds on the explants. Then the adventitious buds are inoculated into the proliferation medium to carry out the subculture proliferation culture, and the described proliferation medium contains 4.0 mg of 6-benzylpurine (6-BA), 1.0 mg of naphthaleneacetic acid (NAA) and 200 mg of coconut milk per liter. ml, 150 mg sodium dihydrogen phosphate, 20 g sucrose and 6 g agar, the balance is 1/2 MS medium, pH 5.8-6.0, culture temperature 28°C, light intensity 2000 lx, light 16 hours/day, every 60 days After subculture once, the multiplication coefficient can reach 3.0 times at the 6th generation, and clustered buds can be obtained.

(4) Cultivation of rooted and strong seedlings

Inoculate clustered shoots into strong seedling medium for strong seedling cultivation, culture temperature is 28°C, light intensity is 2000 lx, light is 16 hours/day, after 60 days, cluster shoots with a height of 3.0 cm are cut into single shoots and transferred to rooting medium for rooting culture , culture temperature 28 ℃, illuminance 2000lx, light 16 hours/day, to form the complete plant of 6 centimeters height after 60 days, described strong seedling culture medium contains 6-benzylpurine (6-BA) 1.5 milligrams per liter, naphthalene Acetic acid (NAA) 0.75 mg, coconut juice 150 ml, sodium dihydrogen phosphate 175 mg, sucrose 20 g and agar 6 g, the balance is 1/2 MS medium, pH 5.8 ~ 6.0, its preparation method is to mix the above medium After the ingredients are evenly mixed, sterilize to obtain a strong seedling medium (the same below); the rooting medium contains 0.5 mg of 6-benzylpurine (6-BA) per liter, 1.0 mg of naphthaleneacetic acid (NAA), coconut milk 200 ml, 200 mg of sodium dihydrogen phosphate, 150 g of banana homogenate, 1.0 g of activated carbon, 20 g of sucrose and 6 g of agar, and the balance is 1/2 MS medium, pH 5.8 to 6.0. The preparation method is to culture the above After the ingredients in the base are mixed evenly, the rooting medium (the same below) is obtained by sterilization.

(6) Transplanting test tube seedlings

Transfer the complete plant with a height of about 6 cm to a greenhouse with natural light to harden the seedlings for 20 days, then take it out of the glass bottle, wash the medium of the root, and transplant it into the golden stone (imported from Japan): bark press In the mixed substrate with a mass ratio of 3:1, the survival rate of transplanting can reach 95% if proper ventilation and sufficient humidity are maintained.

Embodiment 2: the tissue culture of green modiyunzhijun (Paphiopedilum SCBG Yunzhijun)

(1) Material selection and handling

Paphiopedilum SCBG Yunzhijun, which has strong plant growth vigor and round flower shape, was selected as the mother plant. Before 35 days of cutting explant (lateral bud) disinfection, water once and spray blade with 600 times of diluted aqueous solution of carbendazim, water once with 600 times diluted aqueous solution of 35% gram soil bacteria wettable powder after 5 days and Spray the leaves, and after another 5 days, water once with a 3500-fold diluted aqueous solution of 72% agricultural streptomycin soluble powder and spray the leaves. Then every 5 days, the above steps were repeated, and 5 days after the last irrigation of agricultural streptomycin, the lateral buds with a length of 2.5 cm were cut with a scalpel sterilized with a volume fraction of 75% alcohol solution as explants.

(2) explant disinfection

On the ultra-clean workbench, the excised explants were first soaked in 75% alcohol aqueous solution for 20 seconds, then soaked in hypochlorous acid solution with 1.0% available chlorine for 7.5 minutes, rinsed with sterile water for 5 times, and then used Sterilize with 0.1% mercuric chloride aqueous solution for 7.5 minutes, and rinse with sterile water for 5 times. Get the sterilized explants, then cut off the leaves, place the remaining lateral buds in a 1750-fold diluted aqueous solution of medical streptomycin, vibrate in a shaker for 24 hours, and then inoculate them into the induction medium at a culture temperature of 24°C. Illuminance 1500lx, light 12 hours/day, the induction medium contains 7.5 mg of 6-benzylpurine (6-BA), 0.75 mg of naphthaleneacetic acid (NAA), 150 ml of coconut milk, and 175 mg of sodium dihydrogen phosphate per liter. mg, 30 grams of sucrose and 7 grams of agar, the remainder being 1/2 MS medium, pH 5.8～6.0, its preparation method is after the composition in the above-mentioned medium is mixed, sterilized to obtain induction medium (the same below ). The success rate of disinfection is 55%.

(3) Induction of adventitious buds and multiplication of clustered buds

The inoculated explants can be seen to form obvious brown secretions in about 10 days, and will be transferred to a new induction medium in 30 days, and then cultivated for about 28 days to form adventitious buds on the explants. Then the adventitious buds are inoculated into the proliferation medium and carried out the subculture, and the described proliferation medium contains 10 mg of 6-benzylpurine (6-BA), 0.5 mg of naphthaleneacetic acid (NAA) and 100 mg of coconut milk per liter. ml, 200 mg sodium dihydrogen phosphate, 30 g sucrose and 7 g agar, the balance is 1/2 MS medium, pH 5.8-6.0, culture temperature 24°C, light intensity 1500 lx, light 12 hours/day, every 60 days After subculture once, the multiplication coefficient can reach 2.8 times at the 6th generation, and clustered buds can be obtained.

(4) Cultivation of rooted and strong seedlings

Inoculate clustered shoots into strong seedling medium for strong seedling cultivation, culture temperature is 24°C, light intensity is 1500lx, and light is 12 hours/day. After 60 days, cluster shoots with a height of 2.5 cm are cut into single shoots and then transferred to rooting medium for rooting culture. , culture temperature 24 ℃, illuminance 1500lx, light 12 hours/day, to form the complete plant of 5.5 centimeters height after 60 days, described strong seedling culture medium contains 2 milligrams of 6-benzylpurine (6-BA) per liter, naphthalene 1 milligram of acetic acid (NAA), 200 milliliters of coconut milk, 200 milligrams of sodium dihydrogen phosphate, 30 grams of sucrose and 7 grams of agar, and the balance is 1/2 MS medium, pH5.8～6.0; Contains 0.4 mg of 6-benzylpurine (6-BA), 0.75 mg of naphthaleneacetic acid (NAA), 150 ml of coconut milk, 175 mg of sodium dihydrogen phosphate, 100 g of banana homogenate, 0.5 g of activated carbon, 30 g of sucrose and agar 7 grams, the balance is 1/2 MS medium, pH 5.8-6.0;

(6) Transplanting test tube seedlings

Transfer the complete plant with a height of about 5.5 cm from the culture bottle to a greenhouse with natural light to harden the seedlings for 15 days, then take it out of the glass bottle, wash the medium of the root, and transplant it into the golden stone (imported from Japan): bark press In the mixed medium with a mass ratio of 2:1, the survival rate of transplanting can reach 93% with proper ventilation and sufficient humidity.

Embodiment 3: the tissue culture of modi ruby (Paphiopedilum SCBGRedJewel)

(1) Material selection and handling

Paphiopedilum SCBGRed Jewel, a plant with strong growth potential and round flower shape, was selected as the mother plant. Before cutting explant (lateral bud) sterilized 35 days, water once and spray blade with 800 times diluted aqueous solution of carbendazim earlier, water once with 800 times diluted aqueous solution of 35% gram soil bacteria wettable powder after 5 days and Spray the leaves, and after another 5 days, water once with a 4000-fold diluted aqueous solution of 72% agricultural streptomycin soluble powder and spray the leaves. Then every 5 days, the above steps were repeated, and 5 days after the last irrigation of agricultural streptomycin, the lateral buds with a length of 1.5 cm were cut with a scalpel sterilized with a volume fraction of 75% alcohol solution as explants.

(2) explant disinfection

Soak the excised explants in 75% alcohol aqueous solution for 10 seconds on the ultra-clean workbench, then soak them in hypochlorous acid solution with 1.0% available chlorine for 5 minutes, rinse them with sterile water for 4 times, and then use Disinfect with 0.1% mercuric chloride aqueous solution for 5 minutes, and rinse with sterile water for 4 times. Get the sterilized explants, then cut off the leaves, place the remaining lateral buds in a 2000-fold diluted aqueous solution of medical streptomycin, vibrate in a shaker for 24 hours, and then inoculate them into the induction medium. The culture temperature is 26°C, and the light intensity 1800lx, 14 hours/day of light, the induction medium contains 3 mg of 6-benzylpurine (6-BA), 0.5 mg of naphthaleneacetic acid (NAA), 100 ml of coconut milk, and 200 mg of sodium dihydrogen phosphate per liter , 30 grams of sucrose and 6.5 grams of agar, the remainder is 1/2MS medium, pH5.8～6.0, its preparation method is after the composition in the above-mentioned medium is mixed, and sterilization obtains induction medium (the same below) . The success rate of disinfection is 50%.

(3) Induction of adventitious buds and multiplication of clustered buds

The explants after inoculation can be seen to form obvious brown secretions in about 10 days, and will be transferred to a new induction medium at 30 days, and adventitious buds can be formed on the explants after culturing for about 30 days. Then the adventitious buds are inoculated into the proliferation medium to carry out the subculture proliferation culture, and the described proliferation medium contains 3.0 mg of 6-benzylpurine (6-BA), 1.0 mg of naphthaleneacetic acid (NAA) and 200 mg of coconut milk per liter. ml, 150 mg of sodium dihydrogen phosphate, 20 g of sucrose, and 6.5 g of agar, the balance is 1/2 MS medium, pH 5.8-6.0, culture temperature 26°C, illuminance 1800 lx, light 14 hours/day, every 60 days After subculture once, the multiplication coefficient can reach 2.5 times at the 6th generation, and clustered buds can be obtained.

(4) Cultivation of rooted and strong seedlings

Inoculate clustered shoots into strong seedling medium for strong seedling cultivation, culture temperature is 26°C, light intensity is 1800lx, and light is 14 hours/day. After 60 days, cluster shoots with a height of 2 cm are cut into single shoots and then transferred to rooting medium for rooting culture. , culture temperature 26 ℃, illuminance 1800lx, light 14 hours/day, to form the complete plant of 5 centimeters height after 60 days, described strong seedling culture medium contains 6-benzylpurine (6-BA) 1 milligram, naphthalene per liter. 0.5 mg of acetic acid (NAA), 100 milliliters of coconut milk, 150 milligrams of sodium dihydrogen phosphate, 30 grams of sucrose and 6 grams of agar, the balance is 1/2 MS medium, pH5.8～6.0; Contains 0.3 mg of 6-benzylpurine (6-BA), 0.5 mg of naphthaleneacetic acid (NAA), 100 ml of coconut milk, 150 mg of sodium dihydrogen phosphate, 50 g of banana homogenate, 0.1 g of activated carbon, 30 g of sucrose and 6.5 g of agar grams, the balance is 1/2 MS medium, pH 5.8-6.0;

(6) Transplanting test tube seedlings

Transfer the complete plant with a height of about 5 cm from the culture bottle to a greenhouse with natural light to harden the seedlings for 10 days, then take it out of the glass bottle, wash the medium of the root, and transplant it into the golden stone (imported from Japan): bark press In the mixed substrate with a mass ratio of 1:1, the survival rate of transplanting can reach 90% if proper ventilation and sufficient humidity are maintained.

Claims (3)

1. the blue high quality seedling quick breeding method for tissue culture of Supreme Being's class of rubbing pocket, is characterized in that, comprise the following steps:

A, the induction of indefinite bud and the propagation of Multiple Buds: cut the lateral bud of Supreme Being's class pocket orchid that rubs as explant, after sterilization, be inoculated in inducing culture, induce indefinite bud, then the indefinite bud induced is transferred on proliferated culture medium and carry out Multiplying culture, obtain Multiple Buds, described inducing culture and proliferated culture medium are all often liter and contain 6-benzyl purine 3.0 ~ 10.0 milligrams, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0,

B, Rooting and hardening-off culture: the Multiple Buds that propagation obtains is inoculated in strong seedling culture base and carries out strong sprout, obtain sturdy Multiple Buds, then be transferred to after being cut into simple bud in root media and carry out culture of rootage, obtain whole plant, described strong seedling culture base often rises containing 6-benzyl purine 1.0 ~ 2.0 milligrams, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0; Described root media often rises containing 6-benzyl purine 0.3 ~ 0.5 milligram, methyl α-naphthyl acetate 0.5 ~ 1.0 milligram, coconut milk 100 ~ 200 milliliters, sodium dihydrogen phosphate 150 ~ 200 milligrams, banana homogenate 50 ~ 150 grams, active carbon 0.1 ~ 1.0 gram, sucrose 20 ~ 30 grams and 6 ~ 7 grams of agar, surplus is 1/2MS medium, pH5.8 ~ 6.0;

C, whole plant is transferred to natural daylight lower refining seedling, after hardening, clean its root medium and move into and plants metal and stone: cultivate acquisition in the mixed-matrix of bark 1-3:1 in mass ratio and to rub the blue seedling of Supreme Being's class pocket.

2. the blue high quality seedling quick breeding method for tissue culture of Supreme Being's class pocket that rubs according to claim 1, it is characterized in that, described step a is inoculated in inducing culture, and transfer on proliferated culture medium carry out Multiplying culture, being inoculated into by Multiple Buds in strong seedling culture base of step b carries out strong sprout and is transferred in root media after Multiple Buds is cut into simple bud carrying out culture of rootage, its cultivation temperature 24 ~ 28 DEG C, illuminance 1500 ~ 2000lx, illumination 12 ~ 16 hours/day.

3. the blue high quality seedling quick breeding method for tissue culture of Supreme Being's class pocket that rubs according to claim 1, is characterized in that, described cutting rubs the lateral bud of Supreme Being's class pocket orchid as explant, and after sterilization, its explant obtains by the following method:

Select plant strain growth gesture strong, Supreme Being's class pocket orchid that rubs of flower pattern rounding is maternal plant, before 35 days that cut explant sterilization, first water once with 500 ~ 800 times of dilute aqueous solutions of carbendazim and spray blade, within 5 days, water once with 500 ~ 800 times of dilute aqueous solutions of 35% G soil bacterium wetting powder and spray blade afterwards, after 5 days, water once with the dilute aqueous solution of 3000 ~ 4000 times of 72% agricultural streptomycin soluble powder and spray blade, then often 5 days are spent, repeat above-mentioned steps once, last 1 time pouring agricultural streptomycin after 5 days, the lateral bud cutting length 1.5 ~ 2.5 centimetres with the sterile scalpel of volume fraction 75% alcohol water blend is explant.

Its sterilization is specially: first used by the explant cut after soaking 10 ~ 30 seconds in volume fraction 75% alcohol water blend on superclean bench, 5 ~ 10 minutes are soaked with the hypochlorite solution of 1.0% available chlorine, aseptic water washing 4 ~ 5 times, again with mass fraction 0.1% mercuric chloride solution sterilization 5 ~ 10 minutes, aseptic water washing 4 ~ 5 times, then cut away blade, be positioned in 1500 ~ 2000 times of dilute aqueous solutions of medical streptomycin and vibrate 24 hours, the explant after being sterilized.