CN110574683A - Tissue culture and rapid propagation method for Xinyou flower eucalyptus globulus - Google Patents
Tissue culture and rapid propagation method for Xinyou flower eucalyptus globulus Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/001—Culture apparatus for tissue culture
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
- A01H4/008—Methods for regeneration to complete plants
Abstract
The invention relates to the technical field of plant tissue culture, and discloses a tissue culture and rapid propagation method for a new excellent flower eucalyptus globulus, which comprises the steps of explant pretreatment, explant surface disinfection, bud induction, cluster adventitious bud obtaining, rooting culture, seedling hardening and transplanting. The method has the advantages of rapid axillary bud induction, high induction rate, large multiplication factor, thick and strong cluster buds, quick elongation and growth of adventitious buds, high rooting rate, developed root system, thick and strong rooted seedlings, high transplanting survival rate, strong nursery stock growth, low planting and management cost and the like, and provides a stable and efficient in-vitro plant regeneration method for the branch stem segments of the eucalyptus globulus.
Description
Technical Field
The invention relates to the technical field of plant tissue culture, in particular to a tissue culture and rapid propagation method of a new and excellent flower eucalyptus globulus.
background
eucalyptus globulus (Corymbia ptychocarpa subsp. aptycha), known as spring wood in English, also known as Eucalyptus rugosus, belongs to the genus Eucalyptus in the family Myrtaceae, Australia in the original country. The eucalyptus globulus is a medium arbor, the height of the eucalyptus globulus is 8-10m, the initial flowering phase of a sapling is 3-4 years, the flowering phase is 9 months to 3 months in the next year, and the flower color is white, pink, deep red and common pink. The stems of the flowers are provided with a plurality of torch-like single flowers, the diameters of the flowers can reach 7 cm, the flowers are located at the top ends of the lateral branches, and in the withering period, if the flowers are shaken without external force, the petals of the dry flowers are withered slowly, the colors of the flowers are not different from the colors of the fresh flowers, the blooming period is longer, and a very spectacular full-bloom period is formed.
Eucalyptus is cultivated as a main timber forest in China, and the research and development of the gardening ornamental value are less; in foreign countries, eucalyptus has become an important species of flower, garden and landscape trees. In recent years, eucalyptus globulus is successfully introduced and domesticated in China, and begins to be applied as a garden ornamental tree species in southern areas. Although the existing enterprises find the potential market demand of the eucalyptus globulus labill in the development of flower, gardening landscape and fresh cut flower ornamental industry, the ripeness of the seeds of the eucalyptus globulus labill needs more than 10 months, the fruit is similar to a cone, the shell is hard, the height is 2-3 cm, the diameter is 1-2 cm, and the seeds contain 8-10 grains; the seed has more empty and shriveled rate and low germination rate. The seeds of the eucalyptus globulus are imported all the time in China, no seed breeding base exists in China, and the cost of the imported seeds is high. Therefore, the method for cultivating new excellent plant seedlings by the stem-branch vegetative propagation method has important significance.
At present, only 2 documents reporting about the research related to the eucalyptus globulus arcus Roxb are reported at home and abroad, the theoretical basis is deficient, the market of the eucalyptus globulus arcus Roxb seedlings is in short supply, and the popularization and the utilization of the eucalyptus globulus arcus Roxb as a garden ornamental tree and the development process of the fresh cut flower industry are retarded. The regeneration system of the eucalyptus globulus is established by 2 compositions of problems such as the Korean and the Yangqi, but has the following problems: the subculture multiplication material is long in internodes, the average internode length of different clones is 1.6-2.4 cm, and the phenomena that stems of the multiplication material are easy to die and seedlings are weak in the process of inducing rooting are easily caused; secondly, callus is generated at the stem base part in the rooting process, and the later transplanting survival rate is greatly influenced; and the rooting time is long and is as long as about 7 weeks. Therefore, the research uses the stem section of the eucalyptus globulus as an explant to carry out tissue culture rapid propagation technical research and establish a high-efficiency tissue culture rapid propagation technical system, thereby achieving the purposes of short induction period, strong and strong propagation bud, fast growth, high increment rate, high rooting rate, strong bottle seedling and high transplanting survival rate. Provides an effective technical approach for the industrial production and the cultivation of the tissue culture seedlings of the eucalyptus globulus, and has important theoretical and application values.
Disclosure of Invention
Based on the problems, the invention provides a tissue culture and rapid propagation method for the new excellent flower eucalyptus globulus, so that the regeneration system of the eucalyptus globulus has the characteristics of high axillary bud induction rate, rapid propagation, high propagation multiple, rapid growth of adventitious buds, high rooting rate, robust bottle seedlings, high transplanting survival rate and the like, and a new method is provided for tissue culture and industrial seedling culture of the new excellent flower eucalyptus globulus.
In order to solve the technical problems, the invention provides a tissue culture and rapid propagation method of a new excellent flower eucalyptus globulus, which comprises the following steps:
C1: explant pretreatment
Cutting out branches of perennial eucalyptus globulus mother trees, collecting semi-lignified new branches after the sprouts of the cut-out parts of the new branches grow for 45 days, and cutting a stem section which is 5cm long and is provided with a bud and is positioned below the stem tip of the new branches for later use as an explant;
C2: explant surface disinfection
Washing the explant in the step C1 with tap water for 15min, sequentially soaking in sterile water for 1min, disinfecting with a 10% sodium hypochlorite solution for 5min, washing with sterile water for 3-4 times in a sterile super clean bench, soaking in a 0.1% mercuric chloride solution for 4-5 min, and washing with sterile water for 4-5 times to obtain a disinfected explant;
C3: bud induction
C2, inoculating the explant treated in the step C into a bud induction culture medium for culturing for 15-20 days to obtain a bud-induced explant;
C4: obtaining cluster adventitious buds
c3, cutting a single sterile axillary bud with the length of more than 2cm on the explant treated in the step C3, cutting the single sterile axillary bud into axillary bud stem sections with the length of 0.5-1 cm, transferring the axillary bud stem sections to a multiple bud induction culture medium for culturing for 25-30 days to obtain a multiple bud body with the volume of not less than 1cm3, cutting the multiple bud body into small bud blocks with the volume of 0.5cm3, transferring the small bud blocks to a multiple adventitious bud proliferation and differentiation culture medium for culturing for 25-30 days to obtain multiple adventitious buds with the tender stem length of more than 2cm, and repeatedly circulating for 5 generations to obtain a large number of multiple adventitious buds;
C5: rooting culture
C4, cutting tender stems with single bud height not less than 3cm from the clustered adventitious buds, transferring the tender stems into a rooting seedling culture medium for culture, and culturing for 20-30 days under the conditions of natural illumination and constant temperature of 28-32 ℃ to induce the adventitious buds to root so as to obtain rooting seedlings;
C6: hardening and transplanting seedlings
And D, transferring the rooted seedlings obtained in the step C5 to an outdoor greenhouse for culturing for 10 days to obtain tissue culture seedlings, transferring the tissue culture seedlings to a tissue culture bottle for hardening seedlings, unscrewing a bottle cover of the tissue culture bottle for culturing the tissue culture seedlings, hardening the seedlings for 1 day, adding a small amount of tap water into the tissue culture bottle, lifting the cover of the tissue culture bottle for hardening the seedlings for 2 days, taking the tissue culture seedlings out of the tissue culture bottle, transplanting the tissue culture seedlings to a red soil matrix, transplanting while watering, counting the survival rate of each culture matrix seedling after transplanting for 20 days, and then reducing the shading degree and managing according to a conventional seedling management mode.
Further, the bud induction culture medium in the step C3 is an improved MS culture medium, and specifically, 0.5mg/L of 6-BA, 20-30 g/L of sucrose and 8-9 g/L of carrageenan are added in the MS culture medium.
Further, the multiple shoot induction medium in step C4 is modified MS medium, specifically, the MS medium is supplemented with 3 mg/L6-BA and 0.5 mg/LZT.
Further, the adventitious bud proliferation and differentiation culture medium in the step C4 is an improved MS culture medium, and specifically, 0.2-0.8 mg/L6-BA, 0.02-0.06 mg/L IBA, 0-0.03 mg/LZT, 20-30 g/L sucrose, 6-8 g/L carrageenan, 0-0.03 mg/LNAA and 2% fresh coconut water are added in the MS culture medium.
Furthermore, in the adventitious bud proliferation and differentiation medium in the step C4, the additional 6-BA, IBA and ZT are 0.8mg/L, 0.04mg/L and 0.03mg/L, respectively.
Further, the rooting seedling culture medium in the step C5 is an improved MS culture medium, and specifically, 0.2-0.5 mg/L IBA, 0.1-0.2 mg/L LNAA, 15-20 g/L sucrose, 6-8 g/L carrageenan and 6ml/L EDTA-Fe mother liquor which is 200 times of EDTA are added in the MS culture medium.
Further, the IBA and the NAA in the rooting seedling culture medium in the step C5 are 0.5mg/L and 0.2mg/L respectively, and meanwhile, 6-10 mL/L of 200 times of iron salt mother liquor is added in the rooting seedling culture medium in the step C5.
Further, the MS culture medium comprises the following components: 1900mg/LKNO3, 1650mg/LNH4NO3, 332mg/L CaCl 2.2H2O, 370mg/L MgSO 4.7H2O, 170mg/L KH2PO4, 22.3mg/L MnSO 4.4H2O, 8.6mg/LZnSO 4.7H2O, 6.2mg/L H3BO3, 0.83mg/L KI, 0.25mg/L NaMoO 4.2H2O, 0.025mg/LCuSO 4.5H2O, 0.025mg/L CoCl 2.6H2O, 2.78mg/L FeSO 4.7H2O, 37.3mg/LNa 2. EDTA.H2O, 100mg/L Myo-instine, 2 mg/LNiacic/L, 0.1.7H 2-675, Pyzocine/L HCl, 0.8mg/L Ph-5H 2-Na-5-HCl.
Further, when the culture is carried out in the red soil matrix in the step C6, 2 layers of arch-shaped sunshade nets are arranged above the red soil matrix seedbed, the culture process is managed in a two-end ventilation mode, and watering is carried out in three periods of nine o ' clock in the morning, three o ' clock in the afternoon and six o ' clock in the evening.
Compared with the prior art, the invention has the beneficial effects that: the method has the advantages of rapid axillary bud induction, high induction rate, large multiplication factor, thick and strong cluster buds, quick elongation and growth of adventitious buds, high rooting rate, developed root system, thick and strong rooted seedlings, high transplanting survival rate, strong nursery stock growth, low planting and management cost and the like, and provides a stable and efficient in-vitro plant regeneration method for the branch stem segments of the eucalyptus globulus.
Detailed Description
in order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to examples, and the exemplary embodiments and descriptions thereof are only used for explaining the present invention and are not used as limitations of the present invention.
Example (b):
a tissue culture and rapid propagation method of a Xinyou flower eucalyptus globulus comprises the following steps:
c1: explant pretreatment
Cutting out branches of perennial eucalyptus globulus mother trees, collecting semi-lignified new branches after the sprouts of the cut-out parts of the new branches grow for 45 days, and cutting a stem section which is 5cm long and is provided with a bud and is positioned below the stem tip of the new branches for later use as an explant;
c2: explant surface disinfection
Washing the explant in the step C1 with tap water for 15min, sequentially soaking in sterile water for 1min, disinfecting with a 10% sodium hypochlorite solution for 5min, washing with sterile water for 3-4 times in a sterile super clean bench, soaking in a 0.1% mercuric chloride solution for 4-5 min, and washing with sterile water for 4-5 times to obtain a disinfected explant;
C3: bud induction
C2, inoculating the explant treated in the step C into a bud induction culture medium for culturing for 15-20 days to obtain the explant after bud induction, wherein the bud induction culture medium is an improved MS culture medium, and specifically, 0.5 mg/L6-BA, 20-30 g/L sucrose and 8-9 g/L carrageenan are added in the MS culture medium to improve the budding coefficient of the explant, the axillary bud induction rate reaches 97.1%, the axillary bud induction time is shortened, and the multi-bud induction progress is accelerated;
c4: obtaining cluster adventitious buds
c3, cutting a single sterile axillary bud with the length of more than 2cm on the explant treated in the step C3, cutting the single sterile axillary bud into axillary bud stem segments with the length of 0.5-1 cm, transferring the axillary bud stem segments to a multi-bud induction culture medium, culturing for 25-30 days, and obtaining the axillary bud stem segments with the volume of not less than 1cm3Cutting the multi-bud into pieces with a volume of 0.5cm3Transferring the small bud block into a cluster adventitious bud proliferation differentiation culture medium for cultureObtaining cluster adventitious buds with tender stem length more than 2cm after 25-30 days, and repeatedly circulating for 5 generations to obtain a large amount of cluster adventitious buds; the multi-bud induction culture medium is an improved MS culture medium, and particularly 3 mg/L6-BA and 0.5mg/LZT are added in the MS culture medium, so that a large number of adventitious buds can be obtained in a short time under the condition of limited explant materials due to the formation of multi-buds, the adventitious bud induction period is shortened, the adventitious bud induction progress of cluster propagation is accelerated, and the factory seedling raising efficiency is improved; the clustered adventitious bud propagation and differentiation culture medium is an improved MS culture medium, and specifically comprises 0.2-0.8 mg/L of 6-BA, 0.02-0.06 mg/L of IBA, 0-0.03 mg/LZT, 20-30 g/L of sucrose, 6-8 g/L of carrageenan, 0-0.03 mg/LNAA and 2% of fresh coconut water, wherein the 6-BA added to the clustered adventitious bud propagation and differentiation culture medium in the embodiment is 0.8mg/L, the IBA is 0.04mg/L and the ZT is 0.03mg/L, and the clustered adventitious bud culture medium can grow sprouts, can improve the propagation coefficient and is beneficial to seedling strengthening, further obtain a large number of clustered adventitious buds, accelerate the rooting culture progress and further shorten the seedling culture period;
c5: rooting culture
C4, cutting tender stems with single bud height not less than 3cm from the clustered adventitious buds, transferring the tender stems into a rooting seedling culture medium for culture, and culturing for 20-30 days under the conditions of natural illumination and constant temperature of 28-32 ℃ to induce the adventitious buds to root so as to obtain rooting seedlings; the rooting seedling culture medium is an improved MS culture medium, and specifically comprises 0.2-0.5 mg/L IBA, 0.1-0.2 mg/L LNAA, 15-20 g/L sucrose, 6-8 g/L carrageenan and 6 ml/L200-time EDTA-Fe mother liquor which are added into the MS culture medium; in the rooting seedling culture medium in the embodiment, IBA is 0.5mg/L, NAA is 0.2mg/L, and meanwhile, 200 times of 6-10 mL/L of ferric salt mother liquor is added into the rooting seedling culture medium, the rooting rate of the rooting culture medium is more than 95%, the average number of roots is 6 per plant, the yellowing and necrosis rate of the rooting seedling is very low, the seedling shows robust growth, thick stem, dark green leaves and high leaf expansion rate;
c6: hardening and transplanting seedlings
C5, transferring the rooted seedlings obtained in the step C5 to an outdoor greenhouse for culturing for 10 days to obtain tissue culture seedlings, transferring the tissue culture seedlings to a tissue culture bottle for hardening the seedlings, unscrewing a bottle cover of the tissue culture bottle for culturing the tissue culture seedlings, hardening the seedlings for 1 day, adding a small amount of tap water into the tissue culture bottle, lifting the cover of the tissue culture bottle for hardening the seedlings for 2 days, taking the tissue culture seedlings out of the tissue culture bottle, transplanting the tissue culture seedlings to a red soil matrix, transplanting while watering, counting the survival rate of each culture matrix seedling after transplanting for 20 days, and then reducing the shading degree and managing according to a conventional seedling management mode; when the cultivation is carried out in the red soil matrix, 2 layers of arched sunshade nets are built above the red soil matrix seedbed, the cultivation process is managed in a two-end ventilation mode, and watering is carried out at nine morning hours, three afternoon hours and six evening hours.
the composition of the MS medium in this example is as follows: 1900mg/L KNO3, 1650mg/L LNH4NO3, 332mg/L CaCl 2.2H2O, 370mg/L MgSO 4.7H2O, 170mg/L KH2PO4, 22.3mg/L MnSO 4.4H2O, 8.6mg/LZnSO 4.7H2O, 6.2mg/L H3BO3, 0.83mg/L KI, 0.25mg/L NaMoO 4.2H2O, 0.025mg/L CuSO 4.5H2O, 0.025mg/L CoCl 2.6H2O, 2.78mg/L FeSO 4.7H2O, 37.3 mg/L2. EDTA.H2O, 100mg/L Myo-instiol, 2mg/L LNiacic.1.10mg/L, PyXingine, Pyzigine, Pyzrinx HCl/L5.
The invention provides a stable and efficient regeneration method of branch and stem in-vitro plants of eucalyptus globulus, and the cultured seedlings of eucalyptus globulus are suitable for large-scale production, can culture strong eucalyptus globulus with expanded leaves, save the import cost of eucalyptus globulus seeds, and play an important role in improving the ornamental value of eucalyptus globulus.
the above is an embodiment of the present invention. The embodiments and specific parameters in the embodiments are only for the purpose of clearly illustrating the process of verifying the invention and are not intended to limit the scope of the invention, which is defined by the claims, and all the equivalent structural changes made by applying the content of the specification of the invention should be covered by the scope of the invention.
Claims (9)
1. A tissue culture and rapid propagation method of a Xinyou flower eucalyptus globulus is characterized by comprising the following steps:
C1: explant pretreatment
Cutting out branches of perennial eucalyptus globulus mother trees, collecting semi-lignified new branches after the sprouts of the cut-out parts of the new branches grow for 45 days, and cutting a stem section which is 5cm long and is provided with a bud and is positioned below the stem tip of the new branches for later use as an explant;
C2: explant surface disinfection
washing the explant in the step C1 with tap water for 15min, sequentially soaking in sterile water for 1min, disinfecting with a 10% sodium hypochlorite solution for 5min, washing with sterile water for 3-4 times in a sterile super clean bench, soaking in a 0.1% mercuric chloride solution for 4-5 min, and washing with sterile water for 4-5 times to obtain a disinfected explant;
c3: bud induction
C2, inoculating the explant treated in the step C into a bud induction culture medium for culturing for 15-20 days to obtain a bud-induced explant;
C4: obtaining cluster adventitious buds
c3, cutting a single sterile axillary bud with the length of more than 2cm on the explant treated in the step C3, cutting the single sterile axillary bud into axillary bud stem segments with the length of 0.5-1 cm, transferring the axillary bud stem segments to a multi-bud induction culture medium, culturing for 25-30 days, and obtaining the axillary bud stem segments with the volume of not less than 1cm3Cutting the multi-bud into pieces with a volume of 0.5cm3transferring the small bud blocks into a cluster adventitious bud proliferation and differentiation culture medium, culturing for 25-30 days to obtain cluster adventitious buds with tender stem length larger than 2cm, and repeatedly circulating for 5 generations to obtain a large amount of cluster adventitious buds;
C5: rooting culture
C4, cutting tender stems with single bud height not less than 3cm from the clustered adventitious buds, transferring the tender stems into a rooting seedling culture medium for culture, and culturing for 20-30 days under the conditions of natural illumination and constant temperature of 28-32 ℃ to induce the adventitious buds to root so as to obtain rooting seedlings;
C6: hardening and transplanting seedlings
And D, transferring the rooted seedlings obtained in the step C5 to an outdoor greenhouse for culturing for 10 days to obtain tissue culture seedlings, transferring the tissue culture seedlings to a tissue culture bottle for hardening seedlings, unscrewing a bottle cover of the tissue culture bottle for culturing the tissue culture seedlings, hardening the seedlings for 1 day, adding a small amount of tap water into the tissue culture bottle, lifting the cover of the tissue culture bottle for hardening the seedlings for 2 days, taking the tissue culture seedlings out of the tissue culture bottle, transplanting the tissue culture seedlings to a red soil matrix, transplanting while watering, counting the survival rate of each culture matrix seedling after transplanting for 20 days, and then reducing the shading degree and managing according to a conventional seedling management mode.
2. The tissue culture and rapid propagation method of the Xinyou flower Eucalyptus globulus Labill according to claim 1, wherein the bud induction culture medium in the step C3 is an improved MS culture medium, and specifically, 0.5 mg/L6-BA, 20-30 g/L sucrose and 8-9 g/L carrageenan are added in the MS culture medium.
3. The tissue culture and rapid propagation method of the Xinyou flower Eucalyptus globulus Labill according to claim 1, wherein the multiple shoot induction culture medium in step C4 is an improved MS culture medium, specifically, the MS culture medium is added with 3 mg/L6-BA and 0.5mg/L ZT.
4. The tissue culture and rapid propagation method of the Xinyou flower Eucalyptus globulus Labill, according to claim 1, characterized in that the propagation and differentiation medium of the clumpy adventitious buds in the step C4 is an improved MS medium, specifically, the MS medium is added with 0.2-0.8 mg/L6-BA, 0.02-0.06 mg/L IBA, 0-0.03 mg/L ZT, 20-30 g/L sucrose, 6-8 g/L carrageenan, 0-0.03 mg/L LNAA and 2% fresh coconut water.
5. the tissue culture and rapid propagation method of the Xinyou flower Eucalyptus globulus Labill, according to claim 4, wherein the additional 6-BA in the propagation and differentiation medium of the clumpy adventitious buds in the step C4 is 0.8mg/L, IBA is 0.04mg/L, and ZT is 0.03 mg/L.
6. The tissue culture and rapid propagation method of the Xinyou flower eucalyptus globulus, according to claim 1, characterized in that the culture medium of the rooted seedling in step C5 is an improved MS culture medium, and specifically, the MS culture medium is added with 0.2-0.5 mg/L IBA, 0.1-0.2 mg/L NAA, 15-20 g/L sucrose, 6-8 g/L carrageenan and 6ml/L EDTA-Fe mother liquor 200 times.
7. the tissue culture and rapid propagation method of the Xinyou flower Eucalyptus globulus Labill, according to claim 6, characterized in that IBA in the rooting seedling culture medium in the step C5 is 0.5mg/L, NAA is 0.2mg/L, and 6-10 mL/L of 200 times of mother solution of iron salt is added in the rooting seedling culture medium in the step C5.
8. The tissue culture and rapid propagation method of the Xinyou flower Eucalyptus globulus Labill according to any one of claims 2-4 or 6, characterized in that the MS culture medium comprises the following components: 1900mg/L KNO3，1650mg/L NH4NO3，332mg/L CaCl2·2H2O，370mg/L MgSO4·7H2O，170mg/L KH2PO4，22.3mg/L MnSO4·4H2O，8.6mg/L ZnSO4·7H2O，6.2mg/L H3BO3，0.83mg/L KI，0.25mg/L NaMoO4·2H2O，0.025mg/L CuSO4·5H2O，0.025mg/L CoCl2·6H2O，2.78mg/L FeSO4·7H2O，37.3mg/L Na2·EDTA·H2O, 100mg/L Myo-Insitol, 2mg/L Glycine, 0.1mg/L Thiamine HCl, 0.5mg/L Nicotinic acid, 0.5mg/L Pyridoxine HCl, and the Ph of MS medium is 5.8.
9. The tissue culture and rapid propagation method of the Xinyou flower Eucalyptus globulus Labill, according to claim 1, characterized in that, during the culture in the red soil matrix in step C6, 2 layers of arch-shaped sunshade nets are built above the red soil matrix seedbed, the management is carried out in a two-head ventilation mode during the culture process, and watering is carried out at nine o ' clock in the morning, three o ' clock in the afternoon and six o ' clock in the evening.
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Application publication date: 20191217 |