A method for rapid propagation of aseptic seedling by using buds of epimedium
By using flower buds of Rhododendron molle as explants and employing steps such as ethanol disinfection and differentiation culture, the problem of obtaining sterile seedlings of Rhododendron molle quickly has been solved, achieving efficient sterile seedling propagation and promoting Rhododendron molle breeding and research.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG FORESTRY UNIVERSITY
- Filing Date
- 2024-01-11
- Publication Date
- 2026-07-24
AI Technical Summary
Rhododendron molle is difficult to propagate by cuttings and is hard to root. In existing technologies, the leaves and stem segments of Rhododendron molle are seriously contaminated with microorganisms, making it difficult to obtain sterile seedlings quickly, and it is also difficult to regenerate plants from flower organs.
Using flower buds of Rhododendron molle as explants, sterile plants were directly induced through steps such as differentiation culture, subculture culture, proliferation culture, and rooting culture.
It significantly shortened the seedling cycle of aseptic seedlings of Rhododendron molle, improved reproductive efficiency, and provided convenience for Rhododendron molle breeding and genetic transformation research.
Smart Images
Figure CN117652419B_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a method for rapidly propagating sterile seedlings using the flower buds of Rhododendron molle. Background Technology
[0002] Azaleas are one of China's ten traditional and precious flowers. With their diverse colors and long blooming period, they are widely used in landscaping, including planting in the ground, along urban roads, in scenic areas, and in residential communities, earning them the title of "King of Flowers and Trees." *Rhododendron molle (Blume) G. Don*, belonging to the genus *Rhododendron* in the family Ericaceae, is a deciduous shrub and one of the very few yellow-flowered species in the *Rhododendron* genus, possessing high ornamental value. However, *Rhododendron molle* is difficult to propagate by cuttings and struggles to root, severely limiting its widespread application in production. Chinese patent documents CN105830917A and CN106613973A respectively disclose a method for rapid tissue culture propagation of Rhododendron molle, both of which use leaves of sterile Rhododendron molle seedlings as explants for tissue culture. Chinese patent document CN105532467A discloses a method for rapid tissue propagation using axillary buds of Rhododendron molle as explants. These technical methods have initially solved the problem of rapid propagation of Rhododendron molle, but because the surface of Rhododendron molle leaves and stem segments is hairy, microorganisms can easily adhere to them, increasing the difficulty of cleaning. In scientific research and production, it is often necessary to collect leaves or stem segments of *Rhododendron molle* from the wild for tissue culture. However, thorough disinfection of explants using disinfectants is difficult, and after inoculation with culture medium, fungi or bacteria attached to the leaves or stem segments can easily cause repeated contamination, making it difficult to quickly obtain sterile seedlings. Often, it is necessary to subculture the leaves or stem segments multiple times, followed by selection culture, to gradually eliminate bacteria from the explants and finally obtain sterile seedlings. When plants are regenerated through floral organs, the tissue cells are prone to browning, making it difficult to obtain regenerated plants. A method for directly inducing adventitious bud regeneration of *Rhododendron molle* through flower buds has not yet been reported. This invention uses Rhododendron molle flower buds as explants and employs ethanol, a low-toxicity disinfectant, for sterilization. This completes the sterilization process in one step, directly inducing the primary generation of sterile plants. Compared to traditional tissue culture using highly toxic mercuric chloride for sterilization, this method is safer and more environmentally friendly. It also significantly shortens the seedling growth cycle of Rhododendron molle sterile seedlings, providing a new approach for the rapid propagation of superior Rhododendron molle varieties and laying the foundation for Rhododendron molle breeding and genetic transformation research. Summary of the Invention
[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for rapidly propagating sterile seedlings using Rhododendron molle flower buds, which significantly shortens the seedling cultivation cycle of Rhododendron molle sterile seedlings.
[0004] The technical solution adopted in this invention is as follows:
[0005] A method for rapidly propagating sterile seedlings using Rhododendron molle flower buds, comprising the following steps:
[0006] (1) Obtaining sterile flower buds: Select healthy and disease-free Rhododendron plants, collect flower buds, disinfect them, peel off the outer scales of the flower buds, cut off the internal tissue of the flower buds with a sterile scalpel, and inoculate them into a pre-culture medium for culture.
[0007] (2) Differentiation culture: The flower buds from step (1) were transferred to the differentiation medium for adventitious bud induction;
[0008] (3) Subculture: The flower buds from step (2) were inoculated into the subculture medium to continue the induction of callus and adventitious buds;
[0009] (4) Proliferation medium: The budded callus tissue induced to differentiate in step (3) is inoculated onto the proliferation medium for proliferation culture;
[0010] (5) Rooting culture: Inoculate the adventitious shoots proliferated in step (4) into the rooting culture medium and culture under light after inoculation;
[0011] (6) Hardening and transplanting: Select healthy plants from the tissue culture seedlings obtained in step (5) for hardening and transplanting.
[0012] Preferably, in step (1), flower buds with a length of 5-15 mm are selected and sterilized with 60%-80% ethanol solution with 0.1%-1% Silwett added for 10-20 minutes. The culture conditions are: dark culture for 5-15 days, temperature 22-26℃, humidity 50-60%, and the pre-culture medium is: 1 / 2 WPM + 0.1-1.0 mg / L 2,4-D + 25-35 g / L sucrose + 5.0-8.0 g / L agar, pH 5.3-5.7.
[0013] Preferably, the culture conditions in step (2) are: light intensity 10-14 h / day, light intensity 4000-6000 lx, temperature 22-26℃, humidity 50-60%, culture until the flower bud cut ends and the curling intensifies and granular callus tissue begins to appear. The differentiation medium is: WPM + 0.01-0.6 mg / L TDZ + 0.01-0.5 mg / L IBA + 25-35 g / L sucrose + 5.0-8.0 g / L agar + 10 g-20 g / L coconut juice, pH 5.3-5.7.
[0014] Preferably, the culture conditions in step (3) are: light intensity 10-14 h / day, light intensity 4000-6000 lx, temperature 22-26℃, humidity 50-60%, cultured until adventitious buds grow from the callus tissue at the flower bud incision site, wherein the subculture medium is: WPM + 0.1-0.6 mg / L TDZ + 0.1-0.8 mg / L IBA + 25-35 g / L sucrose + 5.0-8.0 g / L agar + 10 g-20 g / L coconut juice, pH 5.3-5.7.
[0015] Preferably, the culture conditions in step (4) are: light intensity of 120-14 h / day, light intensity of 4000-6000 lx, temperature of 22-26℃, humidity of 50-60%, cultured until the callus tissue with adventitious buds grows into clustered buds, and the proliferation medium is: WPM + 2.0-10.0 mg / L ZT + 0.1-1.0 mg / L NAA + 20-30 g / L sucrose + 5.0-8.0 g / L agar, pH 5.3-5.7.
[0016] Preferably, in step (5), the adventitious buds are cultured to a height of 2.0-3.0 cm and have grown fibrous roots. The rooting medium is: 1 / 4 WPM + 0.2-2.0 mg / L NAA + 20-30 g / L sucrose + 4.0-6.0 g / L agar + 1-3 g / L activated carbon, with a pH of 5.3-5.7.
[0017] Preferably, step (6) is as follows: Select tissue culture seedlings with strong root systems and a plant height of 2.0 to 3.0 cm, harden the seedlings for 7 to 10 days, wash off the agar from the roots of the tissue culture seedlings, and then transplant them into a moistened mixed substrate of perlite and vermiculite in a ratio of 1:1 to 4:1. Spray the seedlings regularly, cover them with plastic film for heat preservation and moisture retention, and then gradually remove the film over the next few days to allow ventilation and light. After 25 to 40 days, transplant them into a mixed substrate of peat moss and rice husks in a ratio of 2:1 to 5:1. The transplanting operation is carried out at a temperature of 23 to 26°C. After planting, water thoroughly and pay attention to keeping the substrate moist during daily maintenance.
[0018] This invention uses flower buds of *Rhododendron molle* as explants and obtains sterile *Rhododendron molle* plants through differentiation culture, subculture culture, proliferation culture, rooting culture, and hardening-off transplanting. This solves the problems of incomplete sterilization, easy contamination, and difficulty in obtaining sterile seedlings when using stems and leaves as explants for tissue culture. It provides a new approach for the rapid propagation of sterile *Rhododendron molle* seedlings and creates favorable conditions for *Rhododendron molle* breeding and genetic transformation research. Using flower buds as explants, this invention can obtain a large number of sterile plants in a short time with a high propagation coefficient, which is beneficial for conducting *Rhododendron molle* breeding research and standardized seedling production in enterprises. Attached Figure Description
[0019] Figure 1 To obtain photographs of sterile flower buds;
[0020] Figure 2 Photographs showing the induction of adventitious buds from flower buds;
[0021] Figure 3 A photograph of the adventitious bud proliferation process;
[0022] Figure 4 A photo of seedlings taking root. Detailed Implementation
[0023] The present invention will be further described below with reference to the embodiments, but the description of the embodiments does not limit the scope of protection of the present invention in any way.
[0024] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the invention.
[0025] In the following examples, unless otherwise specified, all reagents and consumables were purchased from conventional reagent manufacturers in the art; unless otherwise specified, the experimental methods and techniques used are conventional methods and techniques in the art.
[0026] Example 1
[0027] The method for rapidly propagating sterile seedlings using Rhododendron molle flower buds includes the following steps:
[0028] (1) Obtaining sterile flower buds: Select robust, disease-free Rhododendron molle plants, collect flower buds 5 mm in length, disinfect with 60% ethanol solution containing 0.1% Silwett for 15 minutes, peel off the outer scales of the flower buds, cut the internal tissue of the flower buds with a sterile scalpel, and inoculate them into a pre-culture medium. Photographs of the flower buds are shown below. Figure 1 As shown. The pre-culture medium was: 1 / 2 WPM + 0.1 mg / L 2,4-D + 25 g / L sucrose + 5.0 g / L agar, pH 5.3; incubated in the dark for 5 days.
[0029] (2) Differentiation culture: The flower buds from step (1) were inoculated into differentiation medium for callus and bud induction. After inoculation, the medium was placed under 10 h / day light, 4000 lx light intensity, 22℃ temperature, and 50% humidity. After 20 days of induction, the cut surface of the flower buds began to swell, with a small amount of granular callus tissue. The differentiation medium was: WPM + 0.1 mg / L TDZ + 0.01 mg / L IBA + 25 g / L sucrose + 5.0 g / L agar + 10 g / L coconut juice, pH 5.3.
[0030] (3) Subculture: After 25 days of flower bud culture in step (2), the buds were inoculated into a subculture medium for adventitious bud induction. After inoculation, the medium was placed under 10 hours of light per day, with a light intensity of 4000 lx, a temperature of 22°C, and a humidity of 50%. After 20 days, buds emerged from the granular callus tissue and gradually grew into green shoots. After 40 days, the adventitious bud differentiation rate was 86.53%. The subculture medium consisted of: WPM + 0.05 mg / L TDZ + 0.1 mg / L IBA + 25 g / L sucrose + 5.0 g / L agar + 10 g / L coconut milk, with a pH of 5.3. A photograph of the flower buds is shown below. Figure 2 As shown, adventitious bud differentiation can be seen after differentiation culture and subculture.
[0031] (4) Proliferation medium: The callus tissue with buds induced in step (3) was inoculated into a proliferation medium for proliferation culture. After inoculation, the light intensity was 4000 lx for 10 h / day, the temperature was 22℃, and the humidity was 50%. After 40 days, the proliferation coefficient of adventitious buds was 6.1. The proliferation medium consisted of WPM + 2.0 mg / L ZT + 0.2 mg / L NAA + 30 g / L sucrose + 5.0 g / L agar, with a pH of 5.4. Photos of the proliferated adventitious buds are shown below. Figure 3 As shown, more robust adventitious buds were successfully induced.
[0032] (5) Rooting Culture: The adventitious shoots proliferated in step (4) were inoculated into the rooting medium. After inoculation, the light intensity was 6000 lx for 14 h / day, the temperature was 22℃, and the humidity was 55%. After 40 days, the rooting rate was 92.32%. The rooting medium consisted of 1 / 4 WPM + 0.2 mg / L NAA + 20 g / L sucrose + 6.0 g / L agar + 1 g / L activated carbon, with a pH of 5.6. Photos of the tissue culture seedlings are shown below. Figure 4 As shown, roots grow from the base of the adventitious buds after rooting culture, and there are no traces of bacterial growth on the surface of the culture medium.
[0033] (6) Hardening off and transplanting: Select tissue culture seedlings with strong root systems induced in step (5) and a plant height of 2.5cm. Open the cap of the culture bottle and harden off the seedlings for 7 days. Then wash off the agar from the roots of the tissue culture seedlings and transplant them into a moistened mixed substrate of perlite:vermiculite = 2:1. Spray the seedlings regularly and cover them with plastic film for warmth and moisture retention. Gradually remove the film over the next few days to allow ventilation and light. After 25 days, transplant them into a mixed substrate of peat moss:rice husk = 3:1. Water thoroughly after planting and keep the substrate moist during daily maintenance. After 30 days, the survival rate of Rhododendron molle was 94%.
[0034] Example 2
[0035] The method for rapidly propagating sterile seedlings using Rhododendron molle flower buds includes the following steps:
[0036] (1) Obtaining sterile flower buds: Select robust, disease-free Rhododendron molle plants, collect flower buds 10 mm in length, disinfect with 75% ethanol solution with 0.4% Silwett for 10 minutes, peel off the outer scales of the flower buds, and use a sterile scalpel to cut out the internal tissue of the flower buds and inoculate them into a pre-medium. The pre-medium was: 1 / 2 WPM + 0.5 mg / L 2,4-D + 30 g / L sucrose + 7.0 g / L agar, pH 5.3; incubate in the dark for 10 days.
[0037] (2) Differentiation culture: The flower buds from step (1) were inoculated into differentiation medium for callus and bud induction. After inoculation, the medium was placed under 12 hours of light per day, with a light intensity of 5000 lx, a temperature of 24°C, and a humidity of 55%. After 20 days of induction, the cut surfaces of the flower buds began to swell, with a small amount of granular callus tissue. The differentiation medium consisted of WPM + 0.3 mg / L TDZ + 0.1 mg / L IBA + 30 g / L sucrose + 7.0 g / L agar + 15 g / L coconut juice, with a pH of 5.5.
[0038] (3) Subculture: After 25 days of flower bud culture in step (2), the buds were inoculated into a subculture medium for bud induction. After inoculation, the medium was placed under a light source of 12 h / day, a light intensity of 5000 lx, a temperature of 24℃, and a humidity of 55%. After 20 days, buds emerged from the granular callus tissue and gradually grew into green shoots. After 40 days, the adventitious bud differentiation rate was 89.23%. The subculture medium was: WPM + 0.1 mg / L TDZ + 0.4 mg / L IBA + 30 g / L sucrose + 7.0 g / L agar + 15 g / L coconut juice, with a pH of 5.5.
[0039] (4) Proliferation medium: The callus tissue with buds induced in step (3) was inoculated into a proliferation medium for proliferation culture. After inoculation, the light intensity was 5000 lx for 12 h / day, the temperature was 24℃, and the humidity was 54%. After 40 days, the adventitious bud proliferation coefficient was 8.3. The proliferation medium consisted of WPM + 6.0 mg / L ZT + 0.6 mg / L NAA + 30 g / L sucrose + 7.0 g / L agar, with a pH of 5.5.
[0040] (5) Rooting culture: The adventitious shoots proliferated in step (4) were inoculated into the rooting medium. After inoculation, the light intensity was 6000 lx for 14 h / day, the temperature was 24℃, and the humidity was 55%. After 40 days, the rooting rate was 94.22%, and there were no traces of bacterial growth on the surface of the medium. The rooting medium was: 1 / 4 WPM + 0.4 mg / L NAA + 30 g / L sucrose + 6.0 g / L agar + 2 g / L activated carbon, with a pH of 5.6.
[0041] (6) Hardening off and transplanting: Select tissue culture seedlings with robust root systems induced in step (5) and a plant height of 2.5cm. Open the cap of the culture bottle and harden off the seedlings for 7 days. Then wash off the agar from the roots of the tissue culture seedlings and transplant them into a moistened mixed substrate of perlite:vermiculite = 2:1. Spray the seedlings regularly and cover them with plastic film for warmth and moisture retention. Gradually remove the film over the next few days to allow ventilation and light. After 25 days, transplant them into a mixed substrate of peat moss:rice husks = 4:1. Water thoroughly after planting and keep the substrate moist during daily maintenance. After 30 days, the survival rate of the Rhododendron molle was 96%.
[0042] Example 3
[0043] The method for rapidly propagating sterile seedlings using Rhododendron molle flower buds includes the following steps:
[0044] (1) Obtaining sterile flower buds: Select robust, disease-free Rhododendron molle plants, collect flower buds 15 mm in length, disinfect with 80% ethanol solution with 1% Silwett for 10 minutes, peel off the outer scales of the flower buds, and use a sterile scalpel to cut out the internal tissue of the flower buds and inoculate them into a pre-medium. The pre-medium was: 1 / 2 WPM + 1.0 mg / L 2,4-D + 35 g / L sucrose + 7.0 g / L agar, pH 5.7; incubate in the dark for 15 days.
[0045] (2) Differentiation culture: The flower buds from step (1) were inoculated into differentiation medium for callus and bud induction. After inoculation, the medium was placed under light for 14 hours / day, with a light intensity of 6000 lx, a temperature of 26℃, and a humidity of 60%. After 20 days of induction, the cut surface of the flower buds began to swell, with a small amount of granular callus tissue. The differentiation medium was: WPM + 0.4 mg / L TDZ + 0.5 mg / L IBA + 35 g / L sucrose + 7.5 g / L agar + 20 g / L coconut juice, with a pH of 5.7.
[0046] (3) Subculture: After 25 days of flower bud culture in step (2), the buds were inoculated into a subculture medium for bud induction. After inoculation, the medium was placed under light for 14 hours / day, with a light intensity of 6000 lx, a temperature of 26℃, and a humidity of 60%. After 20 days, buds emerged from the granular callus tissue and gradually grew into green shoots. After 40 days, the adventitious bud differentiation rate was 87.33%. The subculture medium was: WPM + 0.2 mg / L TDZ + 0.6 mg / L IBA + 35 g / L sucrose + 7.5 g / L agar + 20 g / L coconut juice, with a pH of 5.7.
[0047] (4) Proliferation medium: The callus tissue with buds induced in step (3) was inoculated into a proliferation medium for proliferation culture. After inoculation, the light intensity was 6000 lx for 14 h / day, the temperature was 26℃, and the humidity was 60%. After 40 days, the adventitious bud proliferation coefficient was 7.2. The proliferation medium consisted of WPM + 8.0 mg / L ZT + 0.8 mg / L NAA + 35 g / L sucrose + 7.5 g / L agar, with a pH of 5.7.
[0048] (5) Rooting culture: The adventitious shoots proliferated in step (4) were inoculated into the rooting medium. After inoculation, the light intensity was 6000 lx for 14 h / day, the temperature was 26℃, and the humidity was 60%. After 40 days, the rooting rate was 93.21%, and there were no traces of bacterial growth on the surface of the medium. The rooting medium was: 1 / 4 WPM + 0.6 mg / L NAA + 25 g / L sucrose + 6.0 g / L agar + 3 g / L activated carbon, with a pH of 5.6.
[0049] (6) Hardening off and transplanting: Select tissue culture seedlings with robust root systems induced in step (5) and a plant height of 2.5cm. Open the cap of the culture bottle and harden off for 7 days. Then wash off the agar from the roots of the tissue culture seedlings and transplant them into a moistened mixed substrate of perlite:vermiculite = 3:1. Spray the seedlings regularly and cover them with plastic film for warmth and moisture retention. Gradually remove the film over the next few days to allow ventilation and light. After 25 days, transplant them into a mixed substrate of peat moss:rice husks = 5:1. Water thoroughly after planting and keep the substrate moist during daily maintenance. After 30 days, the survival rate of Rhododendron molle was 95%.
Claims
1. A method for rapidly propagating sterile seedlings using flower buds of Rhododendron molle, comprising the following steps: (1) Obtaining sterile flower buds: Select healthy and disease-free Rhododendron molle plants, collect flower buds, disinfect them, peel off the outer scales of the flower buds, cut off the internal tissue of the flower buds with a sterile scalpel, and inoculate them into the pre-culture medium for culture. Disinfection means disinfecting with 60%~80% ethanol solution with 0.1%~1% Silwett for 10~20 minutes. The pre-culture medium is: 1 / 2 WPM + 0.1~1.0 mg / L 2,4-D + 25~35 g / L sucrose + 5.0~8.0 g / L agar, pH 5.3~5.7; (2) Differentiation culture: The flower buds from step (1) were transferred to the differentiation medium for bud induction. The differentiation medium was: WPM + 0.01~0.6 mg / L TDZ + 0.01~0.5 mg / L IBA + 25~35 g / L sucrose + 5.0~8.0 g / L agar + 10 g~20 g / L coconut juice, pH 5.3~5.7; (3) Subculture: The flower buds from step (2) were inoculated into the subculture medium to continue the induction of callus and buds. The subculture medium was: WPM + 0.1~0.6 mg / L TDZ + 0.1~0.8 mg / L IBA + 25~35 g / L sucrose + 5.0~8.0 g / L agar + 10 g~20 g / L coconut juice, pH 5.3~5.7; (4) Proliferation medium: The callus tissue with buds induced in step (3) was inoculated into the proliferation medium for proliferation culture. The proliferation medium was: WPM + 2.0~10.0 mg / L ZT + 0.1~1.0 mg / L NAA + 20~30 g / L sucrose + 5.0~8.0 g / L agar, pH 5.3~5.7; (5) Rooting culture: The adventitious shoots proliferated in step (4) were inoculated into the rooting medium and cultured under light after inoculation. The rooting medium was: 1 / 4 WPM + 0.2~2.0 mg / L NAA + 20~30 g / L sucrose + 4.0~6.0 g / L agar + 1~3 g / L activated carbon, pH 5.3~5.7; (6) Hardening and transplanting: Select healthy plants from the tissue culture seedlings obtained in step (5) for hardening and transplanting.
2. The method for rapid propagation of sterile seedlings using Rhododendron molle flower buds according to claim 1, characterized in that: In step (1), flower buds with a length of 5-15 mm are selected and cultured under the following conditions: dark culture for 5-15 days, temperature 22-26 ℃, humidity 50-60%.
3. The method for rapid propagation of sterile seedlings using Rhododendron molle flower buds according to claim 2, characterized in that: The cultivation conditions in step (2) are: light exposure 10-14 h / day, light intensity 4000-6000 lx, temperature 22-26 ℃, humidity 50-60%, and the cultivation continues until the flower bud cut surface curls more intensely and granular callus tissue begins to appear.
4. The method for rapid propagation of sterile seedlings using Rhododendron molle flower buds according to claim 3, characterized in that: The cultivation conditions in step (3) are: light 10~14 h / day, light intensity 4000~6000 lx, temperature 22~26 ℃, humidity 50~60%, and cultivation until adventitious buds grow from the callus tissue at the flower bud incision site.
5. The method for rapid propagation of sterile seedlings using Rhododendron molle flower buds according to claim 4, characterized in that: The cultivation conditions in step (4) are: 120-14 h / day of light, 4000-6000 lx of light intensity, 22-26 ℃ of temperature, and 50-60% of humidity, until the callus tissue with adventitious buds grows into clustered buds.
6. The method for rapid propagation of sterile seedlings using Rhododendron molle flower buds according to claim 5, characterized in that: In step (5), the adventitious buds are cultivated until they reach a height of 2.0~3.0 cm and develop fibrous roots.
7. The method for rapidly propagating sterile seedlings using Rhododendron molle flower buds according to claim 6, characterized in that: Step (6) is as follows: Select tissue culture seedlings with strong root systems and a plant height of 2.0~3.0cm, harden them for 7~10 days, wash off the agar from the roots of the tissue culture seedlings, and then transplant them into a moistened mixed substrate of perlite:vermiculite = 1:1~4:
1. Spray the seedlings regularly, cover them with plastic film to keep them warm and moist, and then gradually remove the film over the next few days to allow ventilation and light. After 25~40 days, transplant them into a mixed substrate of peat moss:rice husk = 2:1~5:
1. The transplanting operation is carried out at a temperature of 23~26℃. After planting, water thoroughly and pay attention to keeping the substrate moist during daily maintenance.