A method for promoting induction and growth of single-sided needle hairy roots
By optimizing the transformation of single-sided needle hairy roots through the oblique cutting-cutting method, the problems of long transformation cycle and low efficiency were solved, and efficient and stable hairy root induction and increase of secondary metabolites were achieved, which is suitable for industrial application.
Patent Information
- Application Number
- CN202311515393.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2023-11-15
- Publication Date
- 2025-10-21
- Estimated Expiration
- 2043-11-15
AI Technical Summary
In the existing technology, the transformation cycle of single-sided needle hairy roots is long, the efficiency is low, the content of secondary metabolites is unstable, the operation steps are cumbersome and easy to be contaminated, and it is difficult to meet the needs of industrialization.
The oblique cutting-cutting method is used for Agrobacterium rhizogenes infection. The specific steps include making an oblique cut on the hypocotyl of the nelumbo nucifera seedlings and soaking them in Agrobacterium rhizogenes liquid, then culturing them under specific conditions, using acetosyringone and rifampicin to optimize the liquid, and inserting them into sterile sand to induce hairy roots, simplifying the operation process and improving the transformation efficiency.
It shortens the time for hairy root production, improves conversion efficiency and secondary metabolite content, reduces human pollution, is suitable for industrial cultivation, and the hairy root induction rate can reach more than 75%, and grows faster.
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Figure CN117802144B_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a hairy root induction transformation method mediated by Agrobacterium rhizogenes, and in particular to a hairy root transformation method of scapularis. Background Art
[0002] In 1907, Smith and Townsend discovered that Agrobacterium rhizogenes could infect plants and induce the production of hairy roots. Secondary metabolites are typically present in very low concentrations in plants, often induced by environmental factors. However, the rapid growth and proliferation of transformed hairy roots suggest the potential for a stable increase in plant production of secondary metabolites.
[0003] The medicinal plant Zanthoxylum dissitum Hemsl., also known as "Clamshell Pepper," is a woody vine in the genus Zanthoxylum of the Rutaceae family that grows in mountain forests. It is a near-endangered rare forest medicinal resource. Its roots, stems, and leaves are all used medicinally, with benefits such as promoting blood circulation, dispersing blood stasis, and repairing tendons and bones. It is widely used to treat female ailments, toothache, back pain, limb pain, and arthritis. It is the main ingredient in the traditional Chinese medicines Fuke Qianjin Tablets and Fuke Qianjin Capsules. Zanthoxylum dissitum Hemsl. plants of the Rutaceae family are rich in isoquinoline alkaloids, which exhibit anticancer, antibacterial, analgesic, hepatoprotective, antipyretic, and antitumor properties. They are widely used worldwide to treat a variety of ailments, including digestive and gastrointestinal disorders, bacterial and viral diseases, neurological disorders, and other diseases. However, the long growth cycle of Zanthoxylum dissitum, coupled with the influence of numerous factors such as cultivar, climate change, and pests and diseases, leads to variable alkaloid content. This results in varying quality and a limited supply of Zanthoxylum dissitum. To date, few methods involving the transformation system of hairy roots in Rutaceae plants have been reported. Therefore, it is necessary to find or develop more effective and easier-to-use Agrobacterium rhizogenes-mediated hairy root transformation methods for S. sutchuenensis, providing a convenient way to study the gene functions of S. sutchuenensis and laying the foundation for in-depth research on the genetic transformation of S. sutchuenensis. Summary of the Invention
[0004] To overcome the shortcomings and deficiencies of the prior art, the present invention aims to provide a method for genetic transformation of hairy roots of S. unilateralis mediated by Agrobacterium rhizogenes. The technical problems to be solved are: shortening the hairy root transformation cycle, improving the efficiency of hairy root production, increasing the content of secondary metabolites, reducing the number of operational steps, and minimizing human contamination.
[0005] A method for promoting the induction and growth of hairy roots of unilateral needles, the specific technical solution is as follows:
[0006] The steps include:
[0007] (1) Agrobacterium rhizogenes infects sterile seedlings of nelumbo nucifera;
[0008] (2) After infection, hairy roots of the seedlings of S. unicornis were induced to grow.
[0009] The method described,
[0010] Cut the hypocotyl of the sterile seedling 0.3-0.6 cm above the bottom end (most preferably 0.45-0.55 cm), discard the lower part, soak the cut in the root-inducing Agrobacterium solution added with AS, and then insert the seedling into the culture medium to induce hairy roots.
[0011] There is a relatively obvious dividing line between the primary root and the hypocotyl, and the incision is located at the hypocotyl 0.3-0.6 cm above the dividing line.
[0012] The method described,
[0013] When cutting, cut at an angle to increase the contact area of the explant. The angle of the cut should be 35 to 55 degrees to the hypocotyl, preferably 45 degrees.
[0014] The method described,
[0015] The incision is immersed in the Agrobacterium rhizogenes solution after AS is added for 8-12 minutes (preferably 10 minutes), and then the seedling is inserted into the culture medium to induce hairy roots.
[0016] The method described,
[0017] The Agrobacterium rhizogenes infection solution used in step (1) is prepared as follows:
[0018] Pick a single colony and culture it in Agrobacterium rhizogenes liquid culture medium. 600nm When the OD reaches 0.5-0.7, the bacterial solution is inoculated into a 1-3 times volume of Agrobacterium rhizogenes liquid culture medium, and acetosyringone is added and cultured until the OD 600nm Reach 0.6-0.8; centrifuge the bacterial solution, discard the supernatant, resuspend the precipitate with liquid culture medium, add acetosyringone and culture to obtain the bacterial solution used for infection.
[0019] Furthermore,
[0020] Rifampicin is added to the liquid culture medium of Agrobacterium rhizogenes at a final concentration of 50 to 100 mg / L, preferably YEB liquid culture medium, and acetosyringone is added to the culture medium at a final concentration of 100 to 200 μmol / L, preferably MS liquid culture medium. The precipitate is resuspended, and after adding acetosyringone, the culture is further cultured for 25 to 40 minutes to obtain a bacterial solution for infection.
[0021] Furthermore,
[0022] Agrobacterium rhizogenes stored in a -80°C ultra-low temperature freezer was inoculated into YEB liquid medium with a final concentration of 50 mg / L rifampicin at a ratio of 1:1000 on a clean bench. The medium was cultured at 28°C and shaken at 200 r / min for 14-16 h. 600nm When the OD value reaches 0.5-0.7, the bacterial solution is inoculated into YEB medium containing 50 mg / mL rifampicin at a concentration of 2 times the bacterial solution volume, and acetosyringone is added at a final concentration of 100 μmol / L. The culture is continued for 1-2 hours until the OD value reaches 0.5-0.7. 600nm When the pH value reaches 0.6-0.8, centrifuge at 6000 rpm for 10 min, discard the supernatant, resuspend the precipitate in MS liquid culture medium containing 100 μmol / L acetosyringone, and culture for another 30 min to obtain the bacterial solution used for infection.
[0023] Furthermore,
[0024] The precipitate was resuspended in MS liquid culture medium containing 100 μmol / L acetosyringone and cultured in a shaking incubator at 28°C and 90-150 rpm for 30 min.
[0025] The method described,
[0026] When the infected seedlings are inserted into the culture medium to induce hairy roots, the inoculated nelumbo nucifera seedlings are transplanted into sterile sand with a sand humidity of 80-90%, an air humidity of 60-80%, and a temperature of 24-26°C. The sand is placed under a photoperiod of 14-16 hours and a light intensity of 1500-3500 lx for cultivation until hairy roots grow.
[0027] Furthermore,
[0028] The infected seedlings were inserted into pre-inserted sterile sand and the cut was covered with sand. Each seedling was watered with MS liquid culture medium containing 100-200 μmol / L AS and cultured in a light incubator. After about 30 days, the cut was seen to swell and grow hairy roots.
[0029] Furthermore, the sterile seedling culture of the single-sided needle:
[0030] The seeds of the nelumbo serrata were shelled, treated with gibberellin and disinfected, and then germinated on MS solid culture medium in an incubator at 24-26°C in the dark. After two cotyledons grew out, they were moved into sterile sand and grown in a light cycle to obtain sterile seedlings of the nelumbo serrata.
[0031] Preferably, the gibberellin concentration is 540 mg / L, and the seeds are soaked therein for 5.5 hours.
[0032] The disinfection is to place the seeds in a 0.1% mass fraction mercuric chloride solution (HgCl2) and soak them for disinfection for 10 minutes, and then transfer them into sterile sand for cultivation. The cultivation conditions are a temperature of 24-26°C, a photoperiod of 14-16h, and a light intensity of 1500-3500lx.
[0033] The above operations of the present invention are all carried out under room temperature conditions on a clean bench.
[0034] Identification of the single-sided needle hairy roots of the present invention:
[0035] The genomic DNA of the grown hairy roots was extracted and PCR was used to identify the rolB gene of Agrobacterium rhizogenes.
[0036] Determination of hairy root alkaloid content of the present invention:
[0037] The total alkaloids and magnoline contents in hairy roots were determined by macroporous resin purification and high performance liquid chromatography, respectively.
[0038] The present invention has the following obvious beneficial effects:
[0039] (1) Saves planting substrate and space, suitable for industrial cultivation;
[0040] (2) The oblique cutting-cutting transformation method has a higher transformation efficiency. The transformation efficiency of single-sided needle hairy roots can reach more than 75%, and the hairy roots produced are more and grow faster.
[0041] (3) Shortened time for hairy root production: This method produces hairy roots 7-10 days faster than the traditional leaf disc puncture method;
[0042] (4) After the cuttings and transplanting of the present invention, sterilization is not required, which saves operation steps and time, reduces the contamination of other rhizobia, and lays a foundation for the in-depth study of single-sided needle.
[0043] (5) The present invention has found through research that pouring MS culture medium containing AS into the seedlings of L. philadelphica transplanted back into the sand after infection is more conducive to the production of hairy roots of L. philadelphica and is also beneficial to the growth of the seedlings of L. philadelphica.
[0044] (6) The hairy roots obtained by the method of the present invention have increased content of secondary metabolites. BRIEF DESCRIPTION OF THE DRAWINGS
[0045] Figure 1 In Example 1 of the present invention, the infection and cultivation process of the needle needle seedlings was carried out;
[0046] A. Newly grown seedlings with three leaves, B. Schematic diagram of the oblique cut site of the nelumbo nucifera seedling (circles indicate the range of the oblique cut site), C. Oblique cut treatment of the nelumbo nucifera seedling, D. The incision site is immersed in the infection solution of Agrobacterium rhizogenes, E. The nelumbo nucifera seedlings are cut into sand and cultured in a light incubator, F. The growth of hairy roots after 30 days of culture.
[0047] Figure 2 Example 2 of the present invention, needle seedlings with Agrobacterium rhizogenes infection 30d hairy root diagram;
[0048] Figure 3 The growth of nelumbo nucifera plants and hairy roots without AS-containing MS medium;
[0049] Figure 4 Comparative Example 1 of the present invention, needle leaves infected with Agrobacterium rhizogenes to obtain hairy roots diagram;
[0050] Figure 5 PCR detection results of the rolB gene in hairy roots of S. unilateralis (M: DNA Marker; 1: blank control; 2: negative control; 3-4: positive controls; 5: hairy roots induced by C58C1; 6: hairy roots induced by ATCC10060).
[0051] Figure 6 Determination of alkaloid content in hairy roots of S. unilateralis (A: total alkaloid content, B: magnolamine content.) DETAILED DESCRIPTION
[0052] In order to make those skilled in the art better understand the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments, but they do not constitute a limitation of the present invention. Operations not specifically described therein, such as culture medium, etc., are performed in a conventional manner.
[0053] Example 1
[0054] (1) Cultivation of sterile seedlings of single-sided needle
[0055] The outer shell of the lanceolate seed was broken by external force, and plump seeds with undamaged outer skin were selected and soaked in 540 mg / L gibberellin for 5.5 hours. Then, the seeds were soaked and disinfected in a 0.1% mercuric chloride solution (HgCl2) in a clean bench for 10 minutes. After the disinfection, they were rinsed with sterile water for 3-5 times. The seeds were taken out with tweezers, dried with sterilized filter paper, and half-buried in a culture bottle of MS basal medium without plant growth regulators with the radicle facing downward. The flask was placed in an intelligent artificial climate box and cultured under dark conditions at 25°C for about 7-10 days. After two cotyledons grew out, they were transplanted into sterile sand and grown under conditions of 25°C, a photoperiod of 16h / 8h (day / night), and a light intensity of 2000lx. Sterile lanceolate seedlings were obtained for about 30 days.
[0056] (2) Preparation of Agrobacterium rhizogenes infection solution
[0057] C58C1 Agrobacterium rhizogenes (purchased from Shanghai Weidi Biotechnology Co., Ltd.) stored in a -80°C ultra-low temperature freezer was inoculated into YEB liquid medium with a final concentration of 50 mg / L rifampicin at a ratio of 1:1000, and cultured at 28°C with a shaking incubator at 200 rpm for 14-16 hours. 600 When the OD reaches 0.5-0.7, the bacterial solution was inoculated into YEB medium (containing rif concentration 50 mg / mL) with a volume twice that of the bacterial solution, and AS (acetosyringone) with a final concentration of 100 μmol / L was added. The culture was continued for 1-2 hours until the OD 600 Reach 0.6-0.8. Centrifuge at 6000 rpm for 10 min, discard the supernatant, resuspend the precipitate with MS liquid culture medium containing 100 μmol / L acetosyringone, and culture on a shaker at 28°C and 150 rpm for 30 min to obtain the bacterial solution used for infection.
[0058] (3) Agrobacterium rhizogenes infects the explants of Pinellia ternata
[0059] Select sterile seedlings of nelumbo nucifera with good growth and three leaves, and use a sterilized blade to cut the seedling hypocotyl about 0.5 cm above the bottom end. The oblique cutting can increase the contact area of the explant. Discard the lower part, soak the incision in the resuspended bacterial solution of Agrobacterium rhizogenes after adding AS in step (2) for 10 minutes, then put the seedling into sterile sand with a hole inserted in advance, and cover the incision with sand. Pour 4 ml of MS liquid culture medium containing 100 μmol / L AS into the sand culture medium of each seedling, keep the sand in a high humidity state, with a sand humidity of 90%, place it in a light incubator with an air humidity of 70%, a photoperiod of 16h / 8h (day / night), a light intensity of 2000lx, and a temperature of 26°C for cultivation. After about 30 days, the incision can be seen to swell and grow hairy roots.
[0060] (4) Identification of single-sided needle hairy roots
[0061] The root-inducing rol gene in the T-DNA region of the Ri plasmid in Agrobacterium rhizogenes is closely related to the formation of hairy roots. In order to confirm whether the induced roots are hairy roots, it is necessary to perform molecular level detection on the hairy roots to determine whether the rol gene is inserted into the induced hairy roots. We identified the rolB gene in the T-DNA region of the Ri plasmid in C58C1, and used the DNA of the roots of sterile seedlings of uninfected P. sutchuenensis as a negative control. The rolB gene was amplified from the extracted P. sutchuenensis root DNA (the sequence of rolB-F is: 5′-GCTCTTGCAGTGCTAGATTT-3′; the sequence of rolB-R is: 5′-GAAGGTGCAAGCTACCTCTC-3′). The results are as follows Figure 5 As shown, the target fragment of rolB gene was amplified in all the induced hairy roots, while that in the negative control group was not, indicating that the rolB gene of Agrobacterium rhizogenes had been integrated into the genome of P. sutchuenensis and the induced roots were P. sutchuenensis hairy roots.
[0062] (5) Determination of alkaloid content in hairy roots
[0063] 1. Determination of total alkaloid content in hairy roots by macroporous resin purification method:
[0064] Weigh 0.1g of hairy root dry weight sample and grind it with liquid nitrogen, then ultrasonically extract it with 80% ethanol for 60min. After the ultrasonication, filter it once with filter paper, rinse the filter residue with 80% ethanol, and dilute the filtrate to 25mL for later use; weigh 2g of NKA-9 macroporous resin and soak it in 95% ethanol for 24h; rinse the soaked NKA-9 macroporous resin with deionized water until there is no alcohol smell; use the wet column method, drain the deionized water and pour the sample liquid into the ion exchange column, and perform dynamic adsorption at 2BV / h; after the adsorption is completed, take 20mL of deionized water and wash the impurities adsorbed by the macroporous resin at 3BV / h; after the washing is completed, take 25mL of 80% ethanol and desorb at 2BV / h to collect the liquid; use ultraviolet spectrophotometry to determine the total alkaloid content with solanine as the reference substance, determine the absorbance of the sample at a wavelength of 203nm, and use 80% ethanol as the blank control.
[0065] 2. Determination of magnolamine content in hairy roots by high performance liquid chromatography. The specific conditions are as follows:
[0066] Chromatographic column: Kromasil C18 (4.6 mm × 250 mm; 5 μm); mobile phase: acetonitrile-0.1% phosphoric acid solution (volume ratio 15:85); flow rate 0.6 mL / min; detection wavelength 270 nm; column temperature 30°C; injection volume 10 μL.
[0067] Example 2
[0068] The Agrobacterium strain used in step (2) of this embodiment is ATCC10060, and the specific operation thereof is as follows: Figure 2 Shown is the root growth at the incision after 30 days.
[0069] Comparative Example 1
[0070] The difference from Example 1 is that in step (3), Agrobacterium rhizogenes infects the nelumbo nucifera explants as follows:
[0071] Well-growing sterile seedlings of L. sylvestris were selected, and different explants of L. sylvestris leaves (with petioles) and leaves (without petioles) were taken on a clean bench. After scratching, they were cultured in the dark in an artificial climate box at 25°C on MS solid culture medium for 2 days. Take the above-mentioned single-sided needle explant after dark culture for 2 days and completely immerse it in the root-inducing Agrobacterium suspension prepared in step (2). Shake and infect it on a constant temperature shaker at 28°C and 100rpm for 10 minutes. After the infection, rinse it with sterile water 1-2 times. Take out the explant and dry it. Then move it into MS solid culture medium containing 100μmol / LAS and co-cultivate it at 25°C in the dark for 3 days. Then replace it with MS culture medium containing 500mg / L cefotaxime (Cef) for sterilization. Place it in an environment with a light cycle of 16h / 8h (day / night), a light intensity of 2000lx, and a temperature of 26°C for cultivation. Replace the sterilized culture medium every 3-10 days. After sterilization, replace it with a new MS solid culture medium and continue to culture until hairy roots grow. Figure 4 Shown are the root growth conditions of the leaf explants of Nelumbo nucifera after 40 days.
[0072] Comparative Example 2
[0073] The difference from Example 1 is that in step (3), Agrobacterium rhizogenes infects the nelumbo nucifera explants as follows:
[0074] Select sterile seedlings of nelumbo nucifera with good growth and three leaves, inject the root-inducing Agrobacterium suspension prepared in step (2) into the cotyledonary nodes of the seedlings, pour 4 ml of MS liquid culture medium containing 100 μmol / L AS into the sand matrix of each seedling, and place them in a light incubator at an air humidity of 70%, a photoperiod of 16h / 8h (day / night), a light intensity of 2000lx, and a temperature of 26°C for cultivation.
[0075] Testing of the hairy rooted nephrolepis plants from Examples 1 and 2, 30 days after infection, revealed that multiple hairy roots had grown from the incisions of the plants, showing good growth, with a hairy root induction rate exceeding 75%. In contrast, the traditional leaf disc transformation method in Comparative Example 1, which used seedling leaves as explants and was treated with an Agrobacterium rhizogenes suspension, had a lower conversion rate of approximately 35% compared to the transformation method in Example 1. Furthermore, the time it took to produce hairy roots was 7-10 days slower than in Example 1, and the length of the hairy roots that emerged was also shorter. The conversion rate of hairy roots of nephrolepis spp. using the injection method in Comparative Example 2 was even lower than that of Example 1, with a conversion rate of only approximately 10%. Furthermore, the time it took to produce hairy roots was 25-30 days slower than in Example 1 using the oblique cutting-cutting method. The hairy roots that emerged grew much more slowly than in Example 1, and even stagnated.
[0076] Depend on Figure 1 and Figure 2 It can be seen that the hairy roots can be grown 30 days after the infiltration by the single-sided needle hairy root induction method of the present invention. Figure 6 The alkaloid content of the hairy roots was significantly higher than that of the natural roots of sterile seedlings. The induction rate of the hairy roots of the present invention can reach more than 75%, which is more efficient and has better growth than the traditional leaf disc method and injection method.
[0077] The study found that pouring MS medium containing AS into the infected needle-shaped seedlings planted back in the sand is more conducive to the production and subsequent growth of needle-shaped hairy roots, and is also beneficial to the growth of needle-shaped seedlings. Figure 3 As shown in the figure, the hairy roots of the needlewort that were not poured with MS medium containing AS grew slowly, the root length was shorter, and the growth of the needlewort seedlings was poor, and the leaves turned yellow and fell off.
Claims
1. A method for promoting the induction and growth of hairy roots of unilateral needles, characterized in that: The steps include: Cut the sterile hypocotyl of the single-sided needle seedling 0.3-0.6 cm above the bottom end, discard the lower part, and soak the cut in the root-inducing Agrobacterium solution added with AS. Then, insert the seedling into sterile sand with a humidity of 80-90%. After each seedling is watered with MS liquid culture medium containing 100-200 µmol / L AS, hairy roots are induced.
2. The method according to claim 1, characterized in that Cut obliquely to increase the contact area of the explant.
3. The method according to claim 1, characterized in that The incision was immersed in the Agrobacterium rhizogenes solution with AS added for 8-12 minutes, and then the seedlings were inserted into the culture medium to induce hairy roots.
4. The method according to claim 1, wherein The Agrobacterium rhizogenes infection solution used in step (1) is prepared as follows: Pick a single colony and culture it in Agrobacterium rhizogenes liquid culture medium. 600nm When the OD reaches 0.5-0.7, the bacterial solution is inoculated into a 1-3 times volume of Agrobacterium rhizogenes liquid culture medium, and acetosyringone is added and cultured until the OD 600nm Reach 0.6-0.8; centrifuge the bacterial solution, discard the supernatant, resuspend the precipitate with liquid culture medium, add acetosyringone and culture to obtain the bacterial solution used for infection.
5. The method according to claim 4, characterized in that Rifampicin was added to the liquid culture medium of Agrobacterium rhizogenes at a final concentration of 50-100 mg / L, and acetosyringone was added to the culture medium at a final concentration of 100-200 µmol / L. The precipitate was resuspended in MS liquid culture medium, and acetosyringone was added and cultured for 25-40 minutes to obtain the bacterial solution used for infection.
6. The method according to claim 5, characterized in that The liquid culture medium for Agrobacterium rhizogenes is YEB liquid culture medium.
7. The method according to claim 4, characterized in that Agrobacterium rhizogenes stored in a -80°C ultra-low temperature freezer was inoculated into YEB liquid medium with a final concentration of 50 mg / L rifampicin at a ratio of 1:1000 on a clean bench and cultured in a shaking incubator at 200 r / min at 28°C for 14-16 h. 600nm When the OD reaches 0.5-0.7, the bacterial solution is inoculated into YEB medium containing 50 mg / mL rifampicin at a concentration of 2 times the bacterial solution volume, and acetosyringone is added at a final concentration of 100 μmol / L. The culture is continued for 1-2 hours until the OD reaches 600nm When the concentration reaches 0.6-0.8, centrifuge at 6000 rpm for 10 min, discard the supernatant, resuspend the precipitate in MS liquid culture medium containing 100 µmol / L acetosyringone, and culture for another 30 min to obtain the bacterial solution used for infection.
8. The method according to claim 1, characterized in that When the infected seedlings are inserted into the culture medium to induce hairy roots, the infected nelumbo nucifera seedlings are transplanted into sterile sand, the air humidity is 60-80%, the temperature is 24-26 ℃, and they are cultured under the conditions of a photoperiod of 14-16 h and a light intensity of 1500-3500 lx until hairy roots grow.
9. The method according to claim 8, characterized in that The infected seedlings were inserted into pre-inserted sterile sand and the cut was covered with sand. Each seedling was watered with MS liquid culture medium containing 100-200 µmol / L AS and cultured in a light incubator. After 30 days, the cut was seen to swell and grow hairy roots.
10. The method according to claim 1, characterized in that Sterile seedling culture of single needle needle: The seeds of the nelumbo serrata were shelled, treated with gibberellin and disinfected, and then germinated on MS solid culture medium in an incubator at 24-26°C in the dark. After two cotyledons grew out, they were moved into sterile sand and grown in a light cycle to obtain sterile seedlings of the nelumbo serrata.