CN102511390B - Method and special culture medium for regenerating sterile induced plants of ormosia fordiana seeds - Google Patents
Method and special culture medium for regenerating sterile induced plants of ormosia fordiana seeds Download PDFInfo
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- CN102511390B CN102511390B CN 201110383577 CN201110383577A CN102511390B CN 102511390 B CN102511390 B CN 102511390B CN 201110383577 CN201110383577 CN 201110383577 CN 201110383577 A CN201110383577 A CN 201110383577A CN 102511390 B CN102511390 B CN 102511390B
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Abstract
The invention discloses a method and a special culture medium for regenerating sterile induced plants of ormosia fordiana seeds. The seed germination culture medium in the special culture medium contains Read, 0.2 to 1 mg/L of 6-BA and 0.02 to 0.1 mg/L of NAA; the proliferation culture medium contains Read, 0.5 to 1.5 mg/L of 6-BA, 0.05 to 0.1 mg/L of KT and 0.05 to 0.1 mg/L of NAA; the rooting culture medium contains Read and 0.5 to 1.5 mg/L of IAA; and each of the three culture mediums contains 30 g/L of sucrose and 6.5 g/L of agar, and the pH of each culture medium is 5.8. By the method and the special culture medium, the germination rate of seeds reaches 86 to 100 percent; the proliferation rate reaches 390 to 442 percent; the rooting rate reaches 81 to 92 percent; the survive rate of tempered seedlings reaches 77 to 85 percent; and the invention has practical significance for solving deficiency of ormosia fordiana seed sources and realizing sustainable utilization and large-scaleproduction of the ormosia fordiana seeds.
Description
Technical field
The present invention relates to a seed manure pod red bean seed asepsis and induce the method for plant regeneration, platymiscium field of tissue culture.
Background technology
Fertile pod red bean (
Ormosia fordianaOliv.) be pulse family ormosia arbor, mainly produce Guangdong, Hainan, Guangxi, Yunnan in China, also there are distribution in external Vietnam, Burma, Thailand, Bangladesh, its stem skin, root, leaf can be used as medicine, have clearing heat and detoxicating, the effect of swelling and pain relieving, cure mainly acute hepatitis, wound swells and ache, toothache due to pathogenic wind-fire, burn and scald, there are some researches show the activeconstituents that fertile pod red bean stem, leaf extract, human body pathogenic bacteria streptococcus aureus is had stronger restraining effect, can be developed as new microbiotic; In addition, its texture is perfectly straight, and should be used for construction industry and make top-grade furniture, be medicinal plant and the commerical tree species with higher economic worth and very big potentiality to be exploited.At present, fertile pod red bean is its cultivation technique no matter, or breeding technology, the research report is blank, its provenance is supplied with the main traditional propagation method that adopts seed or cuttage, but reproduction speed is slower, and breeding coefficient is low, be difficult to satisfy the demand of production application, provenance lacks the key factor that becomes the fertile pod red bean Application and Development of restriction.Therefore, the new way that research suits and fertile pod red bean tissue culture propagation technology can become its provenance shortage problem of solution efficiently.
Summary of the invention
The technical problem to be solved in the present invention is to overcome traditional propagation method that prior art adopts seed or cuttage,
Cause reproduction speed slower, breeding coefficient is low, defective and deficiency that provenance lacks, its objective is provide a kind of suitable and efficiently fertile pod red bean seed asepsis induce method and the special culture media thereof of plant regeneration.
For solving above technical problem, the present invention adopts following technical scheme:
1, fertile pod red bean seed asepsis is induced the special culture media of plant regeneration, and it is the seed germination substratum by following prescription, and proliferated culture medium and root media are formed:
(1) prescription of seed germination substratum is: Read minimum medium+6-BA0.2～1mg/L+NAA0.02～0.1 mg/L+ sucrose 30g/L+ agar, 6.5 g/L, Ph=5.8;
(2) prescription of proliferated culture medium is: Read minimum medium+6-BA0.5～1.5mg/L+KT0.05～0.1 mg/L+ NAA0.05～0.1mg/L+ sucrose 30g/L+ agar 6.5 g/L, Ph=5.8;
(3) prescription of root media is: Read minimum medium+IAA0.5～1.5 mg/L+ sucrose 30g/L+ agar, 6.5 g/L, Ph=5.8.
2, preferred fertile pod red bean seed asepsis is induced the special culture media of plant regeneration, and it is the seed germination substratum by following prescription, and proliferated culture medium and root media are formed:
(1) prescription of seed germination substratum is: Read minimum medium+6-BA 0.5 mg/L+NAA 0.05 mg/L+ sucrose 30g/L+ agar 6.5g/L, Ph=5.8;
(2) prescription of proliferated culture medium is: Read minimum medium+6-BA 1 mg/L+KT 0.1 mg/L+NAA 0.1 mg/L+ sucrose 30g/L+ agar 6.5g/L, Ph=5.8;
(3) prescription of root media is: Read minimum medium+IAA 1.0 mg/L++sucrose 30g/L+ agar 6.5g/L, Ph=5.8.
3, fertile pod red bean seed asepsis is induced the method for plant regeneration, carries out according to the following steps:
(1) seed disinfection and processing
Fertile pod red bean seed is soaked 12～18min with 0.12% mercuric chloride solution, soak 8～12min with 2% chlorine bleach liquor again after, use the clear water rinsing, put into 3MNaOH solution soaking 40～80min afterwards, with taking out after the clear water rinsing, peel off kind of a skin again;
(2) seed germination
To be inoculated in the seed germination substratum through step (1) sterilization and the seed of handling, cultivate 24～30 days; The prescription of described seed germination substratum is the prescription of technique scheme 1 described (1) seed germination substratum;
(3) adventitious bud proliferation
The bud that step (2) seed germination is derived separates from bean cotyledon, and whole or the stem section that is cut into band joint are transferred on the proliferated culture medium, cultivate 40 days; The prescription of described proliferated culture medium is the prescription of technique scheme 1 described (2) proliferated culture medium;
(4) take root
The propagation seedling that step (3) is cultivated divides an incision, is inoculated in root media, cultivates 24～31 days; The prescription of described root media is the prescription of technique scheme 1 described (3) root media;
(5) hardening and transplanting
When the seedling for the treatment of step (4) grows 4～6 main roots, culturing bottle is moved in the outdoor booth, it is 70% sunshade hardening off the net in transmittance, took out seedling in 3～7 days, flush away root substratum, be transplanted in the dish of cave, cave dish mesostroma is by leaf-humidified soil: laterite: the mixture that the volume ratio mixed of perlite=2～3.5:1～2:0.5～1 forms, the cave dish is positioned in the hut of growing seedlings, ceiling covers the sunshade net of one deck transmittance 60%, threw off the sunshade net on the 8th day, keeping the mass water content of soil of matrix is 40-50%;
The culture condition of above-mentioned steps (2)～step (4): 22～25 ℃ of culture temperature, light application time 12 h/d, intensity of illumination 1500～2000lx.
4, preferred fertile pod red bean seed asepsis is induced the method for plant regeneration, carries out according to the following steps:
(1) seed disinfection and processing
Fertile pod red bean seed is soaked 15min with 0.12% mercuric chloride solution, soak 10min with 2% chlorine bleach liquor again after, use the clear water rinsing, put into 3MNaOH solution soaking 60min afterwards, with taking out after the clear water rinsing, peel off kind of a skin again;
(2) seed germination
To be inoculated in the seed germination substratum through step (1) sterilization and the seed of handling, cultivate 24 days; The prescription of described seed germination substratum is the prescription of technique scheme 2 described (1) seed germination substratum;
(3) adventitious bud proliferation
The bud that step (2) seed germination is derived separates from bean cotyledon, and whole or the stem section that is cut into band joint are transferred on the proliferated culture medium, cultivate 40 days; The prescription of described proliferated culture medium is the prescription of technique scheme 2 described (2) proliferated culture medium;
(4) take root
The propagation seedling that step (3) is cultivated divides an incision, and basal part of stem is cut flat, is inoculated in root media, cultivates 24 days; The prescription of described root media is the prescription of technique scheme 2 described (3) root media;
(5) hardening and transplanting
When the seedling for the treatment of step (4) grows 4～6 main roots, culturing bottle is moved in the outdoor booth, it is 70% sunshade hardening off the net in transmittance, took out seedling in 7 days, flush away root substratum, be transplanted in the dish of cave, cave dish mesostroma is by leaf-humidified soil: the mixture that the volume ratio mixed of laterite: perlite=3.5:1:0.5 forms, the cave dish is positioned in the hut of growing seedlings, ceiling covering one deck transmittance is 60% sunshade net, threw off the sunshade net on the 8th day, keeping the mass water content of soil of matrix is 40～50%;
The culture condition of step (2)～step (4): culture temperature is 22-25 ℃, and light application time is 12 h/d, and intensity of illumination is 1500-2000lx.
Beneficial effect of the present invention:
Fertile pod red bean seed asepsis of the present invention is induced method and the special culture media of plant regeneration
,Can overcome the hard incrust problem that is not easy to induce sprouting of kind of skin that causes of explant kind skin effectively, make germination rate reach 86～100%; Also make whole and induce its propagation in the plant regeneration process, the rooting rate height, proliferation rate reaches 390～442%; Rooting rate reaches 81～92%; The hardening surviving rate reaches 77～85%, the seedling robust growth, the plant base portion has clump bud propagation, cane joint place has lateral bud to send, quality is good, be the method for the quick breeding of fertile pod red bean seed of the quick breeding of a kind of suitable especially fertile pod red bean seed and high-efficiency high-quality, solved fertile pod red bean provenance and lacked problem, filled up the blank that fertile pod red bean is bred research field.
Embodiment
Present embodiment is done following 3 processing.
Handle 1: fertile pod red bean seed asepsis is induced the method for plant regeneration, carries out according to the following steps:
(1) seed disinfection and processing
Fertile pod red bean seed is soaked 12min with 0.12% mercuric chloride solution, soak 8min with 2% chlorine bleach liquor again after, with clear water rinsing twice, put into 3MNaOH solution soaking 40min afterwards, take out clear water rinsing twice afterwards, peel off kind of a skin;
(2) seed germination
To be inoculated in the seed germination substratum through step (1) sterilization and the seed of handling, cultivate 27 days; The prescription of described seed germination substratum is: Read minimum medium+6-BA0.2mg/L+NAA0.1 mg/L+ sucrose 30g/L+ agar 6.5g/L, Ph=5.8;
(3) adventitious bud proliferation
The bud that step (2) seed germination is derived separates from bean cotyledon, and whole or the stem section that is cut into the 2cm～3cm of band joint are transferred on the proliferated culture medium, cultivate 40 days; The prescription of described proliferated culture medium is: Read minimum medium+6-BA0.5mg/L+KT0.05mg/L+ NAA0.05mg/L+ sucrose 30g/L+ agar 30g/L, Ph=5.8;
(4) take root
The propagation seedling that step (3) is cultivated divides an incision, and basal part of stem is cut flat, is inoculated in root media, cultivates 26 days; The prescription of described root media is: Read minimum medium+IAA0.5mg/L+ sucrose 30g/L+ agar 6.5g/L, Ph=5.8.
(5) hardening and transplanting
When the seedling for the treatment of step (4) grows 4～6 main roots, culturing bottle is moved in the outdoor booth, with transmittance be 70% sunshade hardening off the net, took out seedling in 3 days, flush away root substratum, be transplanted in the dish of cave, cave dish mesostroma is by leaf-humidified soil: the mixture that the volume ratio mixed of laterite: perlite=2:2:0.8 forms, the cave dish is positioned in the hut of growing seedlings, ceiling covering one deck transmittance is 60% sunshade net, threw off the sunshade net on the 8th day, keeping the mass water content of soil of matrix is 40～50 %;
The culture condition of above-mentioned steps (2)～step (4): culture temperature 22-25 ℃, light application time 12 h/d, intensity of illumination 1500-2000lx.
Handle 2: handle 2 except following measure difference, all the other measures are identical with processing 1, repeat no more:
(1) seed disinfection and processing
Fertile pod red bean seed is soaked 18min with 0.12% mercuric chloride solution, soak 12min with 2% chlorine bleach liquor again after, with clear water rinsing twice, put into 3MNaOH solution soaking 80min afterwards, with twice back taking-up of clear water rinsing, peel off kind of a skin again;
(2) seed germination
To be inoculated in the seed germination substratum through step (1) sterilization and the seed of handling, cultivate 30 days; The prescription of described seed germination substratum: Read minimum medium+6-BA1mg/L+NAA0.02 mg/L+ sucrose 30g/L+ agar 6.5 g/L, Ph=5.8;
(3) adventitious bud proliferation
The bud that step (2) seed germination is derived separates from bean cotyledon, and whole or the stem section that is cut into the 2cm～3cm of band joint are transferred on the proliferated culture medium, cultivate 40 days; The prescription of described proliferated culture medium is: Read minimum medium+6-BA1.5mg/L+KT0.07mg/L+ NAA0.08mg/L+ sucrose 30g/L+ agar 6.5 g/L, Ph=5.8;
(4) take root
The propagation seedling that step (3) is cultivated divides an incision, and basal part of stem is cut flat, is inoculated in root media, cultivates 31 days; The prescription of described root media is: Read minimum medium+IAA1.5mg/L+ sucrose 30g/L+ agar 6.5 g/L, Ph=5.8.
(5) hardening and transplanting
When the seedling for the treatment of step (4) grows 4～6 main roots, culturing bottle is moved in the outdoor booth, with transmittance be 70% sunshade hardening off the net, took out seedling in 5 days, flush away root substratum, be transplanted in the dish of cave, cave dish mesostroma is by leaf-humidified soil: the mixture that the volume ratio mixed of laterite: perlite=2.5:1.5:1 forms, the cave dish is positioned in the hut of growing seedlings, ceiling covering one deck transmittance is 60% sunshade net, threw off the sunshade net on the 8th day, keeping the mass water content of soil of matrix is 40～50%;
The culture condition of above-mentioned steps (2)～step (4): 22～25 ℃ of culture temperature, light application time 12 h/d, intensity of illumination 1500-2000lx.
Handle 3: handle 3 except following measure difference, all the other measures are identical with processing 1, repeat no more:
(1) seed disinfection and processing
Fertile pod red bean seed is soaked 15min with 0.12% mercuric chloride solution, soak 10min with 2% chlorine bleach liquor again after, use the clear water rinsing, put into 3MNaOH solution soaking 60min afterwards, with taking out after the clear water rinsing, peel off kind of a skin again;
(2) seed germination
To be inoculated in the seed germination substratum through step (1) sterilization and the seed of handling, cultivate 24 days; The prescription of described seed germination substratum: Read minimum medium+6-BA 0.5 mg/L+NAA 0.05 mg/L+ sucrose 30g/L+ agar 6.5 g/L, Ph=5.8;
(3) adventitious bud proliferation
The bud that step (2) seed germination is derived separates from bean cotyledon, and whole or the stem section that is cut into the 2cm～3cm of band joint are transferred on the proliferated culture medium, cultivate 40 days; The prescription of described proliferated culture medium is: Read minimum medium+6-BA 1 mg/L+KT 0.1 mg/L+NAA 0.1 mg/L+ sucrose 30g/L+ agar 6.5g/L, Ph=5.8;
(4) take root
The propagation seedling that step (3) is cultivated divides an incision, and basal part of stem is cut flat, is inoculated in root media, cultivates 24 days; The prescription of described root media: Read minimum medium+IAA 1.0 mg/L+ sucrose 30g/L+ agar 6.5 g/L, Ph=5.8.
(5) hardening and transplanting
When the seedling for the treatment of step (4) grows 4～6 main roots, culturing bottle is moved in the outdoor booth, with transmittance be 70% sunshade hardening off the net, took out seedling in 7 days, flush away root substratum, be transplanted in the dish of cave, cave dish mesostroma is by leaf-humidified soil: the mixture that the volume ratio mixed of laterite: perlite=3.5:1:0.5 forms, the cave dish is positioned in the hut of growing seedlings, ceiling covering one deck light rate is 60% sunshade net, threw off the sunshade net on the 8th day, keeping the mass water content of soil of matrix is 40～50%;
The culture condition of step (2)～step (4): culture temperature is 22-25 ℃, and light application time is 12 h/d, and intensity of illumination is 1500-2000lx.
The effect of handling 1-processing 3 sees Table 1.
Claims (3)
1. fertile pod red bean seed asepsis is induced the special culture media of plant regeneration, it is characterized in that, described special culture media is the seed germination substratum by following prescription, and proliferated culture medium and root media are formed:
(1) prescription of seed germination substratum is: Read minimum medium+6-BA 0.5 mg/L+NAA 0.05 mg/L+ sucrose 30g/L+ agar 6.5g/L, pH=5.8;
(2) prescription of proliferated culture medium is: Read minimum medium+6-BA 1 mg/L+KT 0.1 mg/L+NAA 0.1 mg/L+ sucrose 30g/L+ agar 6.5g/L, pH=5.8;
(3) prescription of root media is: Read minimum medium+IAA 1.0 mg/L+ sucrose 30g/L+ agar 6.5g/L, pH=5.8.
2. fertile pod red bean seed asepsis is induced the method for plant regeneration, it is characterized in that, carries out according to the following steps:
(1) seed disinfection and processing
Fertile pod red bean seed is soaked 12～18min with 0.12% mercuric chloride solution, soak 8～12min with 2% chlorine bleach liquor again after, use the clear water rinsing, put into 3MNaOH solution soaking 40～80min afterwards, with taking out after the clear water rinsing, peel off kind of a skin again;
(2) seed germination
To be inoculated in the seed germination substratum through step (1) sterilization and the seed of handling, cultivate 24～30 days; The prescription of described seed germination substratum is the prescription of claim 1 described (1) seed germination substratum;
(3) adventitious bud proliferation
The bud that step (2) seed germination is derived separates from bean cotyledon, and whole or the stem section that is cut into band joint are transferred on the proliferated culture medium, cultivate 40 days; The prescription of described proliferated culture medium is the prescription of claim 1 described (2) proliferated culture medium;
(4) take root
The propagation seedling that step (3) is cultivated divides an incision, is inoculated in root media, cultivates 24～31 days; The prescription of described root media is the prescription of claim 1 described (3) root media;
(5) hardening and transplanting
When the seedling for the treatment of step (4) grows 4～6 main roots, culturing bottle is moved in the outdoor booth, it is 70% sunshade hardening off the net in transmittance, took out seedling in 3～7 days, flush away root substratum, be transplanted in the dish of cave, cave dish mesostroma is by leaf-humidified soil: laterite: the mixture that the volume ratio mixed of perlite=2～3.5:1～2:0.5～1 forms, the cave dish is positioned in the hut of growing seedlings, ceiling covers the sunshade net of one deck transmittance 60%, threw off the sunshade net on the 8th day, keeping the mass water content of soil of matrix is 40-50%;
The culture condition of above-mentioned steps (2)～step (4): 22～25 ℃ of culture temperature, light application time 12 h/d, intensity of illumination 1500～2000lx.
3. fertile pod red bean seed asepsis according to claim 2 is induced the method for plant regeneration, it is characterized in that, carries out according to the following steps:
(1) seed disinfection and processing
Fertile pod red bean seed is soaked 15min with 0.12% mercuric chloride solution, soak 10min with 2% chlorine bleach liquor again after, use the clear water rinsing, put into 3MNaOH solution soaking 60min afterwards, with taking out after the clear water rinsing, peel off kind of a skin again;
(2) seed germination
To be inoculated in the seed germination substratum through step (1) sterilization and the seed of handling, cultivate 24 days; The prescription of described seed germination substratum is the prescription of claim 1 described (1) seed germination substratum;
(3) adventitious bud proliferation
The bud that step (2) seed germination is derived separates from bean cotyledon, and whole or the stem section that is cut into band joint are transferred on the proliferated culture medium, cultivate 40 days; The prescription of described proliferated culture medium is the prescription of claim 1 described (2) proliferated culture medium;
(4) take root
The propagation seedling that step (3) is cultivated divides an incision, and basal part of stem is cut flat, is inoculated in root media, cultivates 24 days; The prescription of described root media is the prescription of claim 1 described (3) root media;
(5) hardening and transplanting
When the seedling for the treatment of step (4) grows 4～6 main roots, culturing bottle is moved in the outdoor booth, it is 70% sunshade hardening off the net in transmittance, took out seedling in 7 days, flush away root substratum, be transplanted in the dish of cave, cave dish mesostroma is by leaf-humidified soil: the mixture that the volume ratio mixed of laterite: perlite=3.5:1:0.5 forms, the cave dish is positioned in the hut of growing seedlings, ceiling covering one deck transmittance is 60% sunshade net, threw off the sunshade net on the 8th day, keeping the mass water content of soil of matrix is 40～50%;
The culture condition of step (2)～step (4): culture temperature is 22-25 ℃, and light application time is 12 h/d, and intensity of illumination is 1500-2000lx.
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| CN103444529B (en) * | 2013-08-17 | 2015-04-01 | 福建农林大学 | Method for reproduction and mass propagation of lower axis fragment plants of ormosia hosiei.et wils |
| CN103688855B (en) * | 2013-12-11 | 2016-01-06 | 福建农林大学 | A kind of leaflet red bean isolated seed embryo and plant regeneration method |
| CN116548312A (en) * | 2023-06-25 | 2023-08-08 | 广西壮族自治区中国科学院广西植物研究所 | Red bean rapid propagation medium, application thereof and seedling rapid propagation method |
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Non-Patent Citations (3)
| Title |
|---|
| 李世承, 等.香花槐组织培养及快速无性繁殖.《辽宁大学学报（自然科学版）》.2002,第29卷(第2期),159-163. * |
| 杨丽等.豆科树种无性快速繁殖技术研究与进展.《世界林业研究》.2005,(第02期), |
| 豆科树种无性快速繁殖技术研究与进展;杨丽等;《世界林业研究》;20050415(第02期);全文 * |
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