CN110604056A - A kind of method of Wen Curcuma tissue culture - Google Patents

Abstract

The invention discloses a method for tissue culture of warm curcuma, which mainly includes the steps of germination acceleration in sand bed of tubers, induction of adventitious buds, subculture multiplication, rooting culture, pseudoplanting and the like. By adopting the method of the invention, the pollution rate in the cultivation process is less than 5%, the adventitious bud induction rate reaches more than 129%, the bud multiplication ratio reaches more than 7.5, the survival rate reaches more than 95%, and the volatile oil content reaches more than 5.4%. The method is easy to operate and has remarkable effect, can provide a large amount of high-quality warm curcuma seedlings in a short period of time, and is suitable for industrial promotion and use.

Description

A kind of method of Wen Curcuma tissue culture

technical field

The invention relates to a method for tissue culture of warm turmeric, belonging to the technical field of plant tissue culture.

Background technique

Wenyujin (Curcuma wenyujin Y.H.Chen et C.Ling), also known as Wenzezhu, belongs to Zingiberaceae Curcuma, perennial bulbous herbaceous flowers, plant height 100-120cm, clustered plants, oblong leaves, bright green, can be used for foliage viewing Ornamental plants, suitable for garden cultivation or as large potted plants. The flowering period is from March to May. Cylindrical spikes emerge from the rhizomes, and the purple-red bracts are arranged tightly on the inflorescences. The flowering period lasts for a long time and can be used as flower mirrors and flower beds. The properties of Wen Yujin are round or irregular flakes, 1.5-4cm in diameter, gray-yellow surface, yellow-brown to brown-brown cut surface, often with yellow-white or yellow-brown powder, and many and obvious vascular bundle marks. The aroma is strong, the taste is slightly bitter and pungent. Wenyujin is a well-known authentic medicinal material. Its medicinal parts are mainly rhizomes and tubers, which mainly contain volatile oils and curcuminoids, as well as resins and sugars. It is a variety recorded in multiple editions of the Chinese Pharmacopoeia. Studies have shown that the volatile oil of Curcuma curcuma has anti-phytopathogenic activity, indicating that it has a certain application potential in botanical fungicides.

With the deepening of research, the demand for Wen Yujin resources is also increasing. However, in terms of cultivation and production, turmeric is mainly vegetatively propagated through rhizomes, which consumes a lot of seeds, has a low reproduction coefficient and serious viralization, slow speed, decline in quality, and resources in short supply. In order to solve the problems existing in the production of Curcuma mellifera, it is urgent to develop a rapid propagation technology suitable for Curcuma mellifera. Plant tissue culture technology is a method of rapid propagation using explants such as roots, stems, leaves, flowers, and fruits of plants, which can effectively make up for the shortcomings of traditional cultivation methods. There are many deficiencies, such as poor induction and proliferation effects, etc., so it is urgent to seek a method that is more suitable for the tissue culture of yujin.

Contents of the invention

In view of the deficiencies in the prior art, the present invention provides a method that is more suitable for tissue culture of Curcuma curcuma. This method can obtain a large number of seedlings in a short period of time, rejuvenate excellent new varieties of Curcuma curcuma, and provide certain technical references for large-scale batch propagation of Curcuma curcuma. .

The technical scheme that the present invention takes is as follows:

A method for tissue culture of turmeric, comprising the following steps:

S1: Germination of tubers in sand bed

Take healthy tubers and spread them neatly on clean and fresh river sand, keep the sand bed moist, and after one month, several seedlings will grow on the tubers;

S2: Explant acquisition

Select healthy seedlings, cut off the rhizome, disinfect with 75% alcohol for 30 seconds, then disinfect with 0.1% mercuric chloride for 20 minutes, wash with sterilized water for 3 to 5 times, and inoculate in a solution containing 4 to 5 mg/L 6-BA. MS medium to obtain sterile explants;

S3: Induction of adventitious buds

When the buds grow to 3-5cm, inoculate the buds in MS medium containing 2-5mg/L 6-BA and 0.5-1.0mg/L NAA to induce culture;

S4: Subculture Proliferation

Cut the induced new shoots into small pieces of no more than 5 lateral buds, inoculate them in a medium containing 2-5 mg/L 6-BA and 0.5-1.0 mg/L NAA for proliferation and culture;

S5: rooting culture of test-tube plantlets

After subculture and propagation, inoculate the test-tube plantlets in MS medium containing 0.5-1g/L activated carbon and 0.5-1.0mg/L NAA;

S6: False planting of test-tube seedlings

After rooting and culturing for 1 month, open the bottle cap, make the rooting seedlings fully contact with the air, place them for at least 1 week, take out the test-tube seedlings, and transplant the test-tube seedlings into the river sand substrate for cultivation;

The light conditions of each step are: light intensity 1600-2400lx, light time 10-12h/d, ambient temperature 25-28°C, and ambient humidity 40-90%.

Preferably, the MS medium in steps S2-S5 further contains 30-35 g/L sucrose and 5-6 g/L carrageenan.

Preferably, in step S3, the content of 6-BA is 4-5 mg/L, and the content of NAA is 0.5 mg/L.

Preferably, the MS medium described in step S3 also contains 3-5 g/L fucoidan oligosaccharide, 6.5-10.3 mg/L salicylic acid and 11-18 mg/L methyl jasmonate, and the concentration of calcium chloride in the medium is changed It is 640～680mg/L.

Preferably, in step S4, the content of 6-BA is 5 mg/L, and the content of NAA is 0.5 mg/L.

Preferably, in step S5, the NAA content is 0.5 mg/L.

Preferably, the MS medium described in step S5 also contains 5-10 mg/L methyl jasmonate, and the concentration of calcium chloride is changed to 520-550 mg/L.

Preferably, from step S1 to step S4, under the condition of red and blue LED light for 10 to 12 hours per day, the ratio of red and blue light (4 to 5): 1, the light intensity is 1600 to 2000 lx, the ambient humidity is 40 to 60%, and the ambient temperature is 25 to 28°C; step S5, under the condition of white light for 10-12 hours per day, the light intensity is 1800-2400 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28°C; step S6 is carried out outdoors under natural light conditions, and the ambient humidity is 80-28°C. 90%, the ambient temperature is 25-28°C.

Compared with prior art, the beneficial effect of the present invention is:

Adopting the method of the present invention to carry out the warm curcuma tissue culture, the pollution rate is less than 5%, the induction rate reaches more than 129%, the bud multiplication ratio reaches more than 7.5, the survival rate reaches more than 95%, and the volatile oil content reaches more than 5.4%, and the method of the invention satisfies industrialization application.

The present invention can not only reduce the pollution rate to 2.1%, increase the induction rate to 158%, increase the multiplication factor to 11.37, and more importantly, increase the volatile oil content to 8.22% at the same time, satisfying people's expectations of The actual demand of Wenyujin provides a large amount of high-quality raw materials for the use of Wenyujin.

Description of drawings

Fig. 1: the effect diagram of the tissue culture process of black curcuma in the embodiment of the present invention; A. explant inoculation; B. explant differentiation bud; C. explant differentiation cluster bud; D. proliferation of cluster bud; E. rooted seedlings; F. false planting.

Detailed ways

The present invention will be further described in detail through specific embodiments below.

The experimental methods used in the examples of the present invention are conventional methods unless otherwise specified.

The materials and reagents used in the examples of the present invention can be obtained from commercial sources unless otherwise specified.

Example 1

A method for tissue culture of turmeric, comprising the following steps:

S1: Germination of tubers in sand bed

Take healthy tubers and spread them neatly on clean and fresh river sand, keep the sand bed moist, and after one month, several seedlings will grow on the tubers;

S2: Explant acquisition

Select healthy seedlings, cut off the rhizome, disinfect with 75% alcohol for 30 seconds, then disinfect with 0.1% mercuric chloride for 20 minutes, wash with sterilized water for 3 to 5 times, and inoculate in the seedlings containing 30g/L sucrose and 5g/L karaage. Glue, MS medium of 5mg/L6-BA, to obtain sterile explants;

S3: Induction of adventitious buds

When the buds grow to 3-5cm, inoculate the buds in MS medium containing 30g/L sucrose, 5g/L carrageenan, 5mg/L6-BA and 0.5mg/L NAA to induce culture, and pick one bud per bottle ; The pH of the medium is 5.5-5.8. After 4-5 days, observe the growth of the buds, pick up the polluted bacterial seedlings, and count the pollution rate, and then continue to observe the induction and differentiation of the adventitious buds of Wen turmeric. After 30 days, count the induction of buds Rate. Pollution rate (%) = number of contaminated bottles/total number of bottles × 100%, induction rate of adventitious buds (%) = number of induced lateral buds/number of inoculated buds × 100%.

S4: Subculture Proliferation

Cut the induced new shoots into small pieces of no more than 5 lateral buds, inoculate them in a medium containing 30g/L sucrose, 5g/L carrageenan, 5mg/L 6-BA and 0.5mg/L NAA for proliferation culture , inoculate 1 to 4 small pieces per bottle; subculture once every 20 days. Observe the proliferation and growth of the buds, and count the number of proliferation buds and the multiplication multiple of Curcuma wencurcuma. The number of proliferating shoots is the total number of new shoots induced by the inoculated explants. Proliferation multiple = the number of proliferating buds/the number of inoculated buds.

S5: rooting culture of test-tube plantlets

After subculture, the plantlets were inoculated in MS medium containing 30g/L sucrose, 5g/L carrageenan, 1g/L activated carbon and 0.5mg/L NAA.

S6: False planting of test-tube seedlings

After 1 month of rooting culture, open the bottle cap, make the rooted seedlings fully contact with the air, place them for at least 1 week, take out the test-tube seedlings, rinse the root medium with clean water, and transplant the test-tube seedlings into the river sand substrate for cultivation. After 30 days, the transplanting survival rate of Wen Yujin was counted.

The lighting conditions of each step are: white light source, light intensity of 2000-2200lx, L/D=10:14, ambient temperature of 25-28°C, and ambient humidity of 80-90%.

Example 2

A method for tissue culture of turmeric, comprising the following steps:

S1: Germination of tubers in sand bed

Take healthy tubers and spread them neatly on clean and fresh river sand, keep the sand bed moist, and after one month, several seedlings will grow on the tubers;

S2: Explant acquisition

Select healthy seedlings, cut off the rhizome, disinfect with 75% alcohol for 30 seconds, then disinfect with 0.1% mercuric chloride for 20 minutes, wash with sterilized water for 3 to 5 times, and inoculate in the seedlings containing 35g/L sucrose and 6g/L karaage. Glue, MS medium of 4mg/L6-BA, to obtain sterile explants;

S3: Induction of adventitious buds

When the buds grow to 3-5cm, inoculate the buds in MS medium containing 35g/L sucrose, 6g/L carrageenan, 2mg/L 6-BA and 1.0mg/L NAA to induce culture, and pick one bud per bottle ; The pH of the medium is 5.5-5.8. After 4-5 days, observe the growth of the buds, pick up the polluted bacterial seedlings, and count the pollution rate, and then continue to observe the induction and differentiation of the adventitious buds of Wen turmeric. After 30 days, count the induction of buds Rate. Pollution rate (%) = number of contaminated bottles/total number of bottles × 100%, induction rate of adventitious buds (%) = number of induced lateral buds/number of inoculated buds × 100%.

S4: Subculture Proliferation

Cut the induced shoots into small pieces of no more than 5 lateral buds, inoculate them in a medium containing 35g/L sucrose, 6g/L carrageenan, 2mg/L 6-BA and 1.0mg/L NAA for proliferation culture , inoculate 1 to 4 small pieces per bottle; subculture once every 20 days. Observe the proliferation and growth of the buds, and count the number of proliferation buds and the multiplication multiple of Curcuma wencurcuma. The number of proliferating shoots is the total number of new shoots induced by the inoculated explants. Proliferation multiple = the number of proliferating buds/the number of inoculated buds.

S5: rooting culture of test-tube plantlets

After subculture, the plantlets were inoculated in MS medium containing 35g/L sucrose, 6g/L carrageenan, 0.5g/L activated carbon and 1.0mg/L NAA.

S6: False planting of test-tube seedlings

After 1 month of rooting culture, open the bottle cap, make the rooted seedlings fully contact with the air, place them for at least 1 week, take out the test-tube seedlings, rinse the root medium with clean water, and transplant the test-tube seedlings into the river sand substrate for cultivation. After 30 days, the transplanting survival rate of Wen Yujin was counted.

The lighting conditions of each step are: white light source, light intensity of 2000-2200lx, L/D=10:14, ambient temperature of 25-28°C, and ambient humidity of 40-50%.

Example 3

The difference between embodiment 3 and embodiment 1 is:

The MS medium described in step S3 also contains 3 g/L fucoidan, 6.5 mg/L salicylic acid and 11 mg/L methyl jasmonate, and the calcium chloride concentration in the medium is changed to 640 mg/L.

The MS medium described in step S5 also contains 5 mg/L methyl jasmonate, and the concentration of calcium chloride in the medium is changed to 520 mg/L.

From step S1 to step S4, under the condition of red and blue LED light for 10 hours a day, the ratio of red and blue light is 4:1, the light intensity is 1600-1800lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28°C; step S5, under the condition of white light Illumination for 10 hours a day under white light conditions, the light intensity is 1800-2200lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28°C; step S6, under white light conditions for 10 hours a day, the light intensity is 1800-2200lx, and the ambient humidity is 80-90% , ambient temperature 25 ~ 28 ℃.

Example 4

The difference between embodiment 4 and embodiment 1 is:

The MS medium described in step S3 also contains 5 g/L fucoidan, 10.3 mg/L salicylic acid and 18 mg/L methyl jasmonate, and the calcium chloride concentration in the medium is changed to 680 mg/L.

The MS medium described in step S5 also contains 10 mg/L methyl jasmonate, and the concentration of calcium chloride in the medium is changed to 550 mg/L.

From step S1 to step S4, under the condition of red and blue LED light for 12 hours a day, the ratio of red and blue light is 5:1, the light intensity is 2000-2200lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28°C; step S5, under the white light condition Illuminate for 12 hours a day under white light conditions, the light intensity is 2200-2400lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28°C; step S6, under white light conditions for 12 hours a day, the light intensity is 2200-2400lx, and the ambient humidity is 80-90% , ambient temperature 25 ~ 28 ℃.

Comparative example 1

A method for tissue culture of turmeric, comprising the following steps:

S1: Germination of tubers in sand bed

Take healthy tubers and spread them neatly on clean and fresh river sand, keep the sand bed moist, and after one month, several seedlings will grow on the tubers;

S2: Explant acquisition

Select healthy seedlings, cut off the rhizome, disinfect with 75% alcohol for 20 seconds, then disinfect with 0.2% mercuric chloride for 20 minutes, wash with sterilized water for 3 to 5 times, inoculate in MS culture medium containing 5mg/L 6-BA base to obtain sterile explants;

S3: Induction of adventitious buds

When the buds grow to 3-5cm, inoculate the buds in MS medium containing 2.0mg/L 6-BA and 0.5mg/L NAA to induce culture, and pick one bud per bottle; the pH of the medium is 5.5-5.8. After 4 to 5 days, observe the growth of the buds, pick up the polluted bacterial seedlings, and count the pollution rate, then continue to observe the induction and differentiation of the adventitious buds of warm turmeric, and count the induction rate of the buds after 30 days. Pollution rate (%) = number of contaminated bottles/total number of bottles × 100%, induction rate of adventitious buds (%) = number of induced lateral buds/number of inoculated buds × 100%.

S4: Subculture Proliferation

Cut the induced new shoots into small pieces of no more than 5 lateral buds, inoculate them in a medium containing 30g/L sucrose, 0.4mg/LTDZ, and 0.5mg/L NAA for proliferation and culture, and inoculate 1 to 4 lateral buds per bottle Small block; subculture once every 20d. Observe the proliferation and growth of the buds, and count the number of proliferation buds and the multiplication multiple of Curcuma wencurcuma. The number of proliferating shoots is the total number of new shoots induced by the inoculated explants. Proliferation multiple = the number of proliferating buds/the number of inoculated buds.

S5: rooting culture of test-tube plantlets

After subculture, the plantlets were inoculated in MS medium containing 0.5g/L activated carbon and 0.5mg/L NAA.

S6: False planting of test-tube seedlings

After 1 month of rooting culture, open the bottle cap, make the rooted seedlings fully contact with the air, place them for 1 week, take out the test-tube seedlings, rinse the root medium with clean water, and transplant the test-tube seedlings into the river sand substrate for cultivation. After 30 days, the transplanting survival rate of Wen Yujin was counted.

The lighting conditions of each step are: white light source, light intensity 1300-1500lx, light time 8h/d;

Test example:

(1) Carry out warm curcuma tissue culture by adopting the method of embodiment and comparative example respectively, and count pollution rate, adventitious bud induction rate, adventitious bud multiplication multiple and transplanting survival rate. The results are shown in Table 1.

(2) The seedlings after the artificial planting were transplanted to the field for planting, and their rhizomes were taken after 6 months, and the volatile oil content was determined according to the method of Appendix XD of the 2015 edition of "The Pharmacopoeia of the People's Republic of China". The results are shown in Table 1.

Table 1

pollution rate Bud induction rate Bud multiplier Survival rate Volatile oil content% (mL/g) Example 1 4.3% 137% 9.97 95% 5.57±0.03% Example 2 4.5% 129% 7.50 97% 5.40±0.07% Example 3 2.1% 158% 12.20 97% 7.89±0.05% Example 4 3.4% 155% 11.37 96% 8.22±0.04% Comparative example 1 6.6% 107% 4.12 97% 5.49±0.03%

The result shows that, adopting the method of the present invention to carry out warm curcuma tissue culture, the pollution rate is less than 5%, the induction rate reaches more than 129%, the bud multiplication ratio reaches more than 7.5, the survival rate reaches more than 95%, and the volatile oil content reaches more than 5.4%.

In addition, the present invention found that by improving the culture conditions and medium, not only the pollution rate can be reduced to 2.1%, the induction rate can be increased to 158%, the multiplication factor can be increased to 11.37, and more importantly, the volatile oil content can be increased to 8.22%. .

The above content is a further detailed description of the present invention in conjunction with specific embodiments, and it cannot be assumed that the specific implementation of the present invention is limited to these descriptions. Those of ordinary skill in the technical field to which the present invention belongs can also make some simple deduction or replacement without departing from the concept of the present invention.

Claims (8)

1. A method for culturing Curcuma wenyujin tissue is characterized by comprising the following steps:

s1: tuber sand bed germination acceleration

Paving healthy tubers on clean fresh river sand in a flush manner, keeping a sand bed moist, and growing a plurality of seedlings on the tubers after 1 month;

s2: explant acquisition

Selecting healthy plantlets, cutting off rhizome parts, disinfecting the plantlets with 75% alcohol for 30s, then disinfecting the plantlets with 0.1% mercuric chloride for 20min, washing the plantlets with sterile water for 3-5 times, and inoculating the plantlets in an MS culture medium containing 4-5 mg/L6-BA to obtain sterile explants;

s3: induction of adventitious buds

When the buds grow to 3-5 cm, inoculating the buds into an MS culture medium containing 2-5 mg/L6-BA and 0.5-1.0 mg/L NAA for induction culture;

s4: subculture multiplication

Cutting the new buds obtained by induction into small blocks with less than 5 lateral buds, and inoculating the small blocks into a culture medium containing 2-5 mg/L6-BA and 0.5-1.0 mg/L NAA for enrichment culture;

s5: rooting culture of test-tube plantlets

After subculture, inoculating the test-tube plantlet into an MS culture medium containing 0.5-1 g/L of active carbon and 0.5-1.0 mg/L of NAA for culture;

s6: temporary planting of test-tube plantlet

After rooting culture for 1 month, opening a bottle cap to ensure that the rooted seedlings are fully contacted with air, standing for at least 1 week, taking out the test-tube seedlings, and transplanting the test-tube seedlings into river sand matrix for culture;

the illumination conditions of the steps are as follows: the illumination intensity is 1600-2400 lx, the illumination time is 10-12 h/d, the ambient temperature is 25-28 ℃, and the ambient humidity is 40-90%.

2. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein the MS culture medium in steps S2-S5 further contains sucrose 30-35 g/L and carrageenan 5-6 g/L.

3. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein in step S3, the content of 6-BA is 4-5 mg/L and the content of NAA is 0.5 mg/L.

4. The method for tissue culture of Curcuma wenyujin of claim 1, wherein the MS culture medium obtained in step S3 further comprises alginate oligosaccharide 3-5 g/L, salicylic acid 6.5-10.3 mg/L and methyl jasmonate 11-18 mg/L, and the concentration of calcium chloride in the culture medium is changed to 640-680 mg/L.

5. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein the amount of 6-BA in step S4 is 5mg/L and the amount of NAA in step S is 0.5 mg/L.

6. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein in step S5, NAA content is 0.5 mg/L.

7. The method for tissue culture of Curcuma wenyujin of claim 1, wherein the MS culture medium of step S5 further contains methyl jasmonate in an amount of 5-10 mg/L, and the concentration of calcium chloride is modified to 520-550 mg/L.

8. The method for tissue culture of Curcuma wenyujin according to claim 1, wherein in steps S1 to S4, the Curcuma wenyujin is illuminated under LED red and blue light conditions for 10-12 h per day, the ratio of red and blue light is (4-5): 1, the illumination intensity is 1600-2000 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; step S5, illuminating for 10-12 hours per day under the white light condition, wherein the illumination intensity is 1800-2400 lx, the ambient humidity is 40-60%, and the ambient temperature is 25-28 ℃; and step S6, the method is carried out under outdoor natural illumination conditions, wherein the ambient humidity is 80-90%, and the ambient temperature is 25-28 ℃.