CN117598203B

CN117598203B - Culture medium group for in-vitro regeneration culture of vaneless meconopsis and application and culture method thereof

Info

Publication number: CN117598203B
Application number: CN202410088835.0A
Authority: CN
Inventors: 王可佳; 王美娜; 李健; 王玉; 陈朋; 段晓娟; 陈建兵
Original assignee: ORCHID CONSERVATION & RESEARCH CENTER OF SHENZHEN
Current assignee: ORCHID CONSERVATION & RESEARCH CENTER OF SHENZHEN
Priority date: 2024-01-23
Filing date: 2024-01-23
Publication date: 2024-04-02
Anticipated expiration: 2044-01-23
Also published as: CN117598203A

Abstract

The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium group for in-vitro regeneration culture of vaneless meconopsis, application and a culture method thereof. The invention provides application of the culture medium group in-vitro regeneration culture of the vaneless meconopsis, and the culture medium group can promote rapid proliferation of the vaneless meconopsis after in-vitro regeneration culture. The invention also provides a method for in-vitro regeneration culture of the vaneless crown orchid by using the culture medium group, which is characterized in that the vaneless crown orchid pseudobulb is subjected to 2 times of strong seedling culture in a proliferation stage and a rooting stage respectively, a large number of vaneless crown orchid with strong pseudobulb and developed root system are obtained, greenhouse cultivation management is not needed, and the tissue culture seedlings can be directly transplanted under tree shade after outdoor seedling hardening.

Description

Culture medium group for in-vitro regeneration culture of vaneless meconopsis and application and culture method thereof

Technical Field

The invention belongs to the technical field of plant tissue culture, and particularly relates to a culture medium group for in-vitro regeneration culture of vaneless meconopsis and an application and a culture method thereof.

Background

The vaneless meracil (Eulophia zollingeri) is a typical saprophyte plant of the genus meracil (Eulophia) of the family lanaceae (Orchidaceae), and has both saprophyte and orchid properties. The flower has no green leaves, the ground is pulled out when the flower is flowering, and a plurality of flowers to more than ten yellow brown flowers are thinned; the pseudobulb is medicinal, can be used for treating traumatic injury, blood stasis and pain, traumatic hemorrhage, insect and snake bite and the like, and is a rare orchid with ornamental value and medicinal value.

The vaneless meconopsis is different from autotrophic orchid plants which rely on separate propagation and can only rely on seed propagation. In addition, although the vaneless mersupport has high selfing affinity, pollination depends on insects, and the living environment is high, so that the environment is not changed once the environment is changed. Therefore, the number of the vaneless meracil is rare, and the vannamel is difficult to cultivate manually. The tissue culture technology is used for breeding the vaneless Meihuan orchid, is an effective means for protecting the resources of the vaneless Meihuan orchid, and is an important way for realizing the gardening value and the medicinal value of the vaneless Meihuan orchid. As the only one of the genus Meihuan, the Meihuan has no leaves and buds, and only depends on underground stems to provide nutrition and moisture, so that the realization of the hypertrophy and the efficient proliferation of pseudobulb is a precondition for effectively expanding the number of individuals, but related reports of tissue culture of the Meihuan have not been found at home and abroad.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide a culture medium group for in-vitro regeneration culture of vaneless meconopsis, and application and a culture method thereof. The culture medium group for in-vitro regeneration culture of vaneless meconopsis provided by the invention has the advantages that chlormequat chloride is applied to the proliferation stage and the rooting culture medium, and the pseudobulb is gradually enlarged by culturing the 2 times of strong seedling culture medium, so that efficient proliferation and rooting are effectively realized.

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

the invention provides a culture medium group for in-vitro regeneration culture of vaneless meconopsis, which comprises a strong seedling-proliferation culture medium and a strong seedling-rooting culture medium, wherein:

the culture medium for the strong seedling-proliferation culture takes an MS modified culture medium as a basic culture medium and further comprises the following components: chlormequat chloride 0.5-1.5 mg/L, 6-BA (6-benzylaminopurine) 1.5-2.5 mg/L, NAA (naphthylacetic acid) 0.15-0.25 mg/L, potato 50-150 g/L, sucrose 20-30 g/L and agar 5.5-6.5 g/L;

the culture medium for strong seedling-rooting culture takes MS culture medium as basic culture medium and comprises the following components: 2-5 g/L of peanut, 0.5-1.5 mg/L, NAA of chlormequat chloride, 0.5-1.5 mg/L of potato, 25-100 g/L of banana juice, 0.5-1.5 g/L of activated carbon, 20-30 g/L of sucrose and 5.5-6.5 g/L of agar;

the MS modified culture medium comprises the following components: KNO (KNO) ₃ 2800~2900mg/L、NH ₄ NO ₃ 800~850mg/L、KH ₂ PO ₄ 160~180mg/L、MgSO ₄ ·7H ₂ 350-400 mg/L of O and CaCl ₂ ·2H ₂ O 430~450mg/L。

Preferably, the group of media further comprises germination media and induction media, wherein:

the germination culture medium is based on a 1/2MS culture medium and further comprises the following components: NAA 0.3-0.7 mg/L, 6-BA 0.1-0.3 mg/L, potato 50-150 g/L, sucrose 20-30 g/L and agar 5.5-6.5 g/L;

the culture medium for induction culture is based on a 1/2MS culture medium and further comprises the following components: TDZ (thidiazuron) 0.5-1.5 mg/L, NAA 0.3.3-0.7 mg/L, coconut juice 45-55 mL/L, activated carbon 0.3-0.7 g/L, sucrose 20-30 g/L and agar 5.5-6.5 g/L.

Preferably, the potatoes include fresh potato juice.

The invention provides application of the culture medium group in-vitro regeneration culture of vaneless meconopsis.

The invention also provides a method for in-vitro regeneration culture of vaneless meconopsis by using the culture medium group, which comprises the following steps:

and sequentially transferring the pseudobulb into a strong seedling-proliferation culture medium and a strong seedling-rooting culture medium, and performing strong seedling-proliferation culture and strong seedling-rooting culture to obtain the vaneless meropenia tissue culture seedling.

Preferably, the pseudobulb culturing method comprises the following steps:

inoculating the seeds into a germination culture medium, and performing germination culture to obtain protocorms;

transferring the protocorm into an induction culture medium, and performing induction culture to obtain the pseudobulb.

Preferably, the germination culture temperature is 23-27 ℃, and the culture condition is dark culture.

Preferably, the culture temperature of the induction culture, the strong seedling-proliferation culture and the strong seedling-rooting culture is 23-27 ℃, the humidity is 45-55%, the illumination intensity is 2000-3000 lx, and the illumination period is 12-16 h/d.

Preferably, the method further comprises outdoor hardening after obtaining the vaneless meropenem tissue culture seedling;

the outdoor seedling hardening comprises the following steps: cultivating the vaneless mermaid tissue culture seedling in water, and placing the seedling under shade for 4-10 days after first hardening to obtain a first tissue culture seedling; and transplanting the first tissue culture seedling into a culture medium, and placing under shade of tree to subject the second seedling to hardening for 10-25 d to obtain the second tissue culture seedling.

The beneficial effects are that: the invention provides a culture medium group for in-vitro regeneration culture of vaneless meropenia, which comprises a strong seedling-proliferation culture medium and a strong seedling-rooting culture medium, wherein chlormequat chloride is applied to the proliferation stage and the rooting culture medium, and the culture of the strong seedling culture medium is carried out for 2 times, so that the pseudobulb is gradually enlarged, the efficient proliferation and rooting are effectively realized, the whole growth period is not prolonged, the pseudobulb is ensured to be thicker, and the survival after domestication and transplanting is facilitated.

The invention provides the application of the culture medium group in the in-vitro regeneration culture of the vaneless meconopsis, properly improves the content of nitrate nitrogen in the proliferation stage, reduces the content of ammonium nitrogen, ensures sufficient nitrogen source and effectively avoids browning phenomenon. The invention uses the culture medium group to establish the high-efficiency in-vitro regeneration system of the vaneless meconopsis, and can provide technical support for the protection, development and utilization of wild resources of the vaneless meconopsis.

The invention also provides a method for in-vitro regeneration culture of the vaneless crown orchid by using the culture medium group, which is characterized in that the vaneless crown orchid pseudobulb is subjected to 2 times of strong seedling culture in the proliferation stage and the rooting stage respectively, so that the pseudobulb is gradually and effectively proliferated and rooted while the pseudobulb is gradually and orderly grown, and a large number of vaneless crown orchids with strong pseudobulb and developed root systems are obtained. The isolated culture method does not need to undergo greenhouse cultivation management, and after the tissue culture seedlings are subjected to outdoor seedling hardening, the tissue culture seedlings are gradually adapted to outdoor shade environments, can be directly transplanted under shade, and effectively relieves the greenhouse cultivation management cost and the space use pressure. The invention provides technical support for the effective protection of wild resources of the vaneless meropenia, the realization of gardening value and medicinal value, and provides guidance reference for the in vitro regeneration of other meropenia.

Drawings

FIG. 1 is a diagram showing pseudobulb material for 2 months of induced culture in example 1;

FIG. 2 is a diagram of pseudobulb obtained by culturing strong seedlings and proliferation for 3 months in example 1;

FIG. 3 is a physical diagram of regenerated seedlings subjected to seedling strengthening-rooting culture for 2 months in example 1;

FIG. 4 is a diagram showing the regenerated plantlets washed after being removed from the culture medium in example 1;

FIG. 5 is a physical view of the vaneless Meihan after 2 months of planting under shade in example 1.

Detailed Description

the culture medium for the strong seedling-proliferation culture takes an MS modified culture medium as a basic culture medium and further comprises the following components: 0.5-1.5 mg/L chlormequat chloride, 1.5-2.5 mg/L, NAA 0.15.15-0.25 mg/L6-BA, 50-150 g/L potato, 20-30 g/L sucrose and 5.5-6.5 g/L agar;

the culture medium for strong seedling-rooting culture takes 1/2MS culture medium as basic culture medium and comprises the following components: 2-5 g/L of peanut, 0.5-1.5 mg/L, NAA of chlormequat chloride, 25-100 g/L of potato, 25-100 g/L of banana juice, 0.5-1.5 g/L of activated carbon, 20-30 g/L of sucrose and 5.5-6.5 g/L of agar.

The present invention is not particularly limited to the above-described raw materials, and may be commercially available products known to those skilled in the art, unless otherwise specified.

The culture medium for strong seedling-proliferation culture of the invention is preferably based on an MS modified culture medium, and preferably further comprises the following components: 0.5-1.5 mg/L chlormequat chloride, 1.5-2.5 mg/L, NAA 0.15.15-0.25 mg/L6-BA, 50-150 g/L potato, 20-30 g/L sucrose and 5.5-6.5 g/L agar, and more preferably, the chlormequat chloride comprises the following components: 0.5-1.5 mg/L chlormequat chloride, 1.5-2.5 mg/L, NAA 0.15.15-0.25 mg/L6-BA, 50-150 g/L potato, 20-30 g/L sucrose and 5.5-6.5 g/L agar; wherein the chlormequat chloride is more preferably 0.8-1.2 mg/L, and most preferably 1.0mg/L; the 6-BA is more preferably 1.8-2.2 mg/L, most preferably 2mg/L; the NAA is more preferably 0.18-0.22 mg/L, most preferably 0.2mg/L; the potato is more preferably 80-120 g/L, and most preferably 100g/L; the sucrose is more preferably 22-28 g/L, most preferably 25g/L; the agar is more preferably 5.8-6.2 g/L, most preferably 6g/L. The chlormequat chloride makes the pseudobulb thick and strong; the 6-BA and NAA can promote proliferation; potatoes provide nutrients; sucrose supplements a carbon source; the agar plays a supporting role, the pseudobulb is thicker than the induced state through multiplication culture, and the survival rate after transplanting can be remarkably improved.

The MS modified culture medium preferably comprises the following components: KNO (KNO) ₃ 2800~2900mg/L、NH ₄ NO ₃ 800~850mg/L、KH ₂ PO ₄ 160~180mg/L、MgSO ₄ ·7H ₂ 350-400 mg/L of O and CaCl ₂ ·2H ₂ O430-450 mg/L, and more preferably comprises the following components: KNO (KNO) ₃ 2800~2900mg/L、NH ₄ NO ₃ 800~850mg/L、KH ₂ PO ₄ 160~180mg/L、MgSO ₄ ·7H ₂ 350-400 mg/L of O and CaCl ₂ ·2H ₂ O430-450 mg/L; wherein the KNO ₃ More preferably 2820-2880 mg/L, most preferably 2850mg/L; the NH is ₄ NO ₃ More preferably 820 to 830mg/L, most preferably 825mg/L; the KH ₂ PO ₄ More preferably 165-175 mg/L, most preferably 170mg/L; the MgSO ₄ ·7H ₂ O is more preferably 360-380 mg/L, and most preferably 370mg/L; the CaCl ₂ ·2H ₂ O is more preferably 435 to 445mg/L, most preferably 440mg/L. The book is provided withThe invention improves the MS basic culture medium in the process of strengthening seedlings and proliferating, ensures the nitrogen source supply in the proliferating process by improving the nitrate nitrogen content, reduces the ammonium nitrogen content and effectively inhibits the browning phenomenon of the material.

The culture medium for strong seedling-rooting culture of the invention is preferably based on an MS culture medium, more preferably comprises the following components by removing a large amount of MS culture medium and a small amount of MS culture medium: the peanut oil comprises 2-5 g/L, 0.5-1.5 mg/L, NAA 0.5.5-1.5 mg/L of chlormequat chloride, 25-100 g/L of potato, 25-100 g/L of banana juice, 0.5-1.5 g/L of activated carbon, 20-30 g/L of sucrose and 5.5-6.5 g/L of agar, and further preferably comprises the following components: 2-5 g/L of peanut, 0.5-1.5 mg/L, NAA of chlormequat chloride, 0.5-1.5 mg/L of potato, 25-100 g/L of banana juice, 0.5-1.5 g/L of activated carbon, 20-30 g/L of sucrose and 5.5-6.5 g/L of agar; wherein the amount of the flower is more preferably 2.5-4 g/L, and most preferably 3g/L; the chlormequat chloride is more preferably 0.8-1.2 mg/L, and most preferably 1mg/L; the NAA is more preferably 0.8-1.2 mg/L, and most preferably 1mg/L; the potato is more preferably 40-80 g/L, and most preferably 50g/L; the banana juice is more preferably 40-80 g/L, and most preferably 50g/L; the activated carbon is more preferably 0.8-1.2 g/L, and most preferably 1g/L; the sucrose is more preferably 22-28 g/L, most preferably 25g/L; the agar is more preferably 5.8-6.2 g/L, most preferably 6g/L. The flower of the invention can promote root growth and plant development; chlormequat chloride makes the pseudobulb thick; NAA promotes rooting and plant growth; the potato juice and the banana juice provide nutrient substances, and the strong seedling rooting culture is beneficial to the plant formation of a developed root system.

The potatoes of the present invention preferably include fresh potato juice. The preparation method for preparing the culture medium by using the potatoes preferably comprises the following steps: selecting fresh potato, peeling, cutting into small pieces, placing into a juicer, adding water, and squeezing to obtain potato juice; the mass volume ratio of the potato to the water is preferably 40-60 g:90 to 110mL, more preferably 45 to 55g: 95-105 mL, more preferably 50g:100mL. The preparation method of the banana juice preferably comprises the following steps: selecting fresh bananas, peeling, cutting into small pieces, putting into a juicer, adding water, and squeezing to obtain banana juice; the mass volume ratio of the banana to the water is preferably 40-60 g:90 to 110mL, more preferably 45 to 55g: 95-105 mL, more preferably 50g:100mL.

The group of media according to the invention preferably also comprises germination media and induction media.

The germination culture medium of the invention is preferably based on 1/2MS culture medium, and preferably further comprises the following components: NAA 0.3-0.7 mg/L, 6-BA 0.1-0.3 mg/L, potato 50-150 g/L, sucrose 20-30 g/L and agar 5-6 g/L, and further preferably comprises the following components only: NAA 0.3-0.7 mg/L, 6-BA 0.1-0.3 mg/L, potato 50-150 g/L, sucrose 20-30 g/L and agar 5-6 g/L; wherein NAA is more preferably 0.4-0.6 mg/L, most preferably 0.4mg/L; the 6-BA is more preferably 0.15-0.25 mg/L, most preferably 0.2mg/L; the potato is more preferably 80-120 g/L, and most preferably 100g/L; the sucrose is more preferably 23-28 g/L, most preferably 25g/L; the agar is more preferably 5.8-6.2 g/L, most preferably 6g/L, and the low concentration 6-BA and NAA can be matched to promote germination.

The culture medium for induction culture according to the invention is preferably based on 1/2MS culture medium, and preferably further comprises the following components: TDZ 0.5-1.5 mg/L, NAA 0.3-0.7 mg/L, coconut juice 45-55 mL/L, active carbon 0.3-0.7 g/L, sucrose 20-30 g/L and agar 5.5-6.5 g/L, and more preferably comprises the following components: 0.5-1.5 mg/L, NAA of TDZ, 45-55 mL/L of coconut juice, 0.3-0.7 g/L of activated carbon, 20-30 g/L of sucrose and 5.5-6.5 g/L of agar; wherein the TDZ is 0.8-1.2 mg/L, and most preferably 1.0mg/L; the NAA is more preferably 0.4-0.6 mg/L, and most preferably 0.5mg/L; the coconut juice is more preferably 45-50 mL/L, and most preferably 50mL/L; the activated carbon is more preferably 0.4-0.6 g/L, and most preferably 0.5g/L; the sucrose is more preferably 23-28 g/L, most preferably 25g/L; the agar is more preferably 5.8-6.2 g/L, most preferably 6g/L. The coconut juice of the present invention is preferably fresh coconut juice, which is preferably filtered with gauze.

The invention preferably disinfects the vaneless meconopsis seed. The seeds of the invention are preferably taken from a sterilized capsule. The invention also preferably comprises cleaning the capsule before sterilization, preferably comprising gently wiping the entire capsule with alcohol cotton.

The invention preferably disinfects the cleaned capsules, the disinfecting agent preferably comprises alcohol and sodium hypochlorite, and the cleaned capsules are preferably disinfected by the alcohol and the sodium hypochlorite in sequence.

The volume percentage of the alcohol is preferably 65% -80%, more preferably 70% -75% and most preferably 75%; the disinfection time of the alcohol is preferably 30-90 s, more preferably 50-70 s, and most preferably 60s. The effective chlorine concentration of the sodium hypochlorite is preferably 0.3% -3.5%, more preferably 0.5% -2.5%, and most preferably 2%; the disinfection time of the sodium hypochlorite is preferably 5-10 min, more preferably 6-9 min, and most preferably 8min. The invention preferably uses sterile water for washing after each disinfection, and the washing times are preferably 3-8 times, more preferably 5-6 times.

The invention carries out germination culture on the seeds taken out from the sterilized capsules to obtain protocorms.

The culture temperature of germination culture is preferably 23-27 ℃, more preferably 24-26 ℃ and most preferably 25 ℃; the culture conditions are preferably dark cultures.

The present invention preferably provides pseudobulbs obtained by subjecting the protocorms obtained as described above to induction culture.

The culture temperature of the induction culture is preferably 23-27 ℃, more preferably 24-26 ℃ and most preferably 25 ℃; the humidity is preferably 45% -55%, more preferably 46% -52%, and most preferably 50%; the illumination intensity is preferably 2000-3000 lx, more preferably 2200-2800 lx, and most preferably 2500lx; the illumination period is 12 to 16h/d, more preferably 13 to 15h/d, most preferably 14h/d.

The invention preferably carries out strong seedling-proliferation culture and strong seedling-rooting culture on the obtained pseudobulb in sequence to obtain the vaneless meropenia tissue culture seedling. The invention can strengthen seedlings for 2 times in two stages of proliferation and rooting, can ensure that the pseudobulb becomes thicker and stronger without prolonging the whole growth period, and is more beneficial to survival after domestication and transplanting.

The culture temperature of the induction culture, the strong seedling-proliferation culture and the strong seedling-rooting culture is preferably 23-27 ℃, more preferably 24-26 ℃ and most preferably 25 ℃; the humidity is preferably 45% -55%, more preferably 46% -52%, and most preferably 50%; the illumination intensity is preferably 2000-3000 lx, more preferably 2200-2800 lx; more preferably 2500lx; the illumination period is preferably 12-16 h/d, more preferably 13-15 h/d, and most preferably 14h/d.

The method preferably comprises the step of outdoor hardening after the cultured tissue culture seedlings of the vaneless meconopsis. The outdoor seedling hardening method preferably comprises the following steps: the vaneless mermaid tissue culture seedlings are preferably cultivated in water, and the water depth is preferably 1-2 cm; preferably placing the tissue culture seedling under shade for 4-10 d through first hardening-down to obtain a first tissue culture seedling, more preferably 5-7 d, and most preferably 7d; taking out the first tissue culture seedling, preferably transplanting the first tissue culture seedling into a culture medium, preferably placing under shade, and performing second hardening-off for 10-25 d to obtain a second tissue culture seedling, more preferably 14-21 d, and most preferably 21d; preferably, the second tissue culture seedling is taken out and transplanted under shade. The cultivation substrate according to the invention preferably comprises the following components: the volume ratio of the nutrient soil to the coconut husk is preferably 0.5-1.5:0.5-1.5, more preferably 0.8-1.2:08-1.2, and most preferably 1:1. In the seedling hardening and transplanting stage, the tissue culture seedlings do not need to undergo greenhouse cultivation management, the tissue culture seedlings are gradually adapted to the outdoor shade environment and are directly transplanted under shade, white new roots can grow on the vaneless mersupport pseudo bulbs after being transplanted for about 2 months, the growth condition is good, natural resources can be fully utilized by utilizing the forest shade cultivation, and the greenhouse cultivation management cost and the space use pressure are effectively relieved.

For further explanation of the present invention, a culture medium set for in vitro regeneration culture of vaneless meran, its application and culture method provided by the present invention will be described in detail with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.

Example 1

A set of media for in vitro regeneration culture of vaneless merocallis, the set of media comprising germination media, induction media, strong-proliferation media, and strong-rooting media, wherein:

germination medium: 1/2MS is taken as a basic culture medium and NAA 0.5mg/L, 6-BA 0.2mg/L, potato 100g/L, sucrose 25g/L and agar 6g/L are added; wherein the potato is potato juice, 100mL of the potato juice is added into every 50g of potato, and the potato juice is obtained by squeezing, and the following steps are the same;

induction culture: 1/2MS is taken as a basic culture medium and added with TDZ 1.0mg/L, NAA, coconut juice 50mL/L, activated carbon 0.5g/L, sucrose 25g/L and agar 6g/L; wherein the coconut juice is fresh coconut juice directly taken out of coconut, and is obtained by filtering with gauze;

strong seedling-proliferation medium: 1.0mg/L of chlormequat chloride, 2mg/L, NAA 0.2.2 mg/L of 6-BA, 100g/L of potato, 25g/L of sucrose and 6.0g/L of agar are added on the basis of an improved MS;

modified MS medium: KNO (KNO) ₃ 2850mg/L、NH ₄ NO ₃ 825mg/L、KH ₂ PO ₄ 170mg/L、MgSO ₄ ·7H ₂ O 370mg/L、CaCl ₂ ·2H ₂ O 440mg/L；

Seedling strengthening-rooting culture medium: taking a large amount and a small amount of MS culture medium as a basic culture medium, adding 3g/L of chlormequat chloride, 1mg/L, NAA mg/L of chlormequat chloride, 50g/L of potato, 50g/L of banana juice, 1g/L of activated carbon, 25g/L of sucrose and 6g/L of agar; wherein, the banana juice is obtained by adding 100mL of banana per 50g and squeezing.

A method for efficiently regenerating vaneless meconopsis ex vivo comprises the following steps:

(1) Sterilization of capsules: collecting vaneless meretrix capsules, and lightly wiping dust and stains on the surface by using 75% alcohol cotton; sterilizing with 75% alcohol in an ultra clean bench for 60s, and washing with sterile water for 5 times; sterilizing with sodium hypochlorite with the effective chlorine concentration of 2% for 8min, and washing with sterile water for 6 times; finally, the surface moisture is absorbed by sterile filter paper, a sterile blade is used for cutting the capsule, seeds are sown into a germination culture medium, and the culture medium is placed at the temperature of 25 ℃ for dark culture.

(2) Pseudobulb induction culture: transferring the germinated seeds to an induction culture medium, and placing the culture medium at the temperature of 25 ℃ for light-dark alternate circulation culture, wherein the illumination intensity is 2500lx and the illumination is 14h/d. The pseudobulb was obtained by induction culture for 2 months, as shown in FIG. 1.

(3) Strong seedling-proliferation culture: the induced pseudobulb is transferred to a strong seedling-proliferation culture medium, and is placed at the temperature of 25 ℃ for light-dark alternate circulation culture, the illumination intensity is 2500lx, and the illumination is 14h/d. Strong seedlings-pseudobulb cultivated for 3 months by multiplication, as shown in fig. 2.

(4) Strong seedling-rooting culture: transferring the strong seedling-proliferated pseudobulb into a strong seedling-rooting culture medium, and placing the culture medium at 25 ℃ for light-dark alternate circulation culture, wherein the illumination intensity is 2500lx and the illumination is 14h/d. The regenerated seedlings are obtained by culturing the strong seedlings for 2 months, as shown in fig. 3, the regenerated seedlings are taken out from the culture medium and cleaned, as shown in fig. 4, and new roots are grown on the obtained vaneless mersupport pseudobulb, and the growth condition is good.

(5) Hardening and transplanting: opening the tissue culture bottle cap, thoroughly cleaning the agar culture medium attached to the pseudobulb, injecting water with the depth of 2cm into the bottle, and placing the tissue culture bottle under shade for hardening seedlings for 7d; taking out the tissue culture seedlings, transplanting the tissue culture seedlings into a cave dish filled with nutrient soil and coconut coir (1:1), and placing the cave dish under shade for continuous culture for 21d; taking out the tissue culture seedlings to be planted under shade. The plant of the vaneless meconium majus is obtained after planting under shade for 2 months, and as shown in figure 5, the obtained vaneless meconium majus has developed rooting system and good growth condition.

Example 2

The procedure is as in example 1, except that KNO alone is added to the modified MS medium ₃ Replaced by 2375mg/L, NH ₄ NO ₃ And 1650mg/L.

Example 3

The procedure is as in example 1, except that KNO alone is added to the modified MS medium ₃ Replaced by 3325mg/L, NH ₄ NO ₃ Is replaced by 412.5mg/L.

Comparative example 1

The procedure is as in example 1, except that KNO alone is added to the modified MS medium ₃ Replaced by 1900mg/L, NH ₄ NO ₃ Is replaced by 3300mg/L.

Test example 1

The detection method of the average proliferation coefficient comprises the following steps: counting the number of the new pseudobulbs in 3 months by adopting an observation counting method.

The detection method of the browning rate comprises the following steps: counting the number of the brown pseudobulbs by adopting an observation counting method.

The calculation formula of each parameter is as follows:

average proliferation coefficient = number of pseudobulbs newly added/number of pseudobulbs inoculated.

Browning rate (%) =number of brown pseudobulbs/total number of pseudobulbs inoculated x 100%.

Using the above detection method, examples 1 to 3 and comparative example 1 were compared, i.e., NH in MS minimal medium during proliferation phase ₄ NO ₃ With KNO ₃ The other components were adjusted, unchanged, to explore the effect on proliferation, and the experimental data were analyzed for variables using SPSS 27 and for differential significance testing using Duncan's, with the results shown in table 1.

TABLE 1 different NH ₄ NO ₃ With KNO ₃ Effect of the amount on proliferation of vaneless Meihuanlan

As can be seen from table 1, the proliferation coefficients of example 2, example 3 and comparative example 1 were all not significantly different, wherein the treatment effect of example 1 was the best, the proliferation coefficient of comparative example 1 was significantly different, and the browning control effect was the best; the browning rates of example 2 and example 3 were also significantly reduced compared to comparative example 1, and the plants brown in comparative example 1 gradually died with the extension of the post-culture time. Therefore, the too high salt concentration can cause a large amount of phenols to be generated, cause browning and even cause serious toxicity to culture materials; reducing the salt concentration can reduce phenol overflow and reduce browning. Therefore, the dosages of ammonium nitrate and potassium nitrate are adjusted, firstly, the supply of nitrogen sources is ensured, and proliferation is not affected; secondly, the nitrate concentration is reduced as a whole, so that browning phenomenon is inhibited.

Example 4

The procedure was followed except that the strong seedling-propagation medium was replaced with the modified MS-based medium and chlormequat chloride 0.5mg/L, 6-BA 1mg/L, NAA 0.1mg/L, potato 100g/L, sucrose 25g/L and agar 6.0g/L were added; (4) And during strong seedling-rooting culture, only chlormequat chloride is replaced by 0.5mg/L, NAA and 0.5mg/L.

Example 5

The procedure was followed except that the strong seedling-propagation medium was replaced with the modified MS-based medium and chlormequat chloride 1.5mg/L, 6-BA 2.5mg/L, NAA 0.25.25 mg/L, potato 100g/L, sucrose 25g/L and agar 6.0g/L were added; (4) And during strong seedling-rooting culture, only chlormequat chloride is replaced by 1.5mg/L, NAA to 1.5mg/L.

Comparative example 2

The procedure was followed as in example 5 except that the strong seedling-propagation medium was replaced with MS-based medium and 6-BA 2mg/L, NAA 0.2mg/L, potato 100g/L, sucrose 25g/L and agar 6.0g/L were added; (4) During strong seedling-rooting culture, NAA is replaced by 1mg/L, and chlormequat chloride is omitted.

Test example 2

Proliferation coefficient: counting the number of the new pseudobulbs in 3 months.

Root number: counting the rooting number in 2 months.

Stem thickness: the diameter of the pseudobulb was measured using a vernier caliper.

Transplanting survival rate: counting, and counting the survival number after transplanting for 2 months.

The calculation formula of each parameter is as follows:

proliferation coefficient = number of newly added pseudobulbs/number of pseudobulbs inoculated.

Root number = sum of root numbers of all pseudobulbs/number of pseudobulbs inoculated.

Stem thickness = sum of all pseudobulb diameters/number of pseudobulbs inoculated.

Transplanting survival = number of transplanted pseudobulbs/total number of transplanted pseudobulbs x 100%.

The tissue culture results of examples 1, 4, 5 and comparative example 2 were compared using the above test method, and the test data were subjected to variable analysis using SPSS 27 and differential significance test using Duncan's, and the obtained data are shown in table 2.

TABLE 2 tissue culture results for different media

As can be seen from Table 2, the proliferation factor of example 1, in which chlormequat chloride was added for 2 times of strong seedling treatment in both the proliferation stage and the rooting stage, was as high as 16.11, which is significantly higher than that of comparative example 2 in which only 1 time of strong seedlings were obtained. Although there was no significant difference in the number of roots in the strong seedling-rooting stage, the stem thickness was significantly greater for each example than for comparative example 2. In addition, the embodiment of the invention adopts the improved MS as the proliferation basic culture medium, thereby improving the consumption of nitrate nitrogen, ensuring the supply of nitrogen sources in the vegetative growth period, reducing the consumption of ammonium nitrogen and avoiding the browning phenomenon caused by poisoning materials by high-concentration ammonium salt. The false bulbs of the comparative example 1 which are only subjected to 1-time seedling strengthening are thin and weak, the transplanting survival rate is low after direct outdoor seedling hardening, the false bulbs of the comparative example 1 which are subjected to 2-time seedling strengthening are thick and strong, the root system is strong, the transplanting survival rate is up to 83.25% after outdoor gradual seedling hardening and domestication, and white new roots are generated after 2 months of transplanting, so that the condition is good.

Therefore, the invention provides a method for efficiently regenerating the vaneless meropenem in vitro, aiming at the growth characteristics of the sapropel, the 1 st seedling strengthening culture is started in the proliferation stage, so that the pseudobulb is proliferated for several times and becomes thicker at the same time, and simultaneously, the basic culture medium components are adjusted to ensure the supply of nitrogen sources and avoid the toxic effect of ammonium nitrogen on materials; and 2 times of strong seedling culture are carried out in the rooting stage, so that the root system is developed, the pseudobulb is more robust, and the success rate of outdoor domestication and transplanting is high. The invention can effectively protect the wild resources of the vaneless meconopsis, and has positive benefits in gardening and medicinal application.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, it should be understood that other embodiments may be devised in accordance with the present embodiments without departing from the spirit and scope of the invention.

Claims

1. A culture medium group for in vitro regeneration culture of vaneless meropenia, characterized in that the culture medium group comprises germination medium, induction medium, strong seedling-proliferation medium and strong seedling-rooting medium, wherein:

the germination culture medium is based on 1/2MS culture medium, and the following components are added: NAA 0.3-0.7 mg/L, 6-BA 0.1-0.3 mg/L, potato 50-150 g/L, sucrose 20-30 g/L and agar 5.5-6.5 g/L;

the culture medium for induction culture is based on 1/2MS culture medium, and the following components are added: 0.5-1.5 mg/L, NAA of TDZ, 45-55 mL/L of coconut juice, 0.3-0.7 g/L of activated carbon, 20-30 g/L of sucrose and 5.5-6.5 g/L of agar;

the culture medium for the strong seedling-proliferation culture takes an MS modified culture medium as a basic culture medium, and the following components are also added: 0.5-1.5 mg/L chlormequat chloride, 1.5-2.5 mg/L, NAA 0.15.15-0.25 mg/L6-BA, 50-150 g/L potato, 20-30 g/L sucrose and 5.5-6.5 g/L agar;

the culture medium for strong seedling-rooting culture takes an MS culture medium as a basic culture medium, and the following components are also added: 2-5 g/L of peanut, 0.5-1.5 mg/L, NAA of chlormequat chloride, 0.5-1.5 mg/L of potato, 25-100 g/L of banana juice, 0.5-1.5 g/L of activated carbon, 20-30 g/L of sucrose and 5.5-6.5 g/L of agar;

the MS modified culture medium comprises the following components: KNO (KNO) ₃ 2800~2900mg/L、NH ₄ NO ₃ 800~850mg/L、KH ₂ PO ₄ 160~180mg/L、MgSO ₄ ·7H ₂ 350-400 mg/L of O and CaCl ₂ ·2H ₂ O 430~450mg/L；

The banana juice is obtained by adding 100mL of water into 50g of banana and squeezing;

the potato is potato juice, 100mL of water is added into every 50g of potato, and juice is obtained after squeezing.

2. Use of the culture medium group according to claim 1 for in vitro regeneration culture of vaneless meconopsis.

3. A method for the in vitro regeneration culture of vaneless meconopsis using the group of media according to claim 1, characterized in that it comprises the following steps:

transferring the protocorm into an induction culture medium, and performing induction culture to obtain pseudobulbs;

4. The method according to claim 3, wherein the germination culture is performed at a culture temperature of 23-27 ℃ under dark culture conditions.

5. The method according to claim 3, wherein the culture temperature of the induction culture, the strong seedling-proliferation culture and the strong seedling-rooting culture is 23-27 ℃, the humidity is 45-55%, the illumination intensity is 2000-3000 lx, and the illumination period is 12-16 h/d.

6. The method of claim 3, wherein obtaining the vaneless meconopsis further comprises outdoor acclimatization after the tissue culture seedling;

7. The method of claim 6, wherein the cultivation substrate comprises the following components: nutritional soil and coconut coir; the volume ratio of the nutrient soil to the coconut husk is 0.5-1.5:0.5-1.5.