A pterocarpus santalinus tissue culture medium and a rapid propagation method thereof

Abstract

This invention provides a tissue culture medium for *Pandanus tectorius* leaves and a method for rapid propagation through tissue culture. Using young lateral buds of *Pandanus tectorius* leaves as explants is preferable due to its low contamination rate, rapid initiation of proliferation, and superior surface sterilization method, resulting in low contamination and browning. Adding 0-0.1 mg TDZ and 1-3 mg 6-BA to MS medium for *Pandanus tectorius* leaf bud induction culture rapidly initiates proliferation. Subculture with NAA and 6-BA in MS medium effectively improves the proliferation rate, producing robust and rapidly growing seedlings that effectively balance callus amplification and bud propagation. Adding NAA to 1 / 2 MS medium for *Pandanus tectorius* leaf rooting culture results in high rooting efficiency and good root growth and development. This allows for rapid and large-scale propagation of *Pandanus tectorius* leaf tissue culture seedlings, enabling large-scale production, promoting the vigorous development of *Pandanus tectorius* leaf cultivation, and stimulating the development of related industries such as pharmaceuticals, health foods, and ornamental flowers based on *Pandanus tectorius* leaf raw materials.

Description

Technical Field

[0001] This invention relates to the field of plant tissue culture technology, and in particular to a culture medium for Pandanus leaf tissue and a method for rapid propagation of tissue culture. Background Technology

[0002] Pandanus leaves ( Pandanus amaryllifolius Pandanus (Pandanus chinensis) is a perennial herbaceous plant belonging to the Pandanaceae family and the Pandanus genus. Also known as variegated pandanus, it originated in the Maluku Islands of Indonesia. It was introduced to my country in the 1950s and successfully cultivated in Hainan. Currently, it is mainly cultivated in Hainan and Yunnan, with smaller quantities grown in Guangdong and Taiwan. Pandanus leaves are rich in active ingredients such as squalene, linoleic acid, artemisinin, and sterols. Its main aroma component is 2-acetyl-1-pyrrolline, giving it a unique fragrance reminiscent of rice dumplings, earning it the reputation of "Oriental herb." Because pandanus leaves enhance cell vitality, accelerate metabolism, and improve human immunity, and are a natural food ingredient for making pastries, ice cream, and candies, as well as having numerous applications in beautifying the environment and aromaticating homes, it possesses significant economic value and development potential.

[0003] The market demand for pandan leaves is rapidly increasing, leading to a shortage of seedlings. Currently, pandan leaf propagation mainly involves two methods: stem cutting propagation and root sucker propagation. However, both methods suffer from long cycles and low propagation efficiency. Furthermore, the high price of pandan leaves discourages growers from using this method. Tissue culture-based rapid propagation systems are widely used in agricultural fields such as fruit trees, flowers, and medicinal herbs, offering advantages such as stable traits, batch processing, and rapid propagation. Reports on rapid propagation of pandan leaves using tissue culture are limited. Wang Jingfei et al. constructed a rapid propagation system using the above-ground stems of Pandanus truncatus as explants, obtaining regenerated plants. Ji Xunzhi et al. used above-ground stems, leaves, and lateral buds as explants, and under different culture conditions, only lateral buds produced callus, and the activity of antioxidant enzymes in the callus was tested. This invention uses lateral buds of Pandanus leaves as explants to explore the effects of explant size, surface sterilization time and method, composition of proliferation culture medium, and composition of rooting culture medium on the rapid propagation of Pandanus leaves. It aims to establish a rapid propagation system based on tissue culture, in order to break through and alleviate the current situation of producing large quantities of high-quality Pandanus leaf seedlings, promote the healthy and rapid development of the industry, and provide a theoretical basis for the breeding and propagation of new Pandanus leaf varieties. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a Pandanus leaf tissue culture medium and a rapid propagation method for tissue culture, which can rapidly and cost-effectively propagate high-quality Pandanus leaf tissue culture seedlings.

[0005] The first aspect of the present invention is to provide a subculture medium for Panax notoginseng leaves, using MS as the basic medium, with 0.05-1 mg of NAA and 2-4 mg of 6-BA added per liter of medium.

[0006] Preferably, each liter of pandanus leaf subculture medium contains 0.05 mg of NAA and 2-4 mg of 6-BA.

[0007] More preferably, 0.05 mg of NAA and 3-4 mg of 6-BA are added to each liter of Pandanus leaf subculture medium.

[0008] More preferably, 0.05 mg of NAA and 3 mg of 6-BA are added to each liter of pandanus leaf subculture medium.

[0009] Preferably, the pandanus leaf subculture medium contains fresh pandanus leaf juice or freeze-dried leaf juice powder.

[0010] The juice of fresh pandanus leaves is obtained by juicing fresh pandanus leaves with a small amount of water and then filtering the juice. Freeze-drying the juice yields freeze-dried powder of fresh pandanus leaf juice.

[0011] More preferably, each liter of pandan leaf subculture medium contains 1-2g of fresh pandan leaf juice or freeze-dried powder of leaf juice.

[0012] The second aspect of the present invention is to provide a Panax notoginseng leaf shoot induction medium, which uses MS as the basic medium and adds 0-0.1 mg of TDZ and 1-3 mg of 6-BA per liter of medium.

[0013] Preferably, 0.1 mg of TDZ and 1-3 mg of 6-BA are added to each liter of Panax notoginseng leaf shoot induction medium.

[0014] More preferably, 0.1 mg of TDZ and 2-3 mg of 6-BA are added to each liter of Panax notoginseng leaf shoot induction medium.

[0015] More preferably, 0.1 mg of TDZ and 2 mg of 6-BA are added to each liter of Panax notoginseng leaf shoot induction medium.

[0016] A third aspect of the present invention is to provide a rooting medium for Panax notoginseng leaves, using 1 / 2 MS as the basic medium, with 0.1-0.2 mg of NAA or 0.1-0.2 mg of IBA added per liter of medium.

[0017] Preferably, 0.2 mg of NAA is added to each liter of Pandanus leaf rooting medium.

[0018] A fourth aspect of the present invention is to provide a pandanus leaf tissue culture medium combination, characterized in that it comprises the pandanus leaf subculture medium described in the first aspect of the present invention, the pandanus leaf shoot induction medium described in the second aspect of the present invention, and the pandanus leaf rooting medium described in the third aspect of the present invention.

[0019] The fifth aspect of this invention is to provide a method for rapid propagation of Pandanus leaf tissue culture, using the Pandanus leaf subculture medium described in the first aspect of this invention for subculture.

[0020] Preferably, the rapid propagation method of Pandanus leaf tissue culture includes the following steps: (1) selection of explants: select Pandanus leaf mother plants that are free from obvious diseases, have vigorous growth and vigorous tillering, and take complete lateral buds as explants; (2) sterilization treatment of explant surface;

[0021] (3) Induction and proliferation culture of bud clusters: Inoculate the explant segments or slices into the bud cluster induction culture medium of Panax notoginseng leaves and culture for about 4 weeks; (4) Subculture: Inoculate the buds after bud cluster induction and proliferation culture into the subculture culture medium of Panax notoginseng leaves and culture for about 4 weeks; (5) Rooting culture: Inoculate the young shoots or buds after subculture culture into the rooting culture medium of Panax notoginseng leaves and culture for about 4 weeks; (6) Transplanting of seedlings.

[0022] The selected explants are approximately 0.5-4 cm in size, preferably 2-4 cm, and even more preferably approximately 2 cm.

[0023] When sterilizing the surface of explants, sterilization is performed with 70% ethanol for 30 seconds to 3 minutes, or with 0.1% mercuric chloride for 3 to 10 minutes. Preferably, sterilization is performed with 70% ethanol for 1 to 3 minutes and with 0.1% mercuric chloride for 5 to 10 minutes. More preferably, sterilization is performed with 70% ethanol for 3 minutes and with 0.1% mercuric chloride for 10 minutes.

[0024] More preferably, the selected explants are about 2 cm long, and the surface of the explants is sterilized with 70% ethanol for 3 min and 0.1% mercuric chloride for 10 min.

[0025] Preferably, the transplanting substrate is a mixture of yellow clay and black soil in a weight ratio of 1:1, or a mixture of yellow clay, coconut coir, and black soil in a weight ratio of 1:1:1.

[0026] The beneficial effects of this invention are:

[0027] (1) The Panax notoginseng leaf shoot induction medium of the present invention has a fast start-up proliferation, which can make the explants easily form multiple buds and grow and develop rapidly;

[0028] (2) The subculture medium of Pandanus leaf of the present invention is highly targeted and practical. It can effectively improve the proliferation rate, produce robust seedlings with rapid growth, and can better balance callus expansion and bud propagation. The proliferation rate can reach 5.56 in four weeks. Adding Pandanus leaf powder can further improve the proliferation efficiency, which can increase the proliferation efficiency to 6.42.

[0029] (3) The rooting culture medium for Pandanus leaves of the present invention has a high rooting efficiency, up to 99%, and the roots grow and develop well, which can meet the needs of production.

[0030] (4) The present invention uses young lateral buds of Pandanus leaves as explants, which is better, with less pollution, faster initiation of proliferation, better surface sterilization method, low pollution rate and low degree of browning. The induction culture of Pandanus leaf cluster buds can start proliferation quickly, the subculture culture of Pandanus leaves has high proliferation efficiency, the root culture of Pandanus leaves has high rooting efficiency, and the root growth and development status is good. It can enable the rapid and large-scale propagation of Pandanus leaf tissue culture seedlings, realize large-scale production, promote the vigorous development of Pandanus leaf planting industry, and promote the development of related industries such as pharmaceuticals, health foods, and ornamental flowers based on Pandanus leaf raw materials. Attached Figure Description

[0031] Figure 1 This invention describes the rapid propagation process of *Pandanus orchid* leaf tissue culture. Figure A shows a *Pandanus orchid* leaf lateral bud; Figure B shows the size of a *Pandanus orchid* leaf lateral bud; Figure C shows the induced proliferation culture of *Pandanus orchid* leaf cluster buds; Figure D shows the subculture culture of *Pandanus orchid* leaf multi-bud organisms; Figure E shows a side view of the subculture culture of *Pandanus orchid* leaf multi-bud organisms; Figure F shows the rooting culture of *Pandanus orchid* leaf seedlings; and Figure G shows a side view of the rooting culture of *Pandanus orchid* leaf seedlings. Detailed Implementation

[0032] The present invention will be further described below with reference to the accompanying drawings and specific embodiments to better understand the invention. Where specific techniques or conditions are not specified in the embodiments, they are performed according to the techniques or conditions described in the literature in the art or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be obtained commercially.

[0033] 1. Materials

[0034] Perennial Pandanus leaves from near Baodao New Village, Nada Town, Danzhou, Hainan Province were selected as mother plants and transplanted to the nursery of Hainan Tropical Crops Research Institute Seed Industry Technology Co., Ltd. After a year of continuous observation, mother plants with vigorous growth and strong aroma were selected as starting materials for tissue culture. The botanical characteristics of the plants were identified by researcher Wu Qiong.

[0035] 2. Data and Image Processing and Analysis Methods

[0036] The significance of differences was tested using SAS software, and graphs were plotted using Microsoft Excel. Images were acquired using a Canon EOS 750D camera.

[0037] 3. Methods and Experimental Results

[0038] 3.1 Selection of explants

[0039] Lateral buds from *Panthoceras sorbifolium* leaves were selected as explants. The explants were categorized into three sizes: approximately 0.5-1.0 cm, approximately 2.0 cm, and approximately 3-4 cm. Before collection, shading and rain protection netting were applied. After several consecutive sunny days, healthy, vigorous mother plants with no obvious diseases and abundant tillering were selected from the nursery. Intact lateral buds were removed as explants and brought back to the laboratory for later use.

[0040] 3.2 Sterilization of explant surface

[0041] After the explants were rinsed with tap water for 30 minutes to remove surface dirt and impurities, excess water was absorbed and the explants were placed in a clean bench for later use. Surface sterilization methods: (1) 70% ethanol for 30 seconds, 0.1% mercuric chloride for 3 minutes; (2) 70% ethanol for 1 minute, 0.1% mercuric chloride for 5 minutes; (3) 70% ethanol for 3 minutes, 0.1% mercuric chloride for 10 minutes. After surface sterilization, the explants were rinsed with sterile water 3-5 times, 1-2 minutes each time. Excess water was absorbed with absorbent paper in the clean bench and then inoculated onto MS medium. The number of explants inoculated for each treatment was no less than 30. The contamination rate and browning status were counted one week later.

[0042] Contamination rate (%) = Number of contaminated explants / Number of inoculated explants × 100%.

[0043] Different surface sterilization treatments were applied to explants of different sizes of Panax notoginseng leaves. After inoculation on MS medium for 7 days, the contamination rate and browning degree were statistically analyzed. It was found that the sterilization method (3): 70% ethanol for 3 min and 0.1% mercuric chloride for 10 min was more effective for explants of about 2 cm in size, with a low contamination rate of 2.5% and a moderate browning degree. The browning degree of explants of different sizes was positively correlated with the sterilization time. The smaller the explant, the longer the sterilization time and the more severe the browning degree.

[0044] Table 1. Statistics on contamination rate and browning degree of explants of different sizes after different surface sterilization methods.

[0045]

[0046] 3.3 Bud Cluster Induction and Proliferation Culture

[0047] After surface sterilization and cutting, the leaf suckers of *Panthoceras sorbifolium* were inoculated into different shoot induction media (AF) for shoot induction culture. The shoot induction media (AF) were based on MS medium with appropriate hormones added (as shown in Table 2). Lateral buds of 2 cm in size were used as explants, with no fewer than 25 explants per combination. Four weeks after inoculation, the induction rate and browning degree were recorded. Four weeks later, the proliferation coefficient and shoot differentiation and growth were recorded to screen and optimize the shoot induction and proliferation media.

[0048] The results are shown in Table 3. Statistical analysis revealed that combination E had an induction rate as high as 96.3%, superior to the other five combinations, and also had the highest proliferation coefficient (4.76), the least browning, bright green callus, plump buds, clear leaf veins, bright green leaves, and clear stem nodes. With the increase of 6-BA concentration, both the induction rate and proliferation coefficient increased, and the browning degree was reduced, but it easily led to thin buds, indistinct leaf veins, and elongated stem nodes in the differentiated buds. TDZ could effectively improve the induction rate and proliferation coefficient and reduce the browning degree.

[0049] Proliferation coefficient = number of bud clusters on callus four weeks after inoculation / number of bud clusters on inoculated callus (at least 15 callus with bud clusters are counted for each combination).

[0050] Induction rate = (Number of explants initiated for proliferation / Number of explants inoculated) × 100%.

[0051] Table 2. Hormone concentration composition of different shoot induction media

[0052] A B C D E F TDZ (mg / L) 0 0 0 0.1 0.1 0.1 6-BA (mg / L) 1 2 3 1 2 3

[0053] Table 3. Effects of different shoot induction media on the leaf induction rate of Panax notoginseng.

[0054] Bud induction medium Induction rate (%) Proliferation coefficient browning degree Proliferating bud differentiation and growth A 44.83 2.33 +++++ The callus is yellowish-green, the buds are plump, the veins are clear, the leaves are bright green, and the stem nodes are distinct. B 69.57 3.21 +++ The callus is yellowish-green, the buds are plump, the veins are clear, the leaves are bright green, and the stem nodes are distinct. C 67.86 3.82 +++ The callus is yellowish-green, the buds are thin, the veins are indistinct, the leaves are yellowish-green, and the stems are slender. D 80.00 4.13 ++ The callus is bright green, the buds are plump, the veins are clear, the leaves are bright green, and the stem nodes are distinct. E 96.30 4.76 + The callus is bright green, the buds are plump, the veins are clear, the leaves are bright green, and the stem nodes are distinct. F 83.87 3.96 ++ The callus is bright green, with delicate buds, clear veins, yellowish-green leaves, and slender stems.

[0055] 3.4 Subgeneration

[0056] Using shoot clusters induced and proliferated through bud induction culture as material, they were inoculated into different subculture media (af) for subculture. Subculture media af used MS as the basal medium, with appropriate amounts of hormones added (as shown in Table 4). After four weeks, the initiation proliferation rate and proliferation coefficient were calculated, and the growth status of the shoot clusters was observed. At least 50 shoot clusters were counted for each combination. The results are shown in Table 5. Subculture medium b had the highest proliferation coefficient, reaching 5.56, and produced fewer, bright green, and more compact calluses. The calluses had plump shoot clusters with clear veins, bright green leaves, and clear stem nodes, indicating good seedling development. These results suggest that the hormone concentration in this medium is suitable for use as a subculture medium.

[0057] Our research also found that adding a certain proportion of pandanus leaf juice to the culture medium can further improve the proliferation efficiency. Take 10 grams of fresh pandanus leaves, add a small amount of water, extract the juice, filter, and bring the volume to 100 ml to prepare pandanus leaf juice for later use. It should be understood that in practice, depending on the application, fresh pandanus leaves can also be juiced with a small amount of water, filtered, and freeze-dried to prepare pandanus leaf freeze-dried powder for application.

[0058] Prepare subculture medium g1:

[0059] Subculture medium g: MS medium;

[0060] Subculture medium h: MS as the basal medium, with 10 ml of filtered pandanus leaf juice added per liter of medium (i.e., 1 g of fresh pandanus leaf juice added per liter of medium).

[0061] Subculture medium i: MS as the base medium, with 20 ml of filtered pandanus leaf juice added per liter of medium (i.e., 2 g of fresh pandanus leaf juice added per liter of medium).

[0062] Subculture medium j: MS as the basal medium, with 3 mg of 6-BA and 0.05 mg of NAA added per liter of medium;

[0063] Subculture medium k: MS as the basal medium, with 3 mg of 6-BA, 0.05 mg of NAA and 10 ml of filtered pandanus leaf juice added per liter of medium (i.e., 1 g of fresh pandanus leaf juice added per liter of medium).

[0064] Subculture medium 1: MS as the basal medium, with 3 mg of 6-BA, 0.05 mg of NAA and 20 ml of filtered pandanus leaf juice added per liter of medium (i.e., 2 g of fresh pandanus leaf juice added per liter of medium).

[0065] Using shoots induced and proliferated through bud proliferation culture as material, they were inoculated into subculture medium 1L for subculture. After four weeks, the proliferation rate and growth coefficient were statistically analyzed, and the growth status of the shoots was observed. At least 50 shoots were counted for each combination. The results are shown in Table 6. Adding fresh pandanus leaf juice to the culture medium alone can increase the proliferation coefficient of shoots, but its efficiency is lower than that of hormone alone. The efficiency of hormone alone is significantly higher than that of pandanus leaf powder. Adding pandanus leaf juice to the culture medium can significantly improve the vegetative growth of pandanus seedlings, promote leaf and stem development, and facilitate seedling rooting and transplanting. Adding 2g of fresh pandanus leaf juice per liter of culture medium increases the proliferation efficiency of shoots more than adding 1g of fresh pandanus leaf juice per liter of culture medium. Adding fresh pandanus leaf juice can increase the proliferation efficiency from 5.56 (with hormone alone) to 6.42, significantly accelerating the proliferation process of pandanus leaves.

[0066] Proliferation coefficient = number of bud clusters on callus four weeks after inoculation / number of bud clusters on inoculated callus (at least 15 callus with bud clusters are counted for each combination).

[0067] Table 4 Hormone concentration composition in different subculture media

[0068] a b c d e f NAA (mg / L) 0.05 0.05 0.05 0.1 0.1 0.1 6-BA (mg / L) 2 3 4 2 3 4

[0069] Table 5 Effects of subculture medium af on the proliferation of Panax notoginseng leaf cluster buds

[0070] Subculture medium Proliferation coefficient Proliferating bud differentiation and growth a 4.45 It has few calluses, is pale yellow, has plump buds, clear leaf veins, dark green leaves, and distinct stem nodes. b 5.56 Few callus wounds, bright green color, plump buds, clear leaf veins, bright green leaves, and distinct stem nodes. c 4.82 Numerous callus, yellowish-green in color, slender buds, indistinct leaf veins, yellowish-green leaves, and thin, elongated stems. d 3.13 Few callus wounds, yellowish-green in color, plump buds, clear leaf veins, dark green leaves, and short, thick stems. e 4.30 Numerous callus, yellowish-green in color, plump buds, indistinct leaf veins, bright green leaves, and clearly defined stem nodes. f 3.81 Numerous callus, bright green in color, slender clusters of buds, indistinct leaf veins, yellowish-green leaves, and stem nodes.

[0071] Table 6. Effects of adding Pandanus leaf powder on shoot proliferation in different subculture media

[0072] Subculture medium Proliferation coefficient Proliferating bud differentiation and growth g 1.46 Few calluses, green in color, plump buds, clear leaf veins, dark green leaves, and thick stems. h 1.74 Few calluses, green in color, plump buds, clear leaf veins, dark green leaves, and thick stems. i 1.81 Few calluses, green in color, plump buds, clear leaf veins, dark green leaves, and thick stems. j 5.53 Few callus wounds, yellowish-green in color, plump buds, clear leaf veins, dark green leaves, and short, thick stems. k 6.37 Numerous callus, yellowish-green in color, plump buds, clear leaf veins, dark green leaves, and short, thick stems. l 6.42 Numerous callus, yellowish-green in color, plump buds, clear leaf veins, dark green leaves, and short, thick stems.

[0073] Note: The culture medium j in this table is the same as the subculture medium b in Table 5. The corresponding proliferation coefficients are the results of two experiments, and there are slight differences, which is normal.

[0074] 3.5 Rooting Culture

[0075] Regenerated shoots of approximately 1-1.5 cm in length after subculture were used as materials and inoculated onto different rooting media (R1: 1 / 2 MS; R2: 1 / 2 MS + NAA 0.1 mg / L; R3: 1 / 2 MS + NAA 0.2 mg / L; R4: 1 / 2 MS + IBA 0.1 mg / L; R5: 1 / 2 MS + IBA 0.2 mg / L). After 4 weeks of rooting, the rooting efficiency, average number of roots, and average root length were recorded. At least 50 plants were inoculated onto each rooting medium to screen and optimize the rooting culture conditions for Panax notoginseng leaf tissue culture seedlings.

[0076] Using well-developed young shoots of Pandanus leaves as material, seedlings 1-1.5 cm long with 1-2 stem nodes, unfolded leaves, and clear veins were selected and inoculated into three rooting media, R1-R3, with no fewer than 50 seedlings in each combination. The most suitable media for the rapid propagation of Pandanus leaf seedlings was selected. Four weeks after inoculation into the rooting media, the rooting rate, average number of roots, average root length, and root growth and development were recorded.

[0077] Rooting rate (%) = Number of rooted shoot clusters / Number of inoculated shoot clusters × 100%.

[0078] Table 7. Effects of different culture media on rooting of Panax notoginseng leaf buds

[0079] culture medium Rooting rate Average number of roots per plant Average root length (cm / root) Root growth and development R1 83% 5.03 3.42 Roots are slender and soft, with few branches and few root hairs. R2 98% 7.3 6.13 Thick roots, soft roots, many branches, and many root hairs R3 99% 7.57 6.29 Thick roots, hard roots, many branches, and many root hairs R4 99% 7.36 5.98 Thick roots, soft roots, many branches, and many root hairs R5 99% 7.61 6.32 Thick roots, hard roots, many branches, and many root hairs

[0080] Experiments revealed that media R1, R2, and R3 effectively promoted rooting of seedlings, with rooting rates reaching 83%, 98%, and 99%, respectively. R2 and R3 significantly promoted rooting, exceeding R1. In terms of average number of roots and average root length, R2 and R3 significantly surpassed R1. While there was no difference in average number of roots and average root length between R2 and R3, the roots in R3 were thicker and harder, easily causing the buds to separate from the culture medium, which was detrimental to the further growth of regenerated seedlings. R4 and R5 showed slightly higher rooting rates, average number of roots, and average root length than R2 and R3, but without significant differences. Given that IAA is more expensive than NAA in production, R2 was the better choice as the rooting medium to save production costs. No root browning was observed after 4 weeks in any of the three rooting media. Considering both production costs and other factors, R2 was the most suitable rooting medium for Panax notoginseng leaves.

[0081] 3.6 Seedling Transplanting

[0082] After rooting, the tissue culture seedlings were hardened off outdoors for a week (avoiding direct sunlight; ideally, they should be under the shade of a tree or in a shade structure with 70%-80% shade) before transplanting. Before transplanting, the culture medium adhering to the roots was washed away, and the seedlings were soaked in a 1000-fold diluted 30% carbendazim powder solution for 5-10 minutes. Several common combinations of transplanting substrates were used, including equal proportions of river sand, coconut coir, and black soil; equal proportions of yellow soil and black soil; and equal proportions of yellow soil, coconut coir, and black soil. One month after transplanting, the survival rate was recorded, and root development, stem and leaf development were observed to select the most suitable substrate formula. Statistical analysis showed that a mixture of equal proportions of yellow clay, coconut coir, and black soil was the best substrate, resulting in the highest one-month survival rate of 98.5%. Considering both root development and stem and leaf growth, the seedlings with this combination showed robust growth and rapid, well-developed root systems. Apply a 1 / 1000 compound fertilizer once a week to promote seedling growth. After 3 months, the seedlings will reach a height of 10-12cm and can be sold as mature seedlings.

[0083] Survival rate (%) = Number of surviving seedlings / Number of transplanted seedlings × 100%.

[0084] Table 8: Effects of different substrates on the survival rate and growth of transplanted Panax notoginseng seedlings

[0085] matrix composition Survival rate Root development Stem and leaf development River sand 78.35% Roots are thin and long, with white root tips, few tillers, and no root hairs. The stems are thin and weak, the leaves are light in color, the stem nodes are long, and the leaves are small. Coconut coir: black soil (weight ratio 1:1) 84.27% Roots are slender and long, with orange-yellow root tips, moderate tillering, and few root hairs. The stems are thin and weak, the leaves are pale, and the leaves are small. Loess: Black soil (weight ratio 1:1) 92.64% Thick roots, dark root tips, few tillers, and few root hairs The stem is short and thick, the leaves are thick, the leaf color is dark, and the leaves are small. Loess: Coconut coir: Black soil (weight ratio 1:1:1) 98.5% Thick roots, white root tips, numerous tillers, and abundant root hairs The stem is short and thick, the leaves are thick, the leaf color is dark, and the leaves are large.

[0086] The specific embodiments of the present invention have been described in detail above, but these are merely examples, and the present invention is not limited to the specific embodiments described above. For those skilled in the art, any equivalent modifications and substitutions to this utility model are also within the scope of the present invention. Therefore, all equivalent transformations and modifications made without departing from the spirit and scope of the present invention should be covered within the scope of the present invention.

Claims

1. Application of Pandanus leaf subculture medium in improving the proliferation efficiency of Pandanus leaf shoots after shoot induction and proliferation culture, wherein the Pandanus leaf subculture medium uses MS as the basic medium, and adds 0.05-0.1 mg of NAA and 2-4 mg of 6-BA per liter of medium; and adds 1-2 g of fresh Pandanus leaf juice or freeze-dried leaf juice powder per liter of medium; wherein the fresh Pandanus leaf juice is obtained by adding water to fresh Pandanus leaves, pressing the juice and filtering it; and freeze-drying the leaf juice yields freeze-dried powder of fresh Pandanus leaf juice.

2. Use according to claim 1, wherein Add 0.05 mg NAA and 2-4 mg 6-BA per liter of culture medium.

3. The use according to claim 1, wherein Add 0.05 mg NAA and 3-4 mg 6-BA per liter of culture medium.

4. The use according to claim 1, wherein Add 0.05 mg NAA and 3 mg 6-BA per liter of culture medium.

5. A combination tissue culture medium for Pothos scandens, characterized in that, It includes the *Pandora amurense* leaf subculture medium, *Pandora amurense* leaf shoot induction medium, and *Pandora amurense* leaf rooting medium as described in any one of claims 1-4; the *Pandora amurense* leaf shoot induction medium uses MS as the basal medium, with 0.1 mg of TDZ and 1-3 mg of 6-BA added per liter of medium; the *Pandora amurense* leaf rooting medium uses 1 / 2 MS as the basal medium, with 0.1-0.2 mg of NAA or 0.1-0.2 mg of IBA added per liter of medium.

6. A combination tissue culture medium for Gynura medica Hara as claimed in claim 5, wherein, Add 0.1 mg of TDZ and 2-3 mg of 6-BA to each liter of Panax notoginseng leaf shoot induction medium.

7. The Panax notoginseng leaf tissue culture medium as described in claim 5, characterized in that, Add 0.1 mg TDZ and 2 mg 6-BA to each liter of Pandanus leaf shoot induction medium.

8. The Panax notoginseng leaf tissue culture medium as described in claim 5, characterized in that, Add 0.2 mg of NAA per liter of Pandanus leaf rooting medium.

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