CN114831027A - Dendrobium devonianum tissue culture rapid propagation culture medium combination and application thereof in dendrobium devonianum seedling cultivation - Google Patents
Dendrobium devonianum tissue culture rapid propagation culture medium combination and application thereof in dendrobium devonianum seedling cultivation 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
- A01H4/002—Culture media 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
- A01H4/005—Methods for micropropagation; Vegetative plant propagation using cell or tissue culture techniques
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P60/00—Technologies relating to agriculture, livestock or agroalimentary industries
- Y02P60/20—Reduction of greenhouse gas [GHG] emissions in agriculture, e.g. CO2
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Abstract
The invention belongs to the technical field of plant tissue culture, and particularly relates to a dendrobium candidum tissue culture rapid propagation culture medium combination and application thereof in dendrobium candidum seedling culture. The culture medium combination for the rapid tissue culture of the dendrobium candidum comprises an initial generation induction culture medium, a secondary generation proliferation and differentiation culture medium and a strong seedling rooting culture medium, and a large number of excellent seedlings of the dendrobium candidum can be obtained in a short time by adopting the culture medium combination, so that a foundation is laid for the large-scale planting of the dendrobium candidum.
Description
Technical Field
The invention belongs to the technical field of plant tissue culture, and particularly relates to a dendrobium candidum tissue culture rapid propagation culture medium combination and application thereof in dendrobium candidum seedling culture.
Background
Dendrobium candidum (Dendrobium crystallinum Rchb. F. in Gard. chron.) is a perennial herb of the Orchidaceae (Orchidaceae) of the genus Dendrobium (Dendrobium), and grows on loose bark, trunk or in stone cracks of warm and humid tropical and subtropical deep mountain old forests with elevation of 540-1700 meters. The stem of the dendrobium candidum is upright or inclined, is slightly fleshy and is cylindrical; the flower has beautiful shape, fragrant smell, bright color and high ornamental value, and is known as one of four ornamental flowers. The dendrobium candidum is used as a dendrobium medicinal plant, has the main chemical components similar to those of dendrobium candidum, and has the effects of nourishing yin, promoting the production of body fluid, moistening lung, improving eyesight, resisting cancer, preventing aging and the like. In recent years, due to the discovery of ornamental and medicinal values, the wild resource of the dendrobium candidum is reduced rapidly.
The dendrobium huoshanense has higher commodity development value, but the conventional seedling cultivation method of dendrobium huoshanense consumes long time, has lower multiplication coefficient and has different seedling growth states. And the screening of excellent clone and the rapid propagation of the seedlings thereof are the basis for intensive planting and industrialized development of the dendrobium huoshanense and are also effective modes for species protection and sustainable health development of the dendrobium huoshanense, but the complete and efficient rapid propagation technology of the seedlings of the dendrobium huoshanense is not reported at present.
Disclosure of Invention
The invention aims to provide a dendrobium candidum tissue culture rapid propagation culture medium combination and application thereof in cultivating dendrobium candidum seedlings.
The invention provides a dendrobium huoshanense tissue culture rapid propagation culture medium combination which comprises an initial generation induction culture medium, a successive generation proliferation and differentiation culture medium and a strong seedling rooting culture medium;
the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 0.5-3.0 mg.L of 6-BA -1 、NAA 0.1-1.0mg·L -1 0.1-0.2 wt.% of active carbon, 5-10 wt.% of banana puree and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.6-6.0;
the subculture multiplication and differentiation culture medium is based on MSThe culture medium also comprises 0.5-3.0 mg.L of 6-BA -1 、NAA 0.1-1.0mg·L -1 0.1-0.2 wt.% of active carbon, 5-10 wt.% of banana puree and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.6-6.0;
the strong seedling rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of NAA -1 、ZnSO 4 0.1-1.0mg·L -1 0.1-0.2 wt% of active carbon, 5-10 wt% of banana puree and 15-25 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.6-6.0.
Preferably, the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 1.0-1.5 mg.L of 6-BA -1 、NAA 0.1-0.5mg·L -1 0.1-0.15 wt.% of active carbon, 8-10 wt.% of banana puree and 20-25 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.8-6.0;
the subculture proliferation and differentiation culture medium takes MS as a basic culture medium and also comprises 1.0-2.0 mg.L of 6-BA -1 、NAA 0.3-0.5mg·L -1 0.1-0.15 wt.% of active carbon, 8-10 wt.% of banana puree and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.8-6.0;
the strong seedling rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.5-0.8 mg.L of NAA -1 、ZnSO 4 0.1-0.5mg·L -1 0.1-0.15 wt.% of active carbon, 8-10 wt.% of banana puree and 15-25 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.8-6.0.
Preferably, the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 1.0 mg.L of 6-BA -1 、NAA 0.2mg·L -1 0.1 wt.% of active carbon, 10 wt.% of banana puree and 20 g.L of sucrose -1 And agar 3.4 g.L -1 The pH is 5.8;
the subculture proliferation and differentiation culture medium takes MS as a basic culture medium and also comprises 1.6 mg.L of 6-BA -1 、NAA 0.4mg·L -1 0.1 wt.% of active carbon, 10 wt.% of banana puree and 30 g.L of sucrose -1 And agar 3.4 g.L -1 The pH is 5.8;
the strong seedling rooting culture medium takes 1/2MS as a basic culture medium and also comprisesNAA 0.5mg·L -1 、ZnSO 4 0.2 mg·L -1 0.1 wt.% of active carbon, 10 wt.% of banana puree and 20 g.L of sucrose -1 And agar 3.5 g.L -1 The pH was 5.8.
The invention also provides application of the dendrobium candidum tissue culture rapid propagation culture medium combination in the technical scheme in dendrobium candidum tissue culture.
The invention also provides a method for cultivating the dendrobium candidum seedlings, and the dendrobium candidum tissue culture rapid propagation culture medium combination adopting the technical scheme is adopted to cultivate the dendrobium candidum to obtain the clonal seedlings of the dendrobium candidum.
Preferably, the method comprises the following steps: inoculating dendrobium candidum seeds into a primary induction culture medium, and performing dark culture for 5-10 days and first illumination culture for 20-50 days in sequence to obtain protocorms;
transferring the protocorm into a secondary proliferation and differentiation culture medium for second illumination culture for 40-60 days to obtain a cluster seedling;
transferring the cluster seedling into a strong seedling rooting culture medium, and performing third illumination culture for 30-50 days to obtain a rooted seedling;
and (3) hardening the rooted seedlings to obtain the clonal seedlings of the dendrobium huoshanense.
Preferably, the temperature of the first illumination culture is 28 +/-3 ℃, and the illumination time is 10-15 h.d -1 The illumination intensity is 35 to 45 [ mu ] mol.m -2 ·s -1 。
Preferably, the temperature of the second illumination culture is 28 +/-3 ℃, and the illumination time is 10-15 h.d -1 The illumination intensity is 35 to 45 [ mu ] mol.m -2 ·s -1 。
Preferably, the temperature of the third light culture is 28 +/-3 ℃, and the light time is 10-15 h.d -1 The illumination intensity is 35 to 45 [ mu ] mol/m -2 ·s -1 。
Preferably, the dendrobium candidum seeds are seeds obtained from disinfected plump uncracked dendrobium candidum capsules.
Has the advantages that:
the invention provides a dendrobium candidum tissue culture and rapid propagation culture medium groupThe method comprises a primary generation induction culture medium, a secondary generation proliferation and differentiation culture medium and a strong seedling rooting culture medium, wherein: the formula of the primary induction culture medium is as follows: 1/2MS +6-BA 0.5-3.0 mg.L-1 + NAA 0.1-1.0 mg.L -1 + 0.1-0.2 wt.% of activated carbon + 5-10 wt.% of banana puree + 20-30 g.l of sucrose -1 + agar 3-4 g. L -1 pH5.6-6.0; the formula of the subculture proliferation and differentiation medium is as follows: MS +6-BA 0.5-3.0 mg.L -1 +NAA 0.1-1.0mg·L -1 + 0.1-0.2 wt.% of activated carbon + 5-10 wt.% of banana puree + 20-30 g.l of sucrose -1 + agar 3-4 g. L -1 pH 5.6-6.0; the formula of the strong seedling rooting culture medium is as follows: 1/2MS + NAA 0.1-1.0 mg.L -1 +ZnSO 4 0.1-1.0mg·L -1 + 0.1-0.2 wt.% of activated carbon + 5-10 wt.% of banana puree + 15-25 g.l of sucrose -1 + agar 3-4 g. L -1 And the pH value is 5.6-6.0, a large number of excellent seedlings of the dendrobium candidum can be obtained in a short time by adopting the culture medium combination, and a foundation is laid for large-scale planting of the dendrobium candidum.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the embodiments will be briefly described below.
FIG. 1 shows germination and protocorm induction of Dendrobium devonianum seeds;
FIG. 2 shows the subculture proliferation and differentiation culture of protocorm of Dendrobium devonianum;
FIG. 3 shows the strong seedling and rooting culture of Dendrobium devonianum.
In the specific embodiment of the invention, the "%" in the culture medium composition for tissue culture and rapid propagation of dendrobium represents mass percentage.
Detailed Description
The invention provides a dendrobium candidum tissue culture rapid propagation culture medium combination, which comprises a primary generation induction culture medium, a secondary generation proliferation and differentiation culture medium and a strong seedling rooting culture medium; the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 0.5-3.0 mg.L of 6-BA -1 、NAA 0.1-1.0mg·L -1 0.1-0.2% of active carbon, 5-10% of banana puree and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.6-6.0; the subculture proliferation and differentiation cultureThe culture medium takes MS as a basic culture medium and also comprises 0.5-3.0 mg.L of 6-BA -1 、NAA 0.1-1.0mg·L -1 0.1-0.2% of active carbon, 5-10% of banana puree and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 The strong seedling rooting culture medium with pH of 5.6-6.0 takes 1/2MS as a basal culture medium and also comprises NAA 0.1-1.0 mg.L -1 、ZnSO 4 0.1-1.0mg·L -1 0.1-0.2% of active carbon, 5-10% of banana puree and 15-25 g.L of sucrose -1 And agar 3-4 g.L -1 The pH value is 5.6-6.0.
The formulation of the primary induction culture medium preferably takes 1/2MS as a basal culture medium, and 6-BA in the primary induction culture medium is preferably 1.0-1.5 mg.L -1 More preferably 1.0 mg.L -1 (ii) a The NAA is preferably 0.1-0.5 mg.L -1 More preferably 0.2 mg.L -1 (ii) a The activated carbon is preferably 0.1 to 0.15%, more preferably 0.1%; the banana puree is preferably 8-10%, more preferably 10%; the sucrose is 20-25 g.L -1 More preferably 20 g.L -1 (ii) a The agar is preferably 3-4 g.L -1 More preferably 3.4 g.L -1 . The pH of the primary induction medium of the present invention is preferably 5.8 to 6.0, more preferably 5.8.
The formula of the secondary proliferation and differentiation culture medium is preferably MS as a basal culture medium, and the 6-BA in the secondary proliferation and differentiation culture medium is preferably 1.0-2.0 mg.L -1 More preferably 1.6 mg.L -1 (ii) a The NAA is preferably 0.3-0.5 mg.L -1 More preferably 0.4 mg.L -1 (ii) a The activated carbon is preferably 0.1 to 0.15%, more preferably 0.1%; the banana puree is preferably 8-10%, more preferably 10%; the sucrose is preferably 20-30 g.L -1 More preferably 30 g.L -1 (ii) a The agar is preferably 3-4 g.L -1 More preferably 3.4 g.L -1 . The pH of the secondary proliferation induction medium of the present invention is preferably 5.8 to 6.0, more preferably 5.8.
The formula of the strong seedling rooting culture medium is preferably 1/2MS as a basic culture medium, and the NAA in the strong seedling rooting culture medium is preferably 0.5-0.8 mg.L -1 More preferably 0.5 mg.L -1 (ii) a The ZnSO 4 Preference is given toIs 0.1-0.5 mg.L -1 More preferably 0.2 mg.L -1 (ii) a The activated carbon is preferably 0.1 to 0.15%, more preferably 0.1%; the banana puree is preferably 8-10%, more preferably 10%; the sucrose is preferably 15-25 g.L -1 More preferably 20 g.L -1 (ii) a The agar is preferably 3-4 g.L -1 More preferably 3.5 g.L -1 . The pH value of the strong seedling rooting culture medium is preferably 5.8-6.0, and more preferably 5.8. In the present invention, the ZnSO is present at an appropriate concentration 4 Can promote the growth and rooting of plants and inhibit the formation of calluses or protocorms; the banana puree contains a certain amount of hormone, amino acid and vitamin, and can promote germination of dendrobium seeds and growth of protocorm.
The invention also provides application of the dendrobium candidum tissue culture rapid propagation culture medium combination in the technical scheme in dendrobium candidum tissue culture. The culture medium combination for the rapid tissue culture of the dendrobium huoshanense comprises culture mediums of three growth stages of primary generation induction, protocorm subculture proliferation differentiation and strong seedling rooting, and the culture mediums of all stages are reasonably combined and matched to obtain a large number of excellent seedlings in a short time, so that a foundation is laid for large-scale planting of the dendrobium huoshanense.
The invention also provides a method for cultivating the dendrobium candidum seedlings, and the dendrobium candidum tissue culture rapid propagation culture medium combination adopting the technical scheme is adopted to cultivate the dendrobium candidum to obtain the clonal seedlings of the dendrobium candidum.
According to the invention, the dendrobium huoshanense seeds are preferably inoculated in a primary induction culture medium, and are cultured in a dark environment for 5-10 days and in a first illumination environment for 20-50 days in sequence to obtain protocorms. The dendrobium huoshanense seeds are preferably seeds obtained from disinfected plump uncracked dendrobium huoshanense capsules. The plump uncracked dendrobium candidum capsule is preferably a healthy capsule after pollination for 150 days.
The method for disinfecting the dendrobium candidum capsule preferably comprises the following steps: soaking the dendrobium candidum capsules in laundry powder water with the mass concentration of 0.2%, brushing the soaked dendrobium candidum capsules with a soft brush pen or absorbent cotton, and washing to obtain clean dendrobium candidum capsules; in a volume concentration of75% alcohol solution and HgCl with mass concentration of 0.1% 2 And (3) sequentially carrying out disinfection treatment, continuous oscillation and water washing on the clean dendrobium capilalum capsules to obtain the disinfected dendrobium capilalum capsules. The soaking time is preferably 5-30 min, and more preferably 10 min. The present invention preferably uses running water for the water washing. The disinfection treatment is preferably carried out in an ultra-clean workbench, and the treatment time of the alcohol solution is preferably 30-60 s, and more preferably 60 s; the HgCl 2 The treatment time of (3) is preferably 5 to 10min, more preferably 8 min. The invention preferably adopts sterile water for the rewashing, and the number of rewashing is preferably 3-5, more preferably 5. The re-washing is accomplished, preferably the invention further comprises blotting the water with sterile filter paper.
The invention preferably further comprises the steps of cutting one end of the capsule by using a sterile knife, then longitudinally and gently prying the capsule by using a blade, clamping the uncut end of the capsule, and uniformly spraying fine seeds in the uncut end of the capsule on the surface of a primary induction culture medium for culture. Compared with the conventional method, the method for disinfecting and obtaining the dendrobium huoshanense seeds greatly reduces the pollution rate of the seeds.
The dendrobium candidum is sprayed on the surface of a primary induction culture medium, and then is subjected to dark culture for 5-10 days and first illumination culture for 20-50 days in sequence to obtain protocorms. The dark culture time is preferably 5-10 days, and more preferably 7 days; the temperature of the dark culture is preferably 28. + -. 3 ℃ and more preferably 28 ℃. The seeds can be observed to sprout by dark culture, and the seeds are changed from faint yellow to light green. The first illumination culture period is preferably 20-50 days, more preferably 25-40 days, and even more preferably 30 days; the temperature of the first light culture is preferably 28 +/-3 ℃, and more preferably 28 ℃; the illumination time is preferably 10-15 h.d -1 More preferably 10 h.d -1 (ii) a The light intensity is preferably 35 to 45 [ mu ] mol/m -2 ·s -1 More preferably 40. mu. mol. m -2 ·s -1 . Through the first illumination culture, the dendrobium huoshanense seeds can be observed to develop into protocorms, and the obtained protocorms are fresh green in color and are rawThe length is good.
After the protocorm is obtained, the protocorm is transferred into a secondary proliferation differentiation culture medium for secondary illumination culture for 40-60 days, and a cluster seedling is obtained. The second illumination culture period is further preferably 45-55 days, and more preferably 50 days; the temperature of the second illumination culture is preferably 28 +/-3 ℃, and more preferably 28 ℃; the illumination time is preferably 10-15 h.d -1 More preferably 12 h.d -1 (ii) a The light intensity is preferably 35 to 45 [ mu ] mol/m -2 ·s -1 More preferably 40. mu. mol. m -2 ·s -1 . 4-6cm of cluster seedlings can be obtained through the second illumination culture, and the obtained cluster seedlings are fresh green in leaves and strong in growth.
After the cluster seedlings are obtained, the cluster seedling plantlets are transferred into a strong seedling rooting culture medium, and are subjected to illumination culture for 30-50 days to obtain rooted seedlings. The third illumination culture period is preferably 35-45 days, and more preferably 40 days; the temperature of the third illumination culture is preferably 28 +/-3 ℃, and more preferably 28 ℃; the illumination time is preferably 10-15 h.d -1 More preferably 12 h.d -1 (ii) a The light intensity is preferably 35 to 45 [ mu ] mol/m -2 ·s -1 More preferably 40. mu. mol. m -2 ·s -1 . According to the invention, more than 4cm of clump seedlings are preferably transferred into the strong seedling rooting culture medium, 10 seedlings are preferably transferred into each bottle of strong seedling rooting culture medium, the specification of a bottle for containing the strong seedling rooting culture medium is preferably 650mL, the bottle cover is provided with air holes, and the aperture is preferably 10 mm. The rooted seedlings obtained through the third illumination culture of the invention grow strongly and have high rooting rate.
After the rooting seedling is obtained, the invention carries out seedling hardening on the rooting seedling to obtain the dendrobium candidum clone seedling. The seedling exercising method is not particularly limited, and the conventional seedling exercising method in the field can be adopted.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Example 1
1) Selecting 150d pollinated healthy, plump and uncracked Dendrobium Crystal capsules as explants, soaking in 1% detergent solution for 10min, taking out, washing with flowing tap water, soaking in 75% ethanol on a superclean bench for 60s, taking out, and placing in 0.1% HgCl 2 Soaking for 8min, taking out, and washing with sterile water for 5 times.
2) Inoculating the sterilized explant into a primary culture medium, carrying out dark culture for one week, then observing seed germination, changing the faint yellow color into light green, and continuing illumination culture for 30 days to observe the development of the seed to form protocorm, wherein the obtained protocorm is fresh green in color, good in growth, 20% in pollution rate and 94.6% in seed germination rate;
the formula of the primary induction culture medium is as follows: 1/2MS +6-BA 1.0 mg.L -1 +NAA 0.2mg·L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8. The illumination culture conditions are as follows: the temperature is 28 +/-3 ℃, and the illumination time is 10 h.d -1 The light intensity is 40 mu mol.m -2 ·s -1 。
3) Transferring the obtained protocorm into a subculture proliferation differentiation medium, and performing illumination culture for 50d to obtain clump seedlings with the height of 4-6cm, wherein the proliferation coefficient is 6.0, and the obtained clump seedlings have fresh green leaves and strong growth;
the formula of the subculture proliferation and differentiation medium is as follows: MS +6-BA 1.6 mg.L -1 +NAA 0.4mg·L -1 0.1% of active carbon, 10% of banana puree, 30 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8. The culture conditions were: the temperature is 28 +/-3 ℃, and the illumination time is 12 h.d -1 The light intensity is 40 mu mol.m -2 ·s -1 。
4) Selecting cluster seedlings with the length of more than 4cm, transferring the cluster seedlings into a strong seedling rooting culture medium in a plant division mode, transferring 10 seedlings into each bottle, and performing illumination culture for 40 days to obtain the dendrobium candidum rooted seedlings, wherein the obtained rooted seedlings grow robustly, each plant has 8 roots on average, the average root length is 3.0cm, and the rooting rate is 100% through statistics;
the strong seedling rooting culture methodThe nutrient medium formula is as follows: 1/2MS + NAA 0.5 mg.L -1 +ZnSO 4 0.2 mg·L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.5 g.L -1 pH 5.8. The culture conditions were: the temperature is 28 +/-3 ℃, and the illumination time is 12 h.d -1 The light intensity is 40 mu mol.m -2 ·s -1 。
5) And (4) hardening the obtained rooted seedlings according to a conventional method to obtain the excellent clone seedlings of the dendrobium huoshanense.
Example 2
Example 1 was repeated except that:
modifying the formulation of the subculture proliferation and differentiation medium in the step 3) into: MS +6-BA2.0 mg.L -1 +NAA 0.4mg·L -1 0.1% of active carbon, 10% of banana puree, 30 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8; the seedlings of the clumps obtained by illumination culture for 50d are thicker, the leaves are bright green, and the multiplication coefficient is 5.2. It is shown that the higher the cytokinin 6-BA concentration is, the better the cytokinin 6-BA concentration is, and the proper hormone concentration and proportion are more favorable for the differentiation of protocorms and the propagation of clustered seedlings.
Example 3
Example 1 was repeated except that:
the formula of the strong seedling rooting culture medium in the step 4) is modified as follows: 1/2MS + NAA 0.5 mg.L -1 +ZnSO 4 1.0mg·L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.5 g.L -1 pH 5.8; compared with the example 1, the rooting number, the average root length and the rooting rate are slightly reduced; however, when the strain is cultured for 50 days, the plant grows well, each plant has 5 roots on average, the root length is 2.2cm on average, and the rooting rate is 93 percent. During the culture process, the dendrobium huoshanense has certain tillering capacity while rooting, sometimes can be dedifferentiated to generate callus, and is suitable for ZnSO with concentration 4 Can promote the growth and rooting of plants and inhibit the formation of calluses or protocorms; when ZnSO is present 4 When the concentration is higher, the growth of the root system can be inhibited to a certain extent.
Example 4
Example 2 was repeated except that:
modifying the formulation of the subculture proliferation and differentiation medium in the step 3) into: MS +6-BA 1.0 mg.L -1 +NAA 0.4mg·L -1 0.1% of active carbon, 10% of banana puree, 30 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8; and (5) culturing for 50 days under illumination, wherein the obtained cluster seedlings are relatively thick, the leaves are fresh green, and the multiplication coefficient is 4.5. Compared with example 2, the clumped seedlings have a reduced proliferation coefficient, probably caused by the reduction of 6-BA concentration and the reduction of the cell proliferation and division capacity.
Example 5
Example 2 was repeated except that:
modifying the formulation of the subculture proliferation and differentiation medium in the step 3) into: MS +6-BA 1.6 mg.L -1 +NAA 0.1mg·L -1 0.1% of active carbon, 10% of banana puree, 30 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8; compared with the example 2, the obtained cluster seedlings are thicker, the leaves are bright green, the vitrification phenomenon gradually appears in the later period, and the multiplication coefficient is 4.6. The slight vitrification of the seedlings in the later growth stage may be caused by the decrease of NAA concentration and the increase of the ratio of cytokinin to auxin.
Example 6
Example 2 was repeated except that:
modifying the formula of the primary induction culture medium in the step 2) into: 1/2MS +6-BA 1.0 mg.L -1 +NAA 0.2mg·L -1 0.1% of active carbon, 5% of banana puree, 20 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8. And (4) culturing for 30 days under illumination, wherein the obtained protocorm is light green in color and general in growth, and the seed germination rate is 89.6%. Compared with example 2, the protocorm growth vigor and the seed germination rate are reduced compared with example 1, which is probably caused by the reduction of the concentration of the banana puree, the banana puree contains certain amounts of hormone, amino acid and vitamin, the germination of the dendrobium nobile seeds and the growth of the protocorm can be promoted, and when the concentration of the banana puree is reduced, the content of substances for promoting the growth can be reduced, so that the seed germination rate and the protocorm growth vigor are reduced.
Step 3), transferring the protocorm into a secondary proliferation and differentiation culture medium, example 2The method is modified as follows: MS +6-BA2.0 mg.L -1 +NAA 0.4mg·L -1 0.1% of active carbon, 10% of banana puree, 30 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8; the seedlings of the cluster obtained after 60 days of culture are relatively thick and strong, the leaves are fresh green, and the multiplication coefficient is 4.7. The increase in differentiation-inducing time and the decrease in proliferation coefficient of the tufted seedlings compared to example 2 are probably due to the influence of the decrease in the growth state and viability of the protocorms obtained in step 2) on the secondary proliferative differentiation.
Step 4), selecting clumpy seedlings with the length of more than 4cm, and transferring the clumpy seedlings into a strong seedling rooting culture medium, wherein the formula of the strong seedling rooting culture medium is as follows: 1/2MS + NAA 0.5 mg.L -1 +ZnSO 4 0.2 mg·L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.5 g.L -1 pH 5.8. The culture conditions were: the temperature is 28 +/-3 ℃, and the illumination time is 12 h.d -1 The light intensity is 40 mu mol.m -2 ·s -1 . Transferring 10 plants into each bottle, and performing illumination culture for 40d to obtain the dendrobium candidum rooted seedlings, wherein the obtained rooted seedlings are strong in growth, each plant has 7-8 roots on average, the average root length is 3.0cm, and the rooting rate is 100% by statistics; the formula and conditions of the culture medium are not changed in the step, so that the influence is small.
Example 7
Example 3 was repeated except that:
the formula of the strong seedling rooting culture medium in the step 4) is modified as follows: 1/2MS + NAA 1.0 mg.L -1 +ZnSO 4 0.2mg·L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.5 g.L -1 pH 5.8; after the cultivation for 50 days, the plant grows well, a part of the plant has a certain tillering, the root system is relatively thin, each plant has 5.5 long roots on average, the average root length is 4.0cm, and the rooting rate is 87%. The rooting rate is reduced compared with that of example 1, the root system is thin, and the concentration of NAA is increased, so that the tillering of the plant is promoted, and the growth and development of the root system are inhibited to a certain extent.
Example 8
Example 3 was repeated except that:
modifying the formulation of the subculture proliferation and differentiation medium in the step 3) into: MS +6-BA 0.5mgL -1 +NAA 0.5mg·L -1 0.1% of active carbon, 10% of banana puree, 30 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8; compared with example 3, the protocorm cultured for 60 days has the advantages of less proliferation, light green color, less differentiated seedlings, slow growth speed and a proliferation coefficient of 3.5. It is probably caused by the decrease of 6-BA concentration, the increase of NAA concentration and the suboptimal concentration of hormone.
Example 9
Example 3 was repeated except that:
modifying the formula of the primary induction culture medium in the step 2) into: 1/2MS +6-BA 0.5 mg.L -1 +NAA 0.2mg·L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8. Compared with example 3, the protocorm obtained is light green in color, slow in growth speed and 90.6% in seed germination rate. Probably, the concentration of 6-BA is reduced, and the growth and development speed of protocorm is reduced.
Step 3) transferring the obtained protocorm into a secondary proliferation and differentiation culture medium, wherein the formula of the secondary proliferation and differentiation culture medium is as follows: MS +6-BA 1.6 mg.L -1 +NAA 0.4mg·L -1 0.1% of active carbon, 10% of banana puree, 30 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8. The culture conditions were: the temperature is 28 +/-3 ℃, and the illumination time is 12 h.d -1 The light intensity is 40 mu mol.m -2 ·s -1 . Culturing for 55 days under illumination to obtain clump seedlings with height of 4.5cm, wherein the multiplication coefficient is 5.3, and the obtained clump seedlings have fresh green leaves and strong growth; compared with example 3, the time for obtaining robust seedlings in clumps is increased and the multiplication coefficient is reduced, probably because the longer time is needed for the protocorms obtained in step 2) to grow again.
Comparative example 1
Example 1 was repeated except that:
modifying the formula of the primary induction culture medium in the step 2) into: 1/2MS +6-BA 1.0 mg.L -1 +NAA 0.2mg·L -1 + 0.1% of active carbon + 20 g.L of sucrose -1 + agar 3.4 g. L -1 pH 5.8. Compared with the example 1, the germination rate of the seeds is about 50 percent when the seeds are cultured for 30 days, and the protocorm is light green, small in diameter and slow in growth. Incense stickThe banana puree contains a certain amount of hormone, amino acid and vitamin, can promote the germination of the dendrobium seeds and the growth of the protocorm, and greatly reduces the seed germination rate and the protocorm growth vigor when the banana puree is not included.
Comparative example 2
Example 1 was repeated except that:
the formula of the strong seedling rooting culture medium in the step 4) is modified as follows: 1/2MS + NAA 0.5 mg.L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.5 g.L -1 pH 5.8. Compared with the example 1, the plant grows slimly after being cultured for 50 days, part of the root-growing seedling basal parts are dedifferentiated to generate callus, the callus has redifferentiation capacity, the average rooting rate is 60 percent, and the root system is thinner. Indicating that appropriate concentrations of ZnSO are also included 4 Can promote the growth and rooting of the dendrobium plant and inhibit the formation of callus or protocorm.
Comparative example 3
Example 1 was repeated except that:
the disinfection and sterilization method in the step 1) is modified into the following steps: and after the disinfected dendrobium candidum capsules are obtained, longitudinally splitting pod skins by using a scalpel, scraping and clamping seeds from the pods by using the cooperation of the scalpel and tweezers, and then uniformly inoculating the seeds into a culture medium. After dark culture for one week, the seed pollution rate is found to be about 30%, and the pollution rate is up to 70% after continuous illumination culture for 30 days. The contamination rate was significantly increased compared to example 1, probably because the scalpel and the tweezers contacted the pod skin containing incompletely sterilized microorganisms and contaminated the seeds and the culture medium when the seeds were scraped with the scalpel and the tweezers, resulting in a significant increase in the contamination rate.
Example 10
1) Taking a healthy, plump and uncracked Dendrobium Crystal capsules 150 days after pollination of Dendrobium Crystal as an explant, soaking in a detergent solution with the mass concentration of 1% for 5min, taking out, washing cleanly under flowing tap water, then placing on an ultraclean workbench, soaking for 50s with ethanol with the volume concentration of 75%, taking out, and then placing in HgCl with the mass concentration of 0.1% 2 Soaking for 5min, taking out, and washing with sterile water for 5 times.
2) Inoculating the sterilized explant into a primary culture medium, wherein the germination of seeds can be observed after dark culture for one week, the faint yellow is changed into light green, the development of the seeds can be observed to form protocorms after continuous illumination culture for 30 days, the obtained protocorms are light green, partial protocorms are vitrified, the pollution rate is 52%, and the seed germination rate is 89.8%; the formula of the primary induction culture medium is as follows: 1/2MS +6-BA 3.0 mg.L -1 +NAA 0.5mg·L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8. The illumination culture conditions are as follows: the temperature is 28 +/-3 ℃, and the illumination time is 10 h.d -1 The light intensity is 40 mu mol.m -2 ·s -1 . The contamination rate was increased compared to example 1, probably with less time for disinfection and less ability to kill the microbes carried by the explants well; the seed germination rate is reduced, the protocorm is vitrified, and the 6-BA is possibly increased, so that the cell division is abnormal, and the higher the cytokinin concentration is, the better the cytokinin concentration is.
3) Transferring the obtained protocorm into a subculture proliferation and differentiation culture medium, and culturing for 50d under illumination, wherein the proliferation speed of the protocorm is high, the protocorm at the later stage is in a vitrification state, the differentiated clustered seedlings are short and small and thin, the height of the clustered seedlings is 2-3cm, and the proliferation coefficient is 3.5; the formula of the subculture proliferation and differentiation medium is as follows: MS +6-BA 3.0 mg.L -1 +NAA 0.5mg·L -1 0.1% of active carbon, 10% of banana puree, 30 g.L of cane sugar -1 + agar 3.4 g. L -1 pH 5.8. The culture conditions were: the temperature is 28 +/-3 ℃, and the illumination time is 12 h.d -1 The light intensity is 40 mu mol.m -2 ·s -1 . Indicating that elevated 6-BA concentrations promote protocorm proliferation but do not favor protocorm growth and clump shoot differentiation, indicating that a higher concentration of cytokinin is not preferred.
4) Selecting cluster seedlings with the length of more than 4cm, transferring the cluster seedlings into a strong seedling rooting culture medium in a plant division mode, transferring 10 seedlings into each bottle, and performing illumination culture for 50 days to obtain the dendrobium candidum rooted seedlings, wherein the obtained rooted seedlings grow robustly, each plant has 5 roots on average, the average root length is 2.0cm, and the rooting rate is 92% through statistics; the formula of the seedling strengthening and rooting culture medium is as follows: 1/2MS + NAA 0.1 mg.L -1 +ZnSO 4 0.1 mg·L -1 0.1% of active carbon, 10% of banana puree, 20 g.L of cane sugar -1 + agar 3.5 g.L -1 pH 5.8. The culture conditions were: the temperature is 28 +/-3 ℃, and the illumination time is 12 h.d -1 The light intensity is 40 mu mol.m -2 ·s -1 . The rooting rate and the rooting number are both reduced compared with example 1, the average root length is shortened, and auxin and ZnSO are probably 4 The concentration is lower.
5) And (4) hardening the obtained rooted seedlings according to a conventional method to obtain the excellent clone seedlings of the dendrobium huoshanense.
According to the embodiment, the dendrobium huoshanense tissue culture rapid propagation culture medium combination and the method can obtain excellent clone seedlings of dendrobium huoshanense in a short time, the time from seed germination to protocorm formation is 30 days as fast as possible, the time from seed germination to rooting seedling is 120 days as fast as possible, and the maximum propagation coefficient can reach 6.0; the conventional dendrobium nobile seedling division propagation, the conventional dendrobium nobile seedling division propagation and the conventional dendrobium nobile cutting propagation generally take more than 1 year to obtain the dendrobium nobile seedling, the propagation coefficient is generally 2-3 times, and the seedling growth states are different. Therefore, compared with the conventional method, the method has better time and quality advantages, and lays a foundation for the large-scale planting of the dendrobium candidum.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (10)
1. A culture medium combination for tissue culture and rapid propagation of dendrobium candidum is characterized by comprising a primary generation induction culture medium, a secondary generation proliferation and differentiation culture medium and a strong seedling rooting culture medium;
the primary induction culture medium takes 1/2MS as a basic culture medium and also comprises 0.5-3.0 mg.L of 6-BA -1 、NAA 0.1-1.0mg·L -1 0.1-0.2 wt.% of active carbon, 5-10 wt.% of banana puree and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.6-6.0;
the subculture proliferation and differentiation cultureThe culture medium takes MS as a basic culture medium and also comprises 0.5-3.0 mg.L of 6-BA -1 、NAA 0.1-1.0mg·L -1 0.1-0.2 wt.% of active carbon, 5-10 wt.% of banana puree and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.6-6.0;
the strong seedling rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.1-1.0 mg.L of NAA -1 、ZnSO 4 0.1-1.0mg·L -1 0.1-0.2 wt.% of active carbon, 5-10 wt.% of banana puree and 15-25 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.6-6.0.
2. The dendrobium huoshanense tissue culture rapid propagation culture medium composition as claimed in claim 1, wherein the primary induction culture medium takes 1/2MS as a basic culture medium, and further comprises 6-BA 1.0-1.5 mg-L -1 、NAA 0.1-0.5mg·L -1 0.1-0.15 wt.% of active carbon, 8-10 wt.% of banana puree and 20-25 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.8-6.0;
the subculture proliferation and differentiation culture medium takes MS as a basic culture medium and also comprises 1.0-2.0 mg.L of 6-BA -1 、NAA 0.3-0.5mg·L -1 0.1-0.15 wt% of active carbon, 8-10 wt% of banana puree and 20-30 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.8-6.0;
the strong seedling rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.5-0.8 mg.L of NAA -1 、ZnSO 4 0.1-0.5mg·L -1 0.1-0.15 wt.% of active carbon, 8-10 wt.% of banana puree and 15-25 g.L of cane sugar -1 And agar 3-4 g.L -1 The pH value is 5.8-6.0.
3. The dendrobium huoshanense tissue culture rapid propagation medium composition as claimed in claim 1 or 2, wherein the primary induction medium takes 1/2MS as a basic medium and further comprises 6-BA 1.0 mg-L -1 、NAA 0.2mg·L -1 0.1 wt.% of active carbon, 10 wt.% of banana puree and 20 g.L of sucrose -1 And agar 3.4 g.L -1 The pH is 5.8;
the subculture proliferation and differentiation culture medium takes MS as a basal culture medium,also comprises 1.6 mg.L of 6-BA -1 、NAA 0.4mg·L -1 0.1 wt.% of active carbon, 10 wt.% of banana puree and 30 g.L of sucrose -1 And agar 3.4 g.L -1 The pH is 5.8;
the strong seedling rooting culture medium takes 1/2MS as a basic culture medium and also comprises 0.5 mg.L of NAA -1 、ZnSO 4 0.2mg·L -1 0.1 wt.% of active carbon, 10 wt.% of banana puree and 20 g.L of sucrose -1 And agar 3.5 g. L -1 The pH was 5.8.
4. The application of the dendrobium huoshanense tissue culture rapid propagation culture medium combination of any one of claims 1-3 in dendrobium huoshanense tissue culture.
5. A method for cultivating Dendrobium huoshanense seedlings is characterized in that the Dendrobium huoshanense is cultivated by adopting the dendrobium huoshanense tissue culture rapid propagation culture medium combination of any one of claims 1-3 to obtain the clone seedlings of the Dendrobium huoshanense.
6. The method of claim 5, comprising the steps of:
inoculating dendrobium candidum seeds into a primary induction culture medium, and performing dark culture for 5-10 days and first illumination culture for 20-50 days in sequence to obtain protocorms;
transferring the protocorm into a subculture proliferation and differentiation culture medium for second illumination culture for 40-60 days to obtain a cluster seedling;
transferring the cluster seedling into a strong seedling rooting culture medium, and performing third illumination culture for 30-50 days to obtain a rooted seedling;
and (3) hardening the rooted seedlings to obtain the clonal seedlings of the dendrobium huoshanense.
7. The method according to claim 6, wherein the first light-irradiation culture is performed at a temperature of 28 ± 3 ℃ for 10-15 h-d -1 The illumination intensity is 35 to 45 [ mu ] mol.m -2 ·s -1 。
8. According to the claimsThe method of claim 6 or 7, wherein the temperature of the second light-irradiation culture is 28 + -3 ℃, and the light-irradiation time is 10-15 h.d -1 The illumination intensity is 35 to 45 [ mu ] mol.m -2 ·s -1 。
9. The method according to claim 8, wherein the temperature of the third light-irradiation culture is 28 ± 3 ℃ and the light-irradiation time is 10-15 h-d -1 The illumination intensity is 35 to 45 [ mu ] mol.m -2 ·s -1 。
10. The method according to claim 6, wherein the Dendrobium officinale seed is a seed obtained from a sterilized, plump, uncracked Dendrobium officinale capsule.
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