CN108633376B - A kind of culture method for non-symbiotic germination of Phytophthora grisea seeds - Google Patents
A kind of culture method for non-symbiotic germination of Phytophthora grisea seeds Download PDFInfo
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- 229920001817 Agar Polymers 0.000 claims abstract description 27
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- 230000007226 seed germination Effects 0.000 claims abstract description 26
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 23
- 239000000843 powder Substances 0.000 claims abstract description 20
- 238000012546 transfer Methods 0.000 claims abstract description 18
- 244000060011 Cocos nucifera Species 0.000 claims abstract description 14
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- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 12
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- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 claims description 6
- CGIDKJRJBMFXKV-UHFFFAOYSA-N 6-n'-benzylpurine-6,6-diamine Chemical compound N1=CN=C2N=CN=C2C1(N)NCC1=CC=CC=C1 CGIDKJRJBMFXKV-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 5
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- 239000007640 basal medium Substances 0.000 claims description 5
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 5
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- 239000002363 auxin Substances 0.000 claims description 3
- OKTJSMMVPCPJKN-YPZZEJLDSA-N carbon-10 atom Chemical compound [10C] OKTJSMMVPCPJKN-YPZZEJLDSA-N 0.000 claims description 3
- UQHKFADEQIVWID-UHFFFAOYSA-N cytokinin Natural products C1=NC=2C(NCC=C(CO)C)=NC=NC=2N1C1CC(O)C(CO)O1 UQHKFADEQIVWID-UHFFFAOYSA-N 0.000 claims description 3
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/04—Arranging seed on carriers, e.g. on tapes, on cords ; Carrier compositions
- A01C1/046—Carrier compositions
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01C—PLANTING; SOWING; FERTILISING
- A01C1/00—Apparatus, or methods of use thereof, for testing or treating seed, roots, or the like, prior to sowing or planting
- A01C1/08—Immunising seed
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Abstract
本发明公开一种格力兜兰种子非共生萌发的培养方法，如下步骤：将格力兜兰果荚清洗、消毒灭菌，后在无菌操作台上用无菌剪刀将果荚剪为两段，将种子播散到已灭菌的萌发培养基中，在暗室中25℃暗培养，以出现白色原球茎作为种子萌发的标志。种子产生白色原球茎后，统计格力兜兰种子萌发率。种子萌发形成原球茎后，将其转入光照下继续培养，原球茎全变为绿色后，转接至格力兜兰原球茎转接培养基中继续光照培养。所述的格力兜兰种子萌发培养基以花宝1号为基础培养基，内添加蔗糖和琼脂，并分别添加有活性炭、椰子粉，该格力兜兰种子萌发培养方法及萌发培养基的使用，能促使格力兜兰种子萌发，并提高种子的萌发率，为格力兜兰种子成苗及引种回归奠定基础，有利于格力兜兰保育工作的开展。
The invention discloses a method for culturing the non-symbiotic germination of P. The seeds were sown into sterilized germination medium, and cultivated in a dark room at 25°C, with the appearance of white protocorm as a sign of seed germination. After the seeds produced white protocorm, the germination rate of P. grisea seeds was counted. After the seeds germinate to form protocorms, they are transferred to the light to continue culturing, and after the protocorms are all turned green, they are transferred to the protocorm transfer medium of Glicolaia to continue the light cultivation. Described P. greek seed germination medium is based on Huabao No. 1, added with sucrose and agar, and added with activated carbon and coconut powder respectively, the method for germination and cultivation of P. It can promote the germination of P. grisea seeds and improve the germination rate of the seeds, lay the foundation for the seedlings and introduction and return of P.
Description
The technical field is as follows:
the invention belongs to the field of plant seed propagation, and particularly relates to a non-symbiotic germination culture method for paphiopedilum glaucescens seeds.
Background art:
geliwan (Geliwan)Paphiopedilum gratrixianum) Is a paphiopedilum plant which is mainly distributed in southeast Yunnan, Laos and northern Vietnam and is a terrestrial or stone epiphytic plant. All species of paphiopedilum are 'International convention on trade for endangered species of wild animals and plants' (CITES) appendix I species, and in 'national minimum population wild plant rescue protection engineering planning (2011-2015)', 7 paphiopedilum including gridland are listed as preferential protection objects to carry out rescue protection work. The survival rate of the griffonia cava is extremely low, and the conservation work is not easy to be carried out.
Paphiopedilum seeds are fine like dust and have no endosperm, and cannot provide nutrients necessary for seed germination like most orchids, which is an important reason that paphiopedilum plants are difficult to grow seedlings and are in imminent danger. At present, paphiopedilum propagation mainly aims at non-symbiotic germination of seeds, and a germination culture medium has great influence on the germination rate of the seeds in the non-symbiotic germination of the seeds. Moreover, the method for seed germination of paphiopedilum of different species has no wide adaptability due to the difference of seed structure and maturation time.
Disclosure of Invention
The invention aims to provide a culture method capable of improving the germination rate of paphiopedilum glaucescens seeds aiming at the defects in the prior art, and the culture medium adopted by the method can promote the germination of the paphiopedilum glaucescens seeds and improve the germination rate of the seeds.
The invention is realized by the following technical scheme:
a culture method for non-symbiotic germination of paphiopedilum glaucescens seeds comprises the following steps:
(1) collecting mature but uncracked paphiopedilum glaucescens pods, dipping the pods with a soft brush to wash the surfaces of the paphiopedilum glaucescens pods, washing the surfaces of the paphiopedilum glaucescens pods with washing powder, washing the surfaces of the paphiopedilum glaucescens pods with distilled water on an aseptic workbench for 20-30s, then wiping the surfaces of the paphiopedilum glaucescens pods with alcohol with the mass concentration of 70-75%, soaking and disinfecting the surfaces of the paphiopedilum glaucescens pods with sodium hypochlorite solution with the mass concentration of 5-10% for 15-20 min, washing the pap; also comprises the following steps:
(2) clamping a fruit pod containing the paphiopedilum glaucescens seeds by using sterile forceps, sowing the seeds on the surface of a seed germination culture medium, carrying out dark culture at 25 ℃ until the seeds germinate, and counting the seed germination rate; after the seed germination culture medium generates white protocorms, transferring the white protocorms to the illumination for continuous culture, wherein the illumination time is 12 h/d, and when the protocorms are completely changed into green;
the seed germination culture medium comprises the following components in percentage by weight: 1 g/L Huabao No. 1, 7 g/L agar, 20 g/L sucrose, 0.5 g/L activated carbon powder and 10 g/L coconut powder;
(3) cutting a seed germination culture medium with a completely green protocorm into circular agar blocks with the diameter of about 5 mm by using a sterile knife in an ultraclean workbench, clamping the agar blocks by using sterile forceps, transferring the agar blocks into a paphiopedilum griffithidum protocorm transfer culture medium, uniformly placing 4-5 agar blocks in each transfer culture medium, placing the medium under illumination after the transfer is finished, and continuing illumination culture for 12 h/d;
the formula of the paphiopedilum glaucescens protocorm transfer culture medium is as follows: 1 g/L Huabao No. 1, 7 g/L agar, 20 g/L sucrose, 0.5 g/L activated carbon powder, 10 g/L coconut powder, 1.0 mg/L cytokinin 6-benzylamino adenine (6-BA) and 0.2 mg/L auxin naphthylacetic acid (NAA).
The method for disinfecting the surface of the fruit pod of the paphiopedilum glaucescens in the step (1) comprises the following steps: wiping the surface of the fruit pod with 75% alcohol, soaking and sterilizing the fruit pod with 10% sodium hypochlorite solution for 20 min, and cleaning with a large amount of sterilized distilled water.
And (3) dark culture germination time of the paphiopedilum glaucescens seeds in the step (2) is 54 days.
The seed germination culture medium used by the invention takes 1 g/L Huabao No. 1 as a basic culture medium, 7 g/L agar and 20 g/L sucrose are added into the culture medium, and 0.5 g/L activated carbon powder, 10 g/L coconut powder and a mixture of the activated carbon and the coconut powder are respectively added as the culture medium for the germination of the paphiopedilum glaucescens seeds. The seed germination culture medium provided by the invention can promote germination of paphiopedilum glaucescens seeds in the embodiment list.
In the seed germination culture medium, 1 g/L Huabao, 7 g/L agar and 20 g/L cane sugar are added, and 0.5 g/L active carbon powder and 10 g/L coconut powder (experiment group 3) are simultaneously added, so that the germination of the paphiopedilum glaucum seeds can be obviously promoted, and the germination rate is improved.
The invention adopts mature and uncracked fruit pods, one purpose is to ensure the germination activity of seeds, and the other purpose is to ensure that the seeds are free from pollution, so that the success of a germination experiment can be promoted to the maximum extent.
The protocorm transfer culture medium contains cytokinin 6-benzylamino adenine (6-BA) and auxin naphthylacetic acid (NAA) and can promote the growth and development of protocorms.
The griffonia simplicifolia seed germination culture method and the use of the germination culture medium can promote the griffonia simplicifolia seed to germinate, improve the germination rate of the seeds (table 2), lay a foundation for the griffonia simplicifolia seed seedling and introduction regression, and are favorable for the development of the conservation work of the griffonia simplicifolia.
Drawings
FIG. 1 is a schematic representation of mature but uncracked fruit pods of paphiopedilum glaucescens used in the present invention.
FIG. 2 shows the sowing of paphiopedilum glaucescens seeds on the surface of germination medium and the dark cultivation.
FIG. 3 is a schematic representation of the beginning of germination and formation of white protocorms of paphiopedilum griffithii seeds.
FIG. 4 shows the green color of protocorm when seeds of paphiopedilum glaucescens are cultivated in the light.
FIG. 5 is a schematic representation of the cultivation under light after the grafting of the paphiopedilum glaucescens protocorm.
Detailed Description
The following examples are provided to further illustrate the essence of the present invention, but are not intended to limit the present invention.
Example 1: the invention relates to a culture method for non-symbiotic germination of paphiopedilum glaucum seeds
Preparing a germination culture medium: accurately weighing 1 g of Huabao No. 1 powder, dissolving with 1L of boiling distilled water, adding 20 g of sucrose and 7 g of agar, and ultrasonically dissolving for 1 h to completely dissolve the agar. Pouring 20 ml of the culture medium into a 50 ml triangular flask respectively, repeating the steps for 10 times, and sealing the opening of the triangular flask by using a sealing film.
And (3) disinfection treatment of the culture medium: placing the packaged culture medium in an autoclave, sterilizing at 121 deg.C for 20 min, and naturally cooling in an ultra-clean bench; the sterilized culture medium was used up within 3 days.
And (3) sterilizing the fruit pods of paphiopedilum glaucescens: collecting 1 mature but not cracked paphiopedilum glaucescens pod, dipping the surface with a soft brush to wash the surface, and then washing the surface with a large amount of tap water. The pod surface was cleaned with distilled water in a sterile bench and then wiped with 75% alcohol by mass for 30 s. Soaking in 10% sodium hypochlorite for 20 min for sterilization, and washing with large amount of sterilized distilled water.
Sowing seeds of paphiopedilum glaucescens and non-symbiotic culture: shearing the sterilized fruit pods into two sections by using sterile scissors, clamping one section by using sterile forceps, lightly sowing seeds on the surface of a seed germination culture medium, carrying out dark culture at 25 ℃ until the seeds germinate, and sowing 10 bottles of culture medium on each seed. After the seeds generate white protocorms, calculating the germination rate of the paphiopedilum glaucum seeds, transferring the paphiopedilum glaucum seeds to the illumination for continuous culture, and after the protocorms are completely changed into green, transferring the paphiopedilum glaucum seeds to a paphiopedilum glaucum protocorm transfer culture medium for continuous illumination culture.
The griffonia simplicifolia seed germination medium with the protocorm totally turned green is cut into agar blocks with the diameter of about 5 mm by a sterile knife in an ultraclean workbench, and the agar blocks are clamped and transferred into the griffonia simplicifolia protocorm transfer medium by sterile tweezers, and 4 to 5 agar blocks are uniformly placed in each medium. After the transfer is finished, the cells are cultured under illumination for 12 h/d.
Example 2: the paphiopedilum glaucescens seed germination culture method
Preparing a germination culture medium: accurately weighing 1 g of Huabao No. 1 powder, dissolving with 1L of boiling distilled water, and adding 20 g of sucrose and 7 g of agar as a basic culture medium. After 0.5 g of activated carbon is added into a basic culture medium, ultrasonic dissolution is carried out for 1 hour, so that agar is completely dissolved, and the activated carbon is uniformly dispersed. Pouring 20 ml of the culture medium into a 50 ml triangular flask respectively, repeating the steps for 10 times, and sealing the opening of the triangular flask by using a sealing film.
Disinfection of medium, sterilization of grove pod, sowing of grove seed and non-symbiotic cultivation, transfer of protocorm and cultivation reference example 1.
Example 3: the paphiopedilum glaucescens seed germination culture method
Preparing a germination culture medium: accurately weighing 1 g of Huabao No. 1 powder, dissolving with 1L of boiling distilled water, and adding 20 g of sucrose and 7 g of agar as a basic culture medium. After 10 g of coconut powder was added to the basal medium, the agar was dissolved completely by ultrasonic dissolution for 1 hour. Pouring 20 ml of the culture medium into a 50 ml triangular flask respectively, repeating the steps for 10 times, and sealing the opening of the triangular flask by using a sealing film.
Disinfection of medium, sterilization of grove pod, sowing of grove seed and non-symbiotic cultivation, transfer of protocorm and cultivation reference example 1.
Example 4: the paphiopedilum glaucescens seed germination culture method
Preparing a germination culture medium: accurately weighing 1 g of Huabao No. 1 powder, dissolving with 1L of boiling distilled water, and adding 20 g of sucrose and 7 g of agar as a basic culture medium. After 10 g of coconut powder and 0.5 g of active carbon are added into a basic culture medium at the same time, ultrasonic dissolution is carried out for 1 h, so that agar is completely dissolved, and the active carbon is uniformly dispersed. Pouring 20 ml of the culture medium into a 50 ml triangular flask respectively, repeating the steps for 10 times, and sealing the opening of the triangular flask by using a sealing film.
Disinfection of medium, sterilization of grove pod, sowing of grove seed and non-symbiotic cultivation, transfer of protocorm and cultivation reference example 1.
TABLE 1 Geliwan paphiopedilum seed germination culture medium formula
With reference to the formula of the griffonia simplicifolia germination medium and the seed germination culture method described in examples 1 to 4, the griffonia simplicifolia seeds obtained after the disinfection treatment were inoculated into the germination medium and then cultured, respectively. The germination rate and time of the seeds when the presence of white protocorms was observed was recorded and the results are shown in table 2.
TABLE 2 Gelihood seed Germination time and germination Rate
The control group 1 was a basal medium, no coconut powder and carbon powder were added, the seed germination time was long and the germination rate was low. The carbon powder is added into the experimental group 1, and the culture medium of the coconut powder () of the experimental group 2 can improve the germination rate of the paphiopedilum griffithii seeds and shorten the time required for germination. Meanwhile, the culture medium (experimental group 3) added with carbon powder and coconut powder can obviously shorten the time required by germination of the paphiopedilum glaucescens, and the germination rate is obviously improved.
The embodiments described above are more specific and detailed, but should not be construed as limiting the scope of the invention. Without departing from the concept of the invention, several variations and modifications can be made, which are within the scope of the invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.
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