AU2021101772A4 - Medium combination for tissue cultivation of virus-free seed root tubers (micro-root tubers) of virus-free radix pseudostellariae - Google Patents
Medium combination for tissue cultivation of virus-free seed root tubers (micro-root tubers) of virus-free radix pseudostellariae 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
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
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- C12N5/0025—Culture media for plant cell or plant tissue culture
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Abstract
The present disclosure relates to the technical field of seed propagation, and discloses a
medium combination for tissue cultivation of virus-free seed root tubers (micro-root tubers) of
virus-free Radix pseudostellariae, including a virus-free tissue cultivation seedling induction
5 medium, a virus-free tissue cultivation seedling propagation medium, and a micro-root tuber
induction and swelling medium. The medium combination provided by the present disclosure, when
used to cultivate Radix pseudostellariae root tubers, can shorten the time and cycle for seedling
production and shows advantages such as prominent detoxification effect, high efficiency, simple
operation, and low cost. A propagation medium for tissue cultivation seedlings is 1/2 MS that is
10 supplemented with monopotassium phosphate (MKP), but no hormones, which allows Radix
pseudostellariae seedlings to have more stretched leaves and grow more vigorously. A medium used
for micro-root tubers has a high concentration of sucrose (60 g/L), which allows the rapid induction
of root tubers.
Description
The present disclosure relates to the technical field of seed propagation, and in particular to a
medium combination for tissue cultivation of virus-free seed root tubers (micro-root tubers) of
virus-free Radix pseudostellariae.
Radix pseudostellariaeis a root tuber of Pseudostellariaheterophylla, a perennial herb in the
Caryophyllaceae family. Radix pseudostellariaehas the effects of replenishing qi to invigorate the
spleen, promoting the secretion of saliva or body fluid, and nourishing the lungs, and shows
significant effects on night sweats and anorexia in children and poor health and insomnia in the
elderly. Compared with ginseng, Radix pseudostellariae shows more gentle medicinal properties
and is suitable for children to tonify deficiency and Qi, which thus is also called Pseudostellaria
heterophylla. Radix pseudostellariaeis a tonic traditional Chinese medicine (TCM) that can be used
in health foods, which can be used as a substitute for ginseng or even American ginseng and has a
huge development space in the TCM and the health product industries. The demand for Radix
pseudostellariaehas shown a steady upward trend in recent years.
The cultivation of Radix pseudostellariaeis concentrated in Guizhou, Fujian, Anhui, Shandong,
Jiangsu, etc., and the propagation is conducted mainly by root tubers in production. Due to
long-term asexual propagation by root tubers, viral diseases are common and serious in Radix
pseudostellariae,with a viral disease incidence as high as 90% in some cultivation areas. The viral
disease is the main disease that affects the yield and quality of Radix pseudostellariae, which
significantly influences the income of farmers and the development of the Radix pseudostellariae
industry. There are many types of viruses that infect Radix pseudostellariae, and reported ones
mainly include tobacco mosaic virus (TMV), turnip mosaic virus (TuMV), cucumber mosaic virus
(CMV), etc., which can be hardly controlled by agronomic prevention and treatment measures.
In view of the asexual propagation characteristics of Radix pseudostellariae, cultivating
virus-free seedlings or seed root tubers is a key way to overcome the virus-carrying Radix
pseudostellariaeproduction status at present, and technical personnel have made relevant attempts
and successfully obtained virus-free seedlings or seed root tubers of Radix pseudostellariae. An
existing method for the virus-free cultivation of Radix pseudostellariae involves complicated
operations and techniques, has high technical requirements on operators, and is prone to bacterial
contamination, resulting in high cultivation cost. Moreover, Radix pseudostellariaeis cultivated in
fields at a high planting density of nearly 70,000 plants per mu, which requires a large number of
seedlings, so it is urgent to increase the speed for producing Radix pseudostellariaeseedlings.
Based on the above problems, the present disclosure provides a medium combination for tissue cultivation of virus-free seed root tubers (micro-root tubers) of virus-free Radix pseudostellariae.
The medium combination provided by the present disclosure, when used to cultivate virus-free
Radix pseudostellariae root tubers, can shorten the time and cycle for seedling production and
shows advantages such as prominent detoxification effect, high efficiency, simple operation, and
low cost.
To solve the above technical problem, the present disclosure provides the following technical
solution.
The present disclosure provides a medium combination for tissue cultivation of virus-free seed
root tubers (micro-root tubers) of virus-free Radix pseudostellariae, including a virus-free tissue
cultivation seedling induction medium, a virus-free tissue cultivation seedling propagation medium,
and a micro-root tuber induction and swelling medium.
The virus-free tissue cultivation seedling induction medium is an MS medium supplemented
with 0.5 mg/L to 1 mg/L 6-BA, 0.1 mg/L NAA, 30 g/L sucrose, and 5 g/L agar powder, which has a
pH value of 5.8;
the virus-free tissue cultivation seedling propagation medium is a 1/2 MS medium
supplemented with 85 mg/L monopotassium phosphate (MKP), 30 g/L sucrose, and 5 g/L agar
powder, which has a pH value of 5.8; and
the micro-root tuber induction and swelling medium is an MS medium supplemented with 85 mg/L MKP, 60 g/L sucrose, and 5 g/L agar powder, which has a pH value of 5.8.
Preferably, the virus-free tissue cultivation seedling induction medium may be an MS medium
supplemented with 0.6 mg/L to 0.9 mg/L 6-BA, 0.1 mg/L NAA, 30 g/L sucrose, and 5 g/L agar
powder.
Preferably, the virus-free tissue cultivation seedling induction medium may be an MS medium
supplemented with 0.7 mg/L to 0.8 mg/L 6-BA, 0.1 mg/L NAA, 30 g/L sucrose, and 5 g/L agar
powder.
In the present disclosure, when the medium combination is used for tissue cultivation, the
following solution may preferably be adopted:
A method for tissue cultivation of virus-free Radix pseudostellariaeroot tubers may include the
following steps:
Sl: Treatment and disinfection of explants
Radix pseudostellariaewith robust buds is refrigerated in a refrigeration environment at 1°C to
4°C, and after the buds of Radix pseudostellariaegerminate, germinated buds are cut and used as
explants. The germinated buds are thoroughly rinsed with tap water, air-dried, soaked in alcohol for
30 s and then in 0.1% mercuric chloride for 7 min to 8 min on a clean bench, rinsed 3 to 5 times
with sterile water, and then transferred to filter paper in a sterile petri dish for later use.
S2: Induction of Radix pseudostellariaestem apex
The explants treated in step Si are placed under a stereoscope, a 0.5 mm Radix
pseudostellariae stem apex is cut off from the explants, inoculated on the induction medium, and
then cultivated under the following conditions: cultivation temperature: 15°C to 20°C, light
intensity: 1,500 lx to 2,000 lx, and photoperiod: 10 h/d. After 20 d to 25 d of cultivation, 1 to 2
Radix pseudostellariaeadventitious buds germinate on the Radix pseudostellariaestem apex.
S3: Propagation cultivation of Radix pseudostellariaeseedlings
The Radix pseudostellariae adventitious buds induced in step S2 are transferred to a
propagation medium and cultivated under the following conditions: cultivation temperature: 150 C
to 200 C, light intensity: 1,500 lx to 2,000 lx, and photoperiod: 10 h/d. Radix pseudostellariae
seedlings, after growing tall, are cut into 4 to 5 sections, and the above transferring and propagation
cultivation process is repeated for the Radix pseudostellariaeseedling sections.
S4: Cultivation of Radix pseudostellariaemicro-root tubers in bottles
The Radix pseudostellariaeseedlings obtained in step S3 are transferred to a root tuber medium
and cultivated at 15 0C to 20 0C. After the seedlings are cultivated for 50 d to 60 d with a tissue
cultivation lamp in the cultivation room being off, 2 to 3 root tubers grow from a stem node of
Radix pseudostellariae. After growing to have a diameter of 0.5 cm or more, the root tubers are
collected and cleaned, then refrigerated at 1C to 4C for 20 d to 30 d, directly planted in fields, and
then harvested to be used as Radix pseudostellariaeseed root tubers.
Further, the induction medium in step S2 may be a modified MS medium, specifically an MS
medium supplemented with 0.5 mg/L to 1 mg/L 6-BA, 0.1 mg/L NAA, 30 g/L sucrose, and 5 g/L
agar powder, which has a pH value of 5.8.
Further, the propagation medium in step S3 may be a modified 1/2 MS medium, specifically a
1/2 MS medium supplemented with 85 mg/L MKP, 30 g/L sucrose, and 5 g/L agar powder, which
has a pH value of 5.8.
Further, the transferring and propagation cultivation in step S3 may have a cycle of 18
d/generation to 24 d/generation.
Further, the transferring and propagation cultivation in step S3 may have a cycle of 20
d/generation.
Further, the root tuber medium in step S4 may be a modified MS medium, specifically an MS
medium supplemented with 85 mg/L MKP, 60 g/L sucrose, and 5 g/L agar powder, which has a pH
value of 5.8.
Further, the Radix pseudostellariae seedlings in step S3 may be cut into 4 to 5 Radix
pseudostellariaeseedling sections after growing to have a height of 4 cm to 6 cm.
Compared with the prior art, the present disclosure has the following beneficial effects: The
medium combination provided by the present disclosure, when used for the tissue cultivation of
Radix pseudostellariae root tubers, can shorten the time and cycle for seedling production and shows advantages such as prominent detoxification effect, high efficiency, simple operation, and low cost. A propagation medium is 1/2 MS that is supplemented with MKP, but no hormones, which allows Radix pseudostellariaeseedlings to have more stretched leaves and grow more vigorously. A root tuber induction medium is MS supplemented with MKP and high concentration (60 g/L) of sucrose, which allows root tubers to be more swollen.
In order to make the objectives, technical solutions, and advantages of the present disclosure
more apparent, the present disclosure will be further described in detail below with reference to
examples. The exemplary implementations and descriptions thereof in the present disclosure are
only used to explain the present disclosure, and are not intended to limit the present disclosure.
Example:
A method for tissue cultivation of virus-free Radix pseudostellariae root tubers included the
following steps:
Sl: Treatment and disinfection of explants
Radix pseudostellariaewith robust buds was refrigerated in a refrigeration environment at 1C
to 4°C, and after the buds of Radix pseudostellariaegerminated, germinated buds were cut and used
as explants. The germinated buds were thoroughly rinsed with tap water, air-dried, soaked in
alcohol for 30 s and then in 0.1% mercuric chloride for 7 min to 8 min on a clean bench, rinsed 3 to
5 times with sterile water, and then transferred to filter paper in a sterile petri dish for later use. The
explants treated in this step had a contamination rate not exceeding 10%.
S2: Induction of Radix pseudostellariaestem apex
The explants treated in step Si were placed under a stereoscope, a 0.5 mm Radix
pseudostellariaestem apex was cut off from the explants, inoculated on the induction medium, and
then cultivated under the following conditions: cultivation temperature: 15°C to 20°C, light
intensity: 1,500 lx to 2,000 lx, and photoperiod: 10 h/d. After 20 d to 25 d of cultivation, 1 to 2
Radix pseudostellariaeadventitious buds germinated on the Radix pseudostellariaestem apex. The
induction medium in this step was a modified MS medium, specifically an MS medium
supplemented with 0.5 mg/L to 1 mg/L 6-BA, 0.1 mg/L NAA, 30 g/L sucrose, and 5 g/L agar
powder, with a pH value of 5.8. The 6-BA refers to 6-benzylaminopurine and the NAA refers
naphthaleneacetic acid. An induction rate for Radix pseudostellariae adventitious buds was more
than 90% in this step.
S3: Propagation cultivation of Radix pseudostellariaeseedlings
The Radix pseudostellariae adventitious buds induced in step S2 were transferred to a
propagation medium and cultivated under the following conditions: cultivation temperature: 150 C
to 200 C, light intensity: 1,500 lx to 2,000 lx, and photoperiod: 10 h/d. Radix pseudostellariae
seedlings, after growing tall and strong (growing to have a height of 4 cm to 6 cm in this example),
were cut into 4 to 5 sections, and the above transferring and propagation cultivation process was repeated for the Radix pseudostellariae seedling sections. The transferring and propagation cultivation may have a cycle of 18 d/generation to 24 d/generation, that is, one generation is transferred and propagated every 18 d to 24 d. In this example, the transferring and propagation cultivation had a cycle of 20 d/generation, and a propagation number for a generation increased by
4 to 5 times generation by generation. The propagation medium in this step was a modified 1/2 MS
medium, specifically a 1/2 MS medium supplemented with 85 mg/L MKP, 30 g/L sucrose, and 5
g/L agar powder, with a pH value of 5.8. The modified 1/2 MS medium in this step was not added
with hormones, but added with MKP, which allowed Radix pseudostellariaeseedlings to grow taller
and stronger have more stretched and vigorous leaves.
S4: Cultivation of Radix pseudostellariaemicro-root tubers in bottles
The Radix pseudostellariae seedlings obtained in step S3 were transferred to a root tuber
medium and cultivated at 15°C to 20°C. After the seedlings were cultivated for 60 d (generally 50 d
to 60 d) with a tissue cultivation lamp in the cultivation room being off, 2 to 3 root tubers grew
from a stem node of Radix pseudostellariae. After growing to have a diameter of 0.5 cm or more,
the root tubers were collected and cleaned, then refrigerated at 1C to 4°C for 20 d to 30 d, directly
planted in fields, and then harvested to be used as Radix pseudostellariaeseed root tubers. Most of
the seed root tubers finally harvested were free of viruses, exhibiting a high detoxification rate. The
root tuber medium in this step was a modified 1/2MS medium, specifically an MS medium
supplemented with 85 mg/L MKP, 60 g/L sucrose, and 5 g/L agar powder, with a pH value of 5.8.
The addition of 85 mg/L MKP and 60 g/L sucrose in the medium helped root tubers to swell. In
addition, in this step, there was no need to turn on the tissue cultivation lamp, greatly reducing a
production cost.
The above is an example of the present disclosure. The above example and the specific
parameters in the example are provided merely for clearly expressing a verification process of the
present disclosure, not for limiting the protection scope of the present disclosure. The protection
scope of the present disclosure is still subject to the claims. Any equivalent structural changes made
from the content of the specification of the present disclosure should be included in the protection
scope of the present disclosure.
Claims (3)
1. A medium combination for tissue cultivation of virus-free seed root tubers (micro-root
tubers) of virus-free Radix pseudostellariae, comprising a virus-free tissue cultivation seedling
induction medium, a virus-free tissue cultivation seedling propagation medium, and a micro-root
tuber induction and swelling medium; wherein,
the virus-free tissue cultivation seedling induction medium is an MS medium supplemented
with 0.5 mg/L to 1 mg/L 6-BA, 0.1 mg/L NAA, 30 g/L sucrose, and 5 g/L agar powder, which has a
pH value of 5.8;
the virus-free tissue cultivation seedling propagation medium is a 1/2 MS medium
supplemented with 85 mg/L monopotassium phosphate (MKP), 30 g/L sucrose, and 5 g/L agar
powder, which has a pH value of 5.8; and
the micro-root tuber induction and swelling medium is an MS medium supplemented with
85 mg/L MKP, 60 g/L sucrose, and 5 g/L agar powder, which has a pH value of 5.8.
2. The medium combination for tissue cultivation according to claim 1, wherein, the virus-free
tissue cultivation seedling induction medium is an MS medium supplemented with 0.6 mg/L to 0.9
mg/L 6-BA, 0.1 mg/L NAA, 30 g/L sucrose, and 5 g/L agar powder.
3. The medium combination for tissue cultivation according to claim 1, wherein, the virus-free
tissue cultivation seedling induction medium is an MS medium supplemented with 0.7 mg/L to 0.8 mg/L 6-BA, 0.1 mg/L NAA, 30 g/L sucrose, and 5 g/L agar powder.
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| AU2021101772A AU2021101772A4 (en) | 2021-04-07 | 2021-04-07 | Medium combination for tissue cultivation of virus-free seed root tubers (micro-root tubers) of virus-free radix pseudostellariae |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| AU2021101772A AU2021101772A4 (en) | 2021-04-07 | 2021-04-07 | Medium combination for tissue cultivation of virus-free seed root tubers (micro-root tubers) of virus-free radix pseudostellariae |
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| AU2021101772A4 true AU2021101772A4 (en) | 2021-05-27 |
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2021
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