Water culture rapid rooting method based on bamboo reed tissue culture seedlings

Abstract

The invention discloses a rapid rooting method based on water culture of bamboo reed tissue culture seedlings, which comprises the following steps: inoculating a current-year stem of bamboo reed as an explant into a subculture medium, inducing to generate cluster buds, carrying out water culture rooting on the cluster buds subjected to multiple times of subculture, and finally, directly transplanting and hardening seedlings. The method is beneficial to shortening the tissue culture period, simplifying the experimental process, reducing the production cost and improving the working efficiency, and is suitable for large-scale seedling culture in factories.

Description

Technical Field

[0001] This invention relates to a plant tissue culture propagation technique, specifically a method for rapid rooting of Phragmites australis tissue culture seedlings via hydroponics. Background Technology

[0002] Reed (Phragmites australis) belongs to the Poaceae family, Pooideae subfamily, and Reed genus. It is named for its resemblance to reeds, being hollow and segmented, similar to bamboo. Reed is a perennial herbaceous plant with underground rhizomes. The rhizomes are thick, segmented, and covered with light-colored scales, with a terminal bud (old bud) at the tip and a pair of secondary buds on either side below. The above-ground stems are robust, 2-3 cm in diameter, and can reach a height of 6-8 meters. Reed is highly adaptable, growing in acidic, slightly acidic, neutral, and alkaline clay-sand loam soils. It tolerates poor soil, drought, and is also resistant to flooding and waterlogging. Reed has a high protein content and moderate cellulose content, making it suitable for papermaking, animal feed, energy, and environmental protection industries. Therefore, the market demand for Reed seedlings is increasing.

[0003] Currently, the propagation of *Arundinaria repens* mainly relies on tissue culture for rapid propagation. This rapid propagation system primarily involves inducing callus redifferentiation from stem segments to establish a rapid propagation and regeneration system. However, this method suffers from low rooting rates and long rooting times. Furthermore, the callus tissue undergoes prolonged subculture, resulting in slow differentiation and even the formation of deformed or undifferentiated buds. Therefore, researching rooting methods for *Arundinaria repens* tissue culture seedlings is of great significance for accelerating rapid tissue propagation and ensuring a sufficient supply of seedlings. Summary of the Invention

[0004] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is: a rapid rooting method for hydroponic culture of Reed tuber tissue culture seedlings, which can shorten the tissue culture cycle, simplify the experimental procedure, reduce production costs, improve work efficiency, and is suitable for large-scale seedling production in factories.

[0005] To achieve the above objectives, the present invention provides a method for rapid rooting of Reed rhizome tissue culture seedlings via hydroponics, characterized by the following steps:

[0006] Step 1: Take disease-free, pest-free stem segments with plump buds and axillary buds from the current year's growth of Reed, and disinfect them;

[0007] Step 2: Place the sterilized Reed shoots with axillary buds into the subculture medium to induce clustered shoots. Subculture once every 20 days, and after three consecutive subcultures, clustered shoots with a length of 3-6cm are obtained.

[0008] Step 3: Wash the culture medium adhering to the clump of seedlings with clean water, dry them on filter paper, then put them in the rooting solution for 1 day, and then put them in water with added nutrient solution for hydroponic rooting.

[0009] Step 4: After the hydroponically rooted seedlings are transferred to soil for hardening, seedlings ready for transplanting to the field are obtained.

[0010] Compared with the prior art, the beneficial effects of the present invention are:

[0011] This invention directly utilizes stem segments with axillary buds to induce clustered buds, reducing the callus induction process; the subcultured tissue culture seedlings are directly subjected to outdoor hydroponic rooting, saving time and production costs. Furthermore, the outdoor hydroponic rooting of subcultured tissue culture seedlings solves the need for a sterile environment for tissue culture rooting and enables the conversion of tissue culture seedlings to open rooting culture in a natural environment, solving the problem of easy contamination during sterile operations and reducing the workload of laboratory personnel.

[0012] The method provided by this invention shortens the rooting time for hydroponics by approximately 10-20 days compared to conventional tissue culture rooting. Hydroponics for rooting tissue culture seedlings reduces the need for procedures such as preparing culture media, aseptic inoculation in the laboratory, and culture in a culture room.

[0013] This invention overcomes the drawbacks of traditional Arundo donax stem segment subculture, such as long subculture time, low differentiation rate, and complex and costly root induction procedures in tissue culture bottles. It first induces clustered buds directly from stem segments, then performs multiple subcultures to obtain robust seedlings, and finally allows the tissue-cultured seedlings to root outdoors in hydroponics. This invention not only shortens the tissue culture cycle and simplifies the experimental procedure, but also reduces production costs and improves work efficiency, making it suitable for large-scale factory seedling production. Attached Figure Description

[0014] Figure 1 This is a picture of a tissue culture seedling after multiple subcultures of clustered buds.

[0015] Figure 2 The rooting phenotypes of tissue culture seedlings 3-6cm tall after being treated with different concentrations of rooting powder for 1 day and then hydroponically cultured for 10 days are shown.

[0016] Figure 3 The rooting phenotypes of tissue culture seedlings of different heights after being treated with 0.1 g / L rooting powder for 1 day and then hydroponically cultured for 10 days are shown.

[0017] Figure 4 Rooting phenotypes of tissue culture seedlings 3-6 cm tall after treatment with 0.1 g / L rooting powder for 1 day and hydroponics at different nutrient solution concentrations for 10 days;

[0018] Figure 5 Phenotypic diagram of tissue culture seedlings after hydroponic rooting and 15 days of soil cultivation. Detailed Implementation

[0019] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0020] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0021] In the following examples, all materials and reagents used, unless otherwise specified, are commercially available. The adventitious bud induction medium is MS medium containing 0.7% agar powder, 3% sucrose, and 6-BA:IBA = 4:1.

[0022] Step 1: Take disease-free, pest-free stem segments with plump buds and axillary buds from the current year's growth of sword-leaf reed and disinfect them;

[0023] Step 2: Place the sterilized stem segments of *Arundinaria fragilis* with axillary buds into a subculture medium to induce clustered buds. Subculture once every 20 days, and after three consecutive subcultures, clustered buds with a length of 3-6 cm are obtained.

[0024] Step 3: Wash the culture medium adhering to the clump of buds with clean water, dry them on filter paper, and then treat them in rooting powder solutions of different concentrations (0g / L, 0.05g / L, 0.1g / L, 0.15g / L) for 1 day. Then, place them in 1×Hoagland nutrient solution for hydroponic rooting. The required hydroponic rooting time is 10 days.

[0025] Step 4: After the hydroponically rooted seedlings are transferred to soil for hardening-off for 15 days, seedlings ready for transplanting to the field are obtained.

[0026] In the control group, tissue culture seedlings did not root within 7 days; only 30% of the seedlings showed small white dots on their roots, forming root primordia. In contrast, the rooting powder treatment group showed small white dots on almost all roots, forming root primordia, after 3-5 days. By day 7, adventitious roots emerged from the root primordia and continued to elongate, with a rooting rate of 90% in the treatment group. At day 10, some seedlings in the control group began to root, while the number of adventitious roots in the treatment group continued to increase, and the roots continued to elongate.

[0027] Table 1. Rooting of 3-6cm tall tissue culture seedlings after 10 days of hydroponics following treatment with different rooting powder concentrations.

[0028] Concentration g / L Average number of roots 0 0.9±0.876c 0.05 2.3±1.337b 0.1 3.7±0.949a 0.15 2.3±0.823b

[0029] As shown in Table 1, the optimal rooting powder concentration is 0.1 g / L. Concentrations that are too low or too high will not achieve good rooting results. Therefore, considering factors such as simplifying procedures, reducing workload, and lowering production costs, tissue culture seedlings treated with different concentrations (0.05-0.15 g / L) of rooting powder exhibit significant advantages in outdoor hydroponic rooting, with the optimal concentration being 0.1 g / L.

[0030] Experiment 1

[0031] Tissue culture seedlings of different heights may respond differently to rooting powder treatment. Therefore, this experiment compared the hydroponic rooting effect of tissue culture seedlings of different heights (0-3cm, 3-6cm, 6-8cm) after treatment with 0.1g / L rooting powder for 1 day. Tissue culture seedlings of different heights (0-3cm, 3-6cm, 6-8cm) were treated with 0.1g / L rooting powder for 1 day and then hydroponically cultured for 10 days to observe the hydroponic rooting effect.

[0032] Table 2. Rooting results of tissue culture seedlings of different heights after treatment with rooting powder and hydroponics for 10 days.

[0033] Seedling height / cm Average number of roots 0～3 0.5±0.707b 3～6 3.7±0.949a 6～8 3.3±1.16a

[0034] Table 2 shows that when selecting tissue culture seedlings, those with a height of 3-6 cm are optimal, yielding the highest number of roots. Lower seedlings (0-3 cm) have weaker roots and poorer rooting. Higher seedlings (6-8 cm) are stronger and achieve better rooting, but slightly less effective than the hydroponic rooting of 3-6 cm seedlings. Therefore, tissue culture seedlings with a height of 3-6 cm are recommended for hydroponic rooting.

[0035] Experiment 2

[0036] Different concentrations of hydroponic nutrient solution may affect the rooting effect of tissue culture seedlings. Therefore, in this experiment, 3-6 cm tall tissue culture seedlings treated with 0.1 g / L rooting powder solution for 1 day were hydroponically treated for 10 days in 0, 1 / 10×, 1 / 5×, 1 / 2×, and 1× Hogland nutrient solutions, with other conditions the same as in the previous example, and the hydroponic rooting status of *Arundo donax* tissue culture seedlings in different concentrations of nutrient solution was observed.

[0037] Table 3. Rooting results of tissue culture seedlings after 10 days of hydroponics with different concentrations of nutrient solution.

[0038] Hogland nutrient solution concentration Average number of roots 0 0.9±0.876c 1 / 10× 1.4±0.843bc 1 / 5× 2.2±0.422b 1 / 2× 3.4±0.966a 1× 3.7±0.949a

[0039] As shown in Table 3, the average number of roots is the highest when the Hogland solution concentration is 1 / 2× or 1× Hogland. Therefore, these two concentrations can be used for hydroponic rooting culture of tissue culture seedlings.

[0040] Experiment 3

[0041] Referring to Table 4, a comparison is made between hydroponic rooting, tissue culture rooting, and hydroponic rooting after tissue culture. Direct hydroponic rooting using reed stem segments has seasonal limitations, generally from March to October, requires significant space, and does not require robust seedlings for direct use. Tissue culture rooting can be performed year-round with minimal space requirements, but is prone to contamination, has a long rooting cycle, and requires robust hydroponic cultivation before use. Hydroponic rooting after tissue culture can also be performed year-round with minimal space requirements and a short rooting cycle; rooted seedlings can be directly used for field planting without the need for robust seedlings. Therefore, the hydroponic rooting method after tissue culture is selected.

[0042] Table 4 compares the characteristics of different rooting methods.

[0043] Rooting methods Harvesting season Pollution situation Space requirements Rooting cycle Seedling status Hydroponic rooting March to October none big 7-10d No need for hydroponics to grow strong seedlings Tissue culture rooting annual Easily polluted Small 17-20d Seedlings must be grown hydroponically to be vigorous before use. Tissue culture followed by hydroponic rooting annual none Small 7-10d No need for hydroponics to grow strong seedlings

[0044] In summary, by subculturing sterilized *Arundinaria fragilis* stem segments with axillary buds into a culture medium to induce shoot clustering, and performing three subculturings at 20-day intervals, shoot clusters with a length of 3-6 cm were obtained. These shoots were then treated with a 0.1% rooting powder solution and placed in 1 / 2× or 1× Hoagland nutrient solution for hydroponic rooting. Hydroponic rooting took 10 days, and after 15 days of hardening off by transferring the hydroponically rooted seedlings to soil, seedlings ready for transplanting to the field were obtained. This method of using tissue culture followed by hydroponic rooting overcomes the disadvantages of long subculture times, low differentiation rates, complex and cumbersome rooting induction procedures in tissue culture bottles, and high costs associated with *Arundinaria fragilis* stem segment subculturing.

[0045] In addition, this method, which first induces clustered buds directly from stem segments with axillary buds, then conducts multiple subcultures to obtain robust seedlings, and finally performs outdoor hydroponic rooting of tissue culture seedlings, not only shortens the tissue culture cycle and simplifies the experimental procedures, but also reduces production costs and improves work efficiency, making it suitable for large-scale seedling production in factories.

[0046] The specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art can make various modifications or variations within the scope of the claims, which do not affect the substantive content of the present invention and are all within the protection scope of the present invention patent.

[0047] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention.

Claims

1. A method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics, characterized in that, Includes the following steps: Step 1: Take stem segments of Reed with axillary buds and disinfect them; Step 2: Place the sterilized Reed shoots with axillary buds into the subculture medium to induce clustered shoots. Subculture once every 20 days, and after three consecutive subcultures, clustered shoots with a length of 3-6cm are obtained. Step 3: Wash the culture medium adhering to the clump of seedlings with clean water, dry them on filter paper, then put them in the rooting solution for 1 day, and then put them in water with added nutrient solution for hydroponic rooting. Step 4: After the hydroponically rooted seedlings are transferred to soil for hardening, seedlings ready for transplanting to the field are obtained.

2. The method for rapid rooting of Reed tissue culture seedlings in hydroponics according to claim 1, characterized in that: In steps two and three, the cultivation conditions are: temperature 25℃, light duration 16h, light intensity 18000-20000 lux, and humidity 60%.

3. The method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics according to claim 2, characterized in that: The hydroponic hardening time in step three is 10 days.

4. The method for rapid rooting of Reed tissue culture seedlings in hydroponics according to claim 3, characterized in that: The soil cultivation and hardening-off period in step four is 15 days.

5. The method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics according to claim 4, characterized in that: The rooting powder concentration in the rooting solution of step three is 0-0.15 g / L.

6. The method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics according to claim 5, characterized in that: The rooting solution in step three contains no rooting powder.

7. The method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics according to claim 5, characterized in that: The rooting powder concentration in the rooting solution of step three is 0.05 g / L.

8. The method for rapid rooting of Reed tissue culture seedlings in hydroponics according to claim 5, characterized in that: The rooting powder concentration in the rooting solution of step three is 0.1 g / L.

9. The method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics according to claim 5, characterized in that: The rooting powder concentration in the rooting solution of step three is 0.15 g / L.

10. A method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics according to any one of claims 1-9, characterized in that: The nutrient solution used in step three is Hoagland nutrient solution.

11. The method for rapid rooting of Phragmites australis tissue culture seedlings in hydroponics according to claim 10, characterized in that: The nutrient solution used in step three is 0×Hoagland nutrient solution.

12. The method for rapid rooting of Phragmites australis tissue culture seedlings in hydroponics according to claim 11, characterized in that: The nutrient solution in step three is 1 / 10 × Hoagland nutrient solution.

13. The method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics according to claim 11, characterized in that: The nutrient solution in step three is 1 / 5 × Hoagland nutrient solution.

14. The method for rapid rooting of Reed tuber tissue culture seedlings in hydroponics according to claim 11, characterized in that: The nutrient solution in step three is 1 / 2 × Hoagland nutrient solution.

15. The method for rapid rooting of Phragmites australis tissue culture seedlings in hydroponics according to claim 11, characterized in that: The nutrient solution in step three is 1×Hoagland nutrient solution.

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