Method for rapidly propagating bulbus fargesii by culturing developmental zygotic embryos

Abstract

The invention belongs to the technical field of biology, and particularly relates to a method for rapidly breeding bulbus lilii fagopyri esculenti through culture of incompletely developed zygotic embryos. According to the method, bulbus lilii fagopyri esculenti seeds are used as explants, and the incompletely developed zygotic embryos are contained in the seeds. According to the method, zygote embryos are mainly obtained by adopting two modes: one mode is that the embryos are stripped out under a microscope; the other seed is a complete seed, and directly inserting the part containing the embryo into the culture medium; zygotic embryo culture is adopted in the two culture modes, zygotic embryos are sexual reproduction products, and the reproduction mode can maintain the genetic diversity of the species. By means of the method, the purpose of rapidly expanding the population number can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically relating to a method for rapidly propagating buckwheat-leaved lilies through the culture of incompletely developed zygotes, thereby achieving the goal of rapidly expanding the population size. Background Technology

[0002] Embryo culture technology has many important applications in plant breeding and basic research. Zygote embryos express many genes necessary for embryogenesis and induction (Thorpe & Yeung, 2011), making them a preferred explant for rapid in vitro propagation. This is also why embryogenic callus is easily obtained from immature or mature zygotes in many species. Compared to mature embryos, immature embryos have greater plasticity. Many plants with significant applications, such as *Polygonatum cyrtonema* (Yang et al, 2024), *Lilium tsingtauense* (Ko et al, 2006), *Fritillaria bubiformis* (Carasso & Mucciarelli, 2014), and *Franklinia alatamaha* (Gladfelter et al, 2020), have been regenerated from immature zygotes. In the protection of rare and endangered wild species, it is essential to establish an in vitro germplasm preservation system using embryo culture technology, especially for plants with long seed dormancy periods. The buckwheat-leaved lily is a very representative example.

[0003] *Cardiocrinum cathayanum* (Wils.) Stearn is a plant belonging to the genus *Cardiocrinum* in the family Liliaceae. Other species in this genus include *Cardiocrinum giganteum* (Wall.) Makino, the Yunnan variety *Cardiocrinum giganteum* (Leichtlin ex Elwes) Stearn, and the Japanese giant lily (*Cardiocrinum cordatum* (Thunb.) Makino). *Cardiocrinum cathayanum* is a robust perennial herb, mostly found in the wild, distributed across many parts of my country, growing in sparse forests and thickets on mountain slopes and in valleys at altitudes of 600–1050 m. It has bulbs, and its leaves are divided into basal and cauline leaves, usually ovate-cordate. The inflorescence is a raceme with 3–16 narrowly trumpet-shaped flowers, mostly white with purple stripes, and fragrant. The fruit is a capsule containing numerous flat, reddish-brown seeds with narrow wings. Flowering period is from May to July, and fruiting period is from September to October. The large, elegant flowers of the buckwheat-leaved lily have high ornamental value and have been used as an ornamental plant. Meanwhile, its bulbs are rich in starch and edible; the fruit can be used medicinally (Flora of China, 1980). In folk medicine, the buckwheat-leaved lily is widely used as a herbal remedy, and its bulbs have been used to develop a medicine called "Lily Seven," which has the effects of clearing the lungs, relieving cough, detoxifying, and dispersing blood stasis.

[0004] Domestic and international studies have shown that *Lilium* seeds exhibit deep dormancy, resulting in a long seed propagation cycle. Seed germination requires a prolonged period of fluctuating temperatures, and it takes approximately one and a half years for seeds to germinate after sowing. From seed germination to the formation of a marketable bulb, it takes 3–4 years or 6–8 years, posing significant challenges to production (Kondo et al., 2006; Zhou Zhiguang, 2015). To date, research has only documented propagation methods using buckwheat leaf scales for cuttings (Wang Xiaofei et al., 2011) and using scales as explants for callus induction and adventitious bud differentiation (Li Tingting et al., 2020). However, propagation methods such as scale cuttings and bulb division for *Lilium* plants have low propagation efficiency and may lead to degeneration of ornamental traits, resulting in a decline in the quality and desirable characteristics of *Lilium*, thus reducing its value. Currently, these methods are far from meeting the needs of commercial production and hinder large-scale production, which to some extent affects the promotion and application of *Lilium* (Sun Guofeng et al., 2001).

[0005] Currently, research on tissue culture of *Lilium buckwheatensis* is only in the exploratory stage both domestically and internationally, and there are no reports of propagating this species through immature embryo culture.

[0006] The references mentioned above are as follows:

[0007] Sun Guofeng, Zhang Jinzheng, Zhuang Ping. Introduction and cultivation of Lilium grandiflorum [J]. Chinese Botanical Garden, 2001, 6: 125-128;

[0008] Wang Xiaofei, Zhao Changheng, Liu Maohui, et al. Buckwheat leaf lily scale cutting technique [J]. Practical Forestry Technology, 2011(01)26-28;

[0009] Zhou Zhiguang. Research on propagation techniques and seed dispersal ability of buckwheat-leaved lily [D]. Nanjing Forestry University, 2015;

[0010] Carasso V, Mucciarelli M. In vitro bulblet production and plant regeneration from immature embryos of Fritillaria bubiformis Gren. & Godr. Propagation of Ornamental Plants, 2014, 14(3): 101-111 (Carasso V, Mucciarelli M. In vitro bulblet production and plant regeneration from immature embryos of Fritillaria bubiformis Gren. & Godr. Propagation of Ornamental Plants, 2014, 14(3): 101-111);

[0011] Gladfelter HJ, Johnston J, Wilde HD, et al. Adventitious shoot-based propagation of Franklinia alatamaha for commercial horticulture and restoration[J]. In Vitro Cellular & Developmental Biology-Plant, 2020, 56(6):857–864 (Gladfelter HJ, Johnston J, Wilde HD, et al. Adventitious shoot-based propagation of Franklinia alatamaha for commercial horticulture and restoration[J]. In Vitro Cellular & Developmental Biology-Plant, 2020, 56(6):857-864);

[0012] Ko JA, Kim HS, Kim YS. In vitro bulblet induction and plant regeneration from immature zygotic embryos culture of Lilium tsingtauense Gilg[J]. Korean Journal of Horticultural Science and Technology, 2006, 24(2):285-289;

[0013] Kondo T, Sato C, Baskin JM, et al. Post-dispersal embryo development, germination phenology, and seed dormancy in Cardiocrinum cordatum var. glehnii (Liliaceae s.str.), a perennial herb of the broadleaved deciduous forest in Japan[J]. American Journal of Botany, 2006, 93(6): 849–859;

[0014] Yang GQ, Huang LJ, Jiang D. et al. Development of indirect somatic embryogenesis and plant regeneration system with immature embryos of the cultivated traditional Chinese medicinal herb Polygonatum cyrtonema[J]. Industrial Crops & Products, 2024, 214: 118557. Summary of the Invention

[0015] The technical problem to be solved by the present invention is to provide a method for rapid propagation of buckwheat leaf lily through the culture of incompletely developed zygote embryos.

[0016] To solve the above-mentioned technical problems, the present invention provides a method for rapid propagation of *Lilium buckwheatensis* through the culture of incompletely developed zygotic embryos (i.e., a method for rapid propagation of the rare and endangered plant *Lilium buckwheatensis* through the culture of incompletely developed zygotic embryos): using seeds of *Lilium buckwheatensis* (preferably immature seeds) as explants, wherein the seeds contain incompletely developed zygotic embryos.

[0017] As an improvement to the method of rapid propagation of *Lilium buckwheat-leaf* by culturing incompletely developed zygotes according to the present invention, the following steps are performed sequentially:

[0018] 1) Material collection: Collect buckwheat leaves and large lily capsules;

[0019] Note: Buckwheat leaf lily capsules are preferred, collected in early September;

[0020] The buckwheat leaf lily capsules collected in early September are immature capsules, containing incompletely developed zygotic embryos; even the mature capsules collected in November still contain incompletely developed zygotic embryos; the collected buckwheat leaf lily capsules can be placed in a sealed bag and stored in a refrigerator at 4°C for later use, or they can be used directly in subsequent steps.

[0021] 2) Seed surface sterilization:

[0022] The seeds from the buckwheat leaf lily capsules are removed and sterilized to obtain sterilized seeds;

[0023] 3) Isolation and inoculation of underdeveloped embryos:

[0024] This includes the following two methods:

[0025] Method 1: In vitro embryo separation method:

[0026] The seed embryos were obtained from the sterilized seeds and named as isolated embryos; the isolated embryos were then inoculated into the seed embryo germination medium with the isolated embryos placed horizontally.

[0027] Method 2: Whole Seed Inoculation Method

[0028] Insert the embryonic end of the sterilized seed obliquely into the germination medium of intact seeds;

[0029] That is, in this invention, zygotes are mainly obtained in two ways: one is to peel the embryo out under a stereomicroscope; the other is to directly insert the part containing the embryo into the culture medium as a whole seed.

[0030] 4) Embryo germination:

[0031] ① The isolated embryos inoculated onto the seed embryo germination medium according to Method 1 are first induced and cultured. The induction and culture conditions are: dark culture for 7-10 days; then transferred to light / dark alternating culture for 35±1 days; at least one of the following is obtained accordingly: bipolar zygotic embryo (zygotic embryo that grows both plumule and radicle, with rooted seedling), unipolar zygotic embryo (zygotic embryo that only grows plumule), or zygotic embryo that forms callus tissue.

[0032] ② After sterilization, the seeds inoculated into the complete seed germination medium according to method 2 are first cultured in the dark until the embryo germinates (i.e., granular protrusions appear at the base of the seed, which generally takes about 2 months). The resulting embryo is named seed embryo. Then, it is transferred to light / dark alternating culture and cultured for another 35±1 days (i.e., the culture time is about 5 weeks). The resulting embryo is named somatic embryo (the granular protrusions form clusters of somatic embryos).

[0033] 5) Cultivation of rooted seedlings:

[0034] The bipolar zygote obtained in step 4) is transferred to a rooted seedling culture medium for growth culture; the growth culture is as follows: first, it is cultured in the dark for 2 to 3 days, and then transferred to light / dark alternating culture for 35 ± 1 days. The resulting seedling is named a rooted seedling.

[0035] 6) Induction and proliferation culture of callus tissue:

[0036] The unipolar zygote and the zygote that form callus tissue obtained in step 4) are cut into pieces (about 4-6 mm × 4-6 mm) and transferred to an induction and proliferation culture medium for culture. They are first cultured in the dark for 10-14 days, and then transferred to light / dark alternating culture for 30 ± 1 days to obtain callus tissue.

[0037] Note: At this time, the cut pieces of the unipolar zygote can form granular callus tissue, and the cut pieces of the zygote that form callus tissue can generally increase in volume by 1 to 2 times.

[0038] 7) Differentiation of adventitious buds in callus tissue:

[0039] The callus tissue obtained in step 6) was transferred to differentiation culture medium, wherein the differentiation culture was performed as follows: dark culture for 5-6 days, followed by light / dark alternating culture for 35±1 days.

[0040] After this step is completed, the differentiation rate of adventitious buds in the callus tissue is calculated.

[0041] 8) Proliferation culture of adventitious buds:

[0042] Adventitious shoots differentiated from callus tissue were transferred to adventitious shoot proliferation medium for culture.

[0043] The conditions for the proliferation culture are as follows: first, dark culture for 5-6 days, followed by light / dark alternating culture; when the height of the adventitious buds reaches 2.5-3 cm, they are transferred to rooting medium for rooting.

[0044] 9) Proliferation of somatic embryos:

[0045] The somatic embryos (somatic embryos formed from the germination of complete seed embryos) obtained in step 4) are directly transferred or cut into pieces (about 4-6 mm × 4-6 mm small pieces) and then transferred to somatic embryo proliferation medium for culture.

[0046] The culture conditions are as follows: first, culture in the dark for 5 to 6 days, and then transfer to light / dark alternating culture; when the length of the somatic embryo reaches 2.5 to 3 cm, this step is ended; thus, plants with roots and buds (i.e., the somatic embryo develops into a bipolar embryo) and adventitious buds (only developing into adventitious buds of the above-ground part, i.e., the somatic embryo develops into a unipolar embryo) are obtained accordingly.

[0047] The culture time required for this step is approximately 35 days.

[0048] 10) Rooting:

[0049] The adventitious buds (2.5–3 cm in length) obtained in step 8) and step 9) were transferred to rooting medium for further culture.

[0050] The conditions for rooting culture are as follows: first, culture in the dark for 5 to 7 days, and then transfer to light / dark alternating culture; the rooting culture is ended when 3 to 5 roots with a length of ≥3cm are produced at the base of the adventitious buds; the resulting plants are named rooted plants.

[0051] 11) Transplanting:

[0052] Transplant the rooted seedlings obtained in step 5), the plants with roots and buds obtained in step 9), and the rooted plants obtained in step 10.

[0053] As a further improvement to the method of rapid propagation of buckwheat-leaved lily by culturing incompletely developed zygote embryos according to the present invention:

[0054] The temperature for dark incubation (24-hour complete dark incubation) is 22–24°C;

[0055] The light / dark (12 / 12) alternating culture was performed as follows: during light culture, the light intensity was 35–40 μmol / m. -2 .s -1 The light and dark incubation periods are 12 hours at a temperature of 24–26°C; the dark incubation period is 12 hours at a temperature of 22–24°C; the above light and dark incubation periods are alternated.

[0056] As a further improvement to the method of rapid propagation of buckwheat-leaved lily by culturing incompletely developed zygote embryos according to the present invention:

[0057] The seed embryo germination medium in step 3) of method one is: 1 / 2 MS + KT 0-0.5 mg / L + BA 0-0.5 mg / L + sucrose 25 g / L + agar 7.3-7.4 g / L, pH 5.8-6.0;

[0058] As a preferred option:

[0059] Seed embryo germination medium (medium for germination of incompletely zygotic embryos): 1 / 2 MS + KT 0.5 mg / L + sucrose 25 g / L + agar 7.4 g / L, pH 5.8;

[0060] Seed embryo germination medium (medium for germination of incompletely zygotic embryos): 1 / 2 MS + BA 0.5 mg / L + sucrose 25 g / L + agar 7.4 g / L, pH 5.8;

[0061] The complete seed germination medium in step 3) of method two is: 1 / 2 MS + CPPU 0-0.2 mg / L + sucrose 25 g / L + fresh coconut water 100 ml / L + agar 6.9-7.0 g / L;

[0062] As a preferred option:

[0063] The complete seed germination medium was: 1 / 2 MS + CPPU 0.2 mg / L + sucrose 25 g / L + fresh coconut water 100 ml / L + agar 7.0 g / L;

[0064] Step 5) The rooted seedling culture medium is B5 medium + BA 0-0.2 mg / L + NAA 0-0.5 mg / L + IBA 0-0.5 mg / L;

[0065] As a preferred option, the rooted seedling culture medium is any of the following:

[0066] B5 medium + BA 0.2 mg / L + NAA 0.5 mg / L;

[0067] B5 medium + BA 0.2 mg / L + IBA 0.5 mg / L;

[0068] The induction and proliferation medium in step 6) is: MS medium + BA 0-1 mg / L + NAA 0-0.6 mg / L + IAA 0-0.4 mg / L;

[0069] Preferably, the induction and proliferation culture medium is any one of the following:

[0070] MS medium + BA 1 mg / L + NAA 0.3 mg / L;

[0071] MS medium + BA 0.1 mg / L + NAA 0.6 mg / L + IAA 0.4 mg / L;

[0072] The differentiation medium in step 7) is: B5 medium + BA 0-1 mg / L + 2,4-D 0-0.5 mg / L + TDZ 0-1 mg / L;

[0073] Preferably, the differentiation medium is any of the following:

[0074] B5 medium + BA 1 mg / L + 2,4-D 0.5 mg / L;

[0075] B5 medium + TDZ 1 mg / L;

[0076] The adventitious bud proliferation medium in step 8) is: B5 medium + BA 0-0.2 mg / L + KT 0-1 mg / L + IBA 0-0.5 mg / L + NAA 0-0.5 mg / L;

[0077] As a preferred option, the adventitious bud proliferation medium is any one of the following:

[0078] B5 medium + BA 0.2 mg / L + KT 1 mg / L + IBA 0.2 mg / L;

[0079] B5 medium + BA 0.2 mg / L + KT 1 mg / L + NAA 0.2 mg / L;

[0080] The somatic embryo proliferation medium for step 9) is: B5 medium + BA 0-0.1 mg / L + KT 0-0.5 mg / L + NAA 0-0.1 mg / L;

[0081] As a preferred option, the somatic embryo proliferation medium is: B5 medium + BA 0.1 mg / L + KT 0.5 mg / L + NAA 0.1 mg / L.

[0082] The rooting medium for step 10) is any of the following:

[0083] 1 / 2 MS (all elements halved) + sucrose 25 g / L + agar 7.3–7.4 g / L + activated charcoal 0.5 g / L + IBA 0–0.5 mg / L + NAA 0–0.5 mg / L; pH 5.8–6.0.

[0084] 1 / 2 B5 (all elements halved) + sucrose 25 g / L + agar 6.6–6.7 g / L + activated charcoal 0.5 g / L + IBA 0–0.5 mg / L + NAA 0–0.5 mg / L, the pH of the culture medium is 5.8–6.0;

[0085] Preferably, the rooting medium is any one of the following:

[0086] 1 / 2 B5 + NAA 0.5 mg / L + sucrose 25 g / L + agar 6.6 g / L + activated charcoal 0.5 g / L, pH 5.8

[0087] 1 / 2B5 + IBA 0.5mg / l + sucrose 25g / l + agar 6.6g / l + activated charcoal 0.5g / l, pH 5.8.

[0088] As a further improvement to the method of rapid propagation of buckwheat-leaved lily by culturing incompletely developed zygote embryos according to the present invention:

[0089] Step 8) is as follows:

[0090] When the adventitious buds differentiated from the callus tissue obtained in step 7) are small adventitious buds with a base of <3mm, the cut small adventitious buds are directly transferred to the adventitious bud proliferation culture medium.

[0091] When the adventitious buds differentiated from the callus tissue obtained in step 7) are medium-sized adventitious buds with a base of 3mm to 10mm, the medium-sized adventitious buds are cut in half longitudinally from the center and then transferred to the adventitious bud proliferation culture medium.

[0092] When the adventitious buds differentiated from the callus tissue obtained in step 7) are large adventitious buds with a base >10mm, the large adventitious buds are cut into four longitudinal sections from the center and then transferred to the adventitious bud proliferation culture medium.

[0093] As a further improvement to the method of rapid propagation of buckwheat-leaved lily by culturing incompletely developed zygote embryos of the present invention, step 11) is: transplanting on a mixed substrate of peat moss: perlite: garden soil = 2:1:1 (volume ratio).

[0094] As a further improvement to the method of rapid propagation of buckwheat-leaved lily by culturing incompletely developed zygotic embryos of the present invention, step 2) is as follows: the capsule is first washed with water, then the pericarp is wiped with alcohol cotton, the pericarp is cut open, the seeds are taken out, the seeds are first soaked in 75% alcohol for 20-30 seconds, rinsed with sterile water, then sterilized with sodium hypochlorite solution containing 0.5% available chlorine for 6-8 minutes, and rinsed with sterile water; sterilized seeds are obtained.

[0095] This invention, through extensive experimentation, utilizes immature buckwheat-leaved lily seeds collected in September (at which point the embryo occupies only 5-10% of the seed length) to develop a mature method that successfully achieves seed germination. It utilizes sterile seedlings to induce callus tissue and differentiate adventitious buds, or leverages somatic embryos from germinating seeds for rapid proliferation. Both methods can quickly cultivate a large number of test-tube seedlings, providing a practical development project for the protection of this rare and endangered plant and for landscaping purposes.

[0096] During the invention process, it was discovered that the germination of seed embryos, callus induction and proliferation, differentiation of adventitious shoots from callus, proliferation of adventitious shoots and their rooting all require the use of various culture media with different ion concentrations (1 / 2 MS, MS, B5). The use of MS and B5 media alternately can reduce the browning and vitrification of the culture material, which is beneficial to plant growth.

[0097] In this invention: rapid propagation of seedlings is achieved through in vitro culture of the zygote, which is a product of sexual reproduction; seedlings are formed from intact seeds without removing the embryo, although the process is simpler. Both culture methods utilize zygote culture, which is a product of sexual reproduction, thus preserving the genetic diversity of the species.

[0098] This invention utilizes underdeveloped zygotic embryos for rapid propagation of *Lilium buckwheatense*, a method not addressed in current technologies. Existing methods, such as cuttings or tissue culture using bulbs or leaves of *Lilium buckwheatense*, only produce plants with largely identical genetic backgrounds, and the propagation rate is slow, failing to achieve the goal of rapid proliferation.

[0099] This invention has the following technical advantages:

[0100] (1) This invention overcomes the defects of incomplete embryo development, long dormancy time, and low germination rate of buckwheat-leaved lily seeds under natural conditions. After the capsules are harvested, there is no need to break the seed dormancy. The seed surface is sterilized, and the zygotic embryo is cut out or the whole seeds are directly inoculated for rapid propagation. Rapid propagation can be achieved in a short period of time to obtain a large number of buckwheat-leaved lily seedlings. This allows the production of buckwheat-leaved lily to be carried out under artificially controlled conditions, without being restricted by factors such as season, climate, and soil.

[0101] (2) The proliferation rate is fast. The zygote with incomplete development only takes 6 months from germination to transplantation, which is convenient for industrial production.

[0102] (3) It can preserve the germplasm resources of buckwheat leaf lily, and at the same time help protect wild resources and reduce damage to natural resources.

[0103] In summary, through extensive experimentation, this invention has developed a mature method that has successfully achieved rapid tissue culture propagation of buckwheat leaf lily, enabling the rapid cultivation of a large number of seedlings and providing a practical development project for the landscaping and pharmaceutical industries. Attached Figure Description

[0104] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0105] Figure 1 A diagram illustrating the rapid propagation of immature embryos of *Lilium buckwheatensis* via tissue culture.

[0106] Figure 1 middle:

[0107] A: Flowering period of the buckwheat-leaf lily plant;

[0108] B: Buckwheat leaf lily fruit collected in September;

[0109] C: The buckwheat leaf lily seeds used for cultivation have a membranous wing on the outside and the lower part of the triangle in the middle pointed to by the arrow as an immature embryo.

[0110] D: Immature embryos separated for culture were cultured on 1 / 2 MS medium containing 0.5 mg / L KT, 25 g / L sucrose, and 7.4 g / L agar at pH 5.8 for germination.

[0111] E. The immature embryo grows for 5 weeks and forms a seedling, which develops roots downward and buds upward.

[0112] F. Germinated seedlings without roots were cut and transferred to MS medium containing 1 mg / L BA, 0.3 mg / L NAA, 30 g / L sucrose, and 7.2 g / L agar at pH 5.8 for callus induction culture.

[0113] G. Adventitious shoots formed by the differentiation of callus tissue were transferred to a medium containing B5+BA 1mg / l+2,4-D 0.5mg / l+sucrose 30g / l+agar 6.4g / l, pH 5.8;

[0114] H. Adventitious buds 3-4 cm in length rooted on a medium containing 1 / 2 B5 (all elements halved) + NAA 0.5 mg / L + sucrose 25 g / L + agar 6.6 g / L, pH 5.8.

[0115] I. Transplant the rooted plants onto a substrate of peat moss: perlite: garden soil (2:1:1) to ensure survival.

[0116] What followed was (not shown in the image):

[0117] Whole seeds, without removing the embryo, were used to form somatic embryos on a medium containing 1 / 2 MS + 0.2 mg / L CPPU + 100 ml / L fresh coconut water + 25 g / L sucrose + 7.0 g / L agar.

[0118] Somatic embryos were divided into small pieces and transferred to B5 medium. The medium was supplemented with 0.1 mg / L BA, 0.5 mg / L KT, 0.1 mg / L NAA, 30 g / L sucrose, and 6.4 g / L agar at pH 5.8. The somatic embryos further developed into bipolar or unipolar plants.

[0119] Unipolar plants formed 7–10 robust adventitious roots on a medium containing 1 / 2 B5 + NAA 0.5 mg / L + sucrose 25 g / L + agar 6.6 g / L, pH 5.8. Detailed Implementation

[0120] The present invention will be further described below with reference to specific embodiments, but the scope of protection of the present invention is not limited thereto:

[0121] The abbreviations mentioned in the text are shown in Table 1 below.

[0122] Table 1. List of Abbreviations

[0123]

[0124] In the following cases:

[0125] The temperature for dark incubation (24-hour complete dark incubation) is 22–24°C;

[0126] During alternating light / dark culture (22–26℃): For light culture, the light intensity should be 35–40 μmol / m. -2 .s -1The light and dark incubation periods are 12 hours at a temperature of 24–26°C; the dark incubation period is 12 hours at a temperature of 22–24°C; the above light and dark incubation periods are alternated.

[0127] B5 medium (Gamborg B5 Basal Medium, 1968), formulation reference: Gamborg OL, Miller RA, Ojima K. Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research, 1968, 50(1), 151-158. DOI:10.1016 / 0014-4827(68)90403-5. Furthermore, the B5 medium contains: 30 g / L sucrose, 6.4–6.5 g / L agar, and a pH of 5.8–6.0.

[0128] This invention uses conventional MS medium and 1 / 2 MS medium (all elements are reduced by half).

[0129] Furthermore, in MS medium: sucrose 30 g / L, agar 7.1–7.2 g / L; pH 5.8–6.0.

[0130] Example 1: A method for rapid propagation of *Lilium buckwheatensis* through the culture of incompletely developed zygotes, comprising the following steps:

[0131] 1) Material collection: Buckwheat leaf capsules of large lily were collected in early September. The capsules can be used directly for subsequent steps, or they can be placed in a sealed bag and stored in a refrigerator at 4°C for later use.

[0132] Note: The buckwheat leaf lily capsules collected in early September are immature capsules containing underdeveloped zygotic embryos. Even the mature capsules collected in November still contain underdeveloped zygotic embryos.

[0133] 2) Seed surface sterilization

[0134] Place the capsules in a small beaker, add 100-150 ml of tap water containing detergent (1-2 drops of detergent per 100 ml of tap water), wash thoroughly, and then rinse under running water for 15 minutes. Wipe the pericarp with cotton wool soaked in 75% (volume%) alcohol. Then, on a clean bench, cut open the pericarp, remove the seeds, and first soak them in 75% alcohol for 20-30 seconds. Rinse them 3-5 times with sterile water, then sterilize them by soaking them in a sodium hypochlorite solution containing 0.5% (mass%) available chlorine for 6-8 minutes, and rinse them 3-5 times with sterile water. The resulting seeds are named the sterilized seeds.

[0135] 3) Isolation and inoculation of underdeveloped zygotes

[0136] This includes the following two methods:

[0137] Method 1: In vitro embryo dissection method (obtaining in vitro embryos by dissection):

[0138] Place the sterilized seeds obtained in step 2) in a petri dish lined with sterile filter paper and blot off the surface moisture. Under a stereomicroscope in a laminar flow hood, hold the seed with sterile forceps in one hand and use a sterile scalpel in the other hand to squeeze out the seed embryo from the tip of the triangular seed. The squeezed-out seed embryo is named the in vitro embryo. Inoculate the in vitro embryo into a petri dish containing seed embryo germination medium and place it horizontally for induction culture.

[0139] The seed embryo germination medium was: 1 / 2 MS + KT 0-0.5 mg / L + BA 0-0.5 mg / L + sucrose 25 g / L + agar 7.3-7.4 g / L, pH 5.8-6.0;

[0140] That is, the seed embryo germination medium is based on 1 / 2 MS (all elements are halved), with the addition of KT 0-0.5 mg / L, BA 0-0.5 mg / L, sucrose 25 g / L, agar 7.3-7.4 g / L, and pH 5.8-6.0.

[0141] Method 2: Whole Seed Inoculation Method

[0142] Place the sterilized seeds obtained in step 2) in a petri dish lined with sterile filter paper, absorb the surface moisture, and insert the seed with the embryo end obliquely into the intact seed germination medium.

[0143] The complete seed germination medium consisted of 1 / 2 MS + CPPU 0–0.2 mg / L + sucrose 25 g / L + coconut water 100 ml / L + agar 6.9–7.0 g / L, with a natural pH.

[0144] Coconut water is the water extracted from fresh coconuts (11-12 months old) after they have been cracked open.

[0145] Note: In the complete seed germination medium of the present invention, the amount of agar is appropriately reduced due to the addition of coconut water.

[0146] 4) Embryo germination:

[0147] ① The isolated embryos inoculated onto the seed embryo germination medium according to Method 1 are first induced and cultured under the following conditions: dark culture for 7-10 days; then transferred to light / dark alternating culture for 35±1 days; at least one of the following is obtained: bipolar zygotic embryo (zygotic embryo that grows both plumule and radicle), unipolar zygotic embryo (zygotic embryo that grows only plumule), or zygotic embryo that forms callus tissue.

[0148] Note: Due to reasons such as damage caused during embryo removal or different levels of development within the embryo, even when cultured on the same seed embryo germination medium, the embryos develop to different degrees after turning green; some form bipolar zygotes (zygotes that grow both plumules and radicles), some form unipolar zygotes (zygotes that grow only plumules), and some form zygotes that form callus (embryos swell and form callus).

[0149] Generally speaking, after being cultured in the dark, the isolated embryos are transferred to light / dark alternating culture for 5 to 7 days, after which they begin to turn green, the cotyledons elongate, and then the radicle elongates; about 2 to 3 adventitious roots emerge from the base of the hypocotyl.

[0150] ② After sterilization, the seeds inoculated into the complete seed germination medium according to method 2 are first cultured in the dark until the embryo germinates (granular protrusions appear at the base of the seed), which takes about 2 months; the resulting embryo is named seed embryo; then the seed embryo is placed in light / dark alternating culture and cultured for another 35±1 days, the resulting embryo is named somatic embryo (granular protrusions form clusters of somatic embryos) the height of the somatic embryo is less than 2cm.

[0151] 5) Cultivation of rooted seedlings:

[0152] The bipolar zygote obtained in step 4) is transferred to the rooted seedling culture medium for growth culture (i.e., growth and strengthening culture of rooted seedlings). The growth culture conditions in step 5) are as follows: first, dark culture for 2 to 3 days, and then light / dark alternating culture for 35 ± 1 days; the resulting seedlings are named rooted seedlings; at this time, the plant height can generally reach 3 to 4 cm, and the radicle length can reach ≥ 3 cm.

[0153] The culture medium for rooted seedlings is B5 medium + BA 0-0.2 mg / L + NAA 0-0.5 mg / L.

[0154] 6) Induction and proliferation culture of callus tissue

[0155] The unipolar zygote and the zygote that form callus tissue obtained in step 4) are cut into pieces (about 5mm × 5mm pieces) and transferred to induction and proliferation medium for culture: first cultured in the dark for 10 to 14 days, and then cultured in light / dark alternating culture for 30 ± 1 days to obtain callus tissue.

[0156] Note: At this time, the cut pieces of the unipolar zygote can form granular callus tissue, and the cut pieces of the zygote that form callus tissue can generally increase in volume by 1 to 2 times.

[0157] The induction and proliferation culture medium is: MS medium + BA 0-1 mg / L + NAA 0-0.6 mg / L + IAA 0-0.4 mg / L.

[0158] 7) Differentiation of adventitious buds in callus tissue:

[0159] The callus obtained in step 6) was transferred to a differentiation medium for culture: first cultured in the dark for 5-6 days, and then cultured in alternating dark and light for 35±1 days. At this time, adventitious buds differentiated on the callus.

[0160] Differentiation medium: B5 medium + BA 0-1 mg / L + 2,4-D 0-0.5 mg / L + TDZ 0-0.5 mg / L;

[0161] After step 7) is completed, the differentiation rate of adventitious buds in the callus tissue is calculated.

[0162] Adventitious bud differentiation rate of callus = number of callus blocks with adventitious bud differentiation / total number of inoculated callus blocks.

[0163] 8) Proliferation culture of adventitious buds:

[0164] After the adventitious shoots differentiated from the callus were cut from the callus block, they were transferred to an adventitious shoot proliferation medium for culture; the details are as follows:

[0165] Small adventitious buds (base of adventitious bud <3mm) differentiated from callus tissue were separated from the callus tissue block and directly transferred to adventitious bud proliferation medium.

[0166] Medium-sized adventitious buds differentiated from callus tissue (adventitious bud base swollen to 3mm-10mm) are longitudinally and evenly divided in two through the center of the adventitious bud and then transferred to adventitious bud proliferation medium.

[0167] Large adventitious buds differentiated from callus tissue (adventitious bud base swollen to >10mm) were longitudinally and evenly divided into four parts through the center of the adventitious bud and then transferred to adventitious bud proliferation medium.

[0168] The aforementioned base of the adventitious bud refers to the transverse diameter of the base of the adventitious bud.

[0169] The height of the small, medium, and large adventitious buds mentioned above is less than 2 cm.

[0170] The conditions for propagation culture are as follows: first, culture in the dark for 5 to 6 days, and then transfer to light / dark alternating culture; when the height of the adventitious buds reaches 2.5 to 3 cm, transfer to rooting medium for rooting.

[0171] The adventitious bud proliferation medium in step 8) is: B5 medium + BA 0-0.1 mg / L + NAA 0-0.5 mg / L + 2,4-D 0-0.5 mg / L.

[0172] 9) Proliferation of somatic embryos:

[0173] The somatic embryos (somatic embryos formed from the germination of complete seed embryos) obtained in step 4) were cut into small pieces of 5mm×5mm and transferred to somatic embryo proliferation medium for culture.

[0174] The culture conditions are as follows: first, culture in the dark for 5 to 6 days, then transfer to light / dark alternating culture; when the length of the somatic embryo reaches 2.5 to 3 cm, this step is ended; the culture time required for this step is about 35 days.

[0175] Somatic embryo proliferation medium: B5 medium + BA 0~0.1mg / l + KT 0~0.5mg / l + NAA 0~0.1mg / l.

[0176] At this point, some somatic embryos develop into bipolar embryos (complete plants with roots and buds), while others develop into unipolar embryos (developing only as adventitious buds in the above-ground parts).

[0177] 10) Rooting

[0178] The adventitious buds with a length of approximately 2.5–3 cm obtained in step 8) and the adventitious buds with unipolar embryo development obtained in step 9) were transferred to rooting medium for rooting culture.

[0179] The rooting medium is any one of the following:

[0180] 1 / 2 MS (all elements halved) + sucrose 25 g / L + agar 7.3–7.4 g / L + activated charcoal 0.5 g / L + IBA 0–0.5 mg / L + NAA 0–0.5 mg / L; pH 5.8–6.0.

[0181] 1 / 2 B5 (all elements halved) + sucrose 25 g / L + agar 6.6–6.7 g / L + activated charcoal 0.5 g / L + IBA 0–0.5 mg / L + NAA 0–0.5 mg / L, the pH of the culture medium is 5.8–6.0;

[0182] The conditions for rooting culture are as follows: first, culture in the dark for 5 to 7 days, and then transfer to light / dark alternating culture; the rooting culture is ended when 3 to 5 roots with a length of ≥3cm are produced at the base of the adventitious buds; the resulting plants are named rooted plants.

[0183] 11) Transplanting (transplanting rooted plants)

[0184] Place the culture containers containing the rooted seedlings obtained in steps 5), 9), and 10) at room temperature for 5–7 days (hardening off the seedlings under natural temperature and light conditions). Then, open the containers and leave them for 1–2 days. After that, remove the rooted plants and transplant them into a mixed substrate with a volume ratio of peat moss:perlite:garden soil of 2:1:1. The cultivation conditions after transplanting are in an artificial climate chamber at a temperature of 22–24°C. For the first 1–2 weeks, maintain a relative humidity of 70–80%, followed by 50–60% for the next 3–8 weeks, and then switch to natural temperature and humidity.

[0185] Experiment 1: Following the method described in Example 1 above, the specific conditions are set as follows:

[0186] Step 3) Use the "ex vivo embryo peeling method" of Method 1:

[0187] Seed embryo germination medium (medium for germination of incompletely zygotic embryos): 1 / 2 MS + KT 0.5 mg / L + sucrose 25 g / L + agar 7.4 g / L, pH 5.8;

[0188] Step 4): The isolated embryos inoculated onto the seed embryo germination medium according to Method 1 were first induced and cultured under the following conditions: 10 days in the dark; then transferred to light / dark (12 / 12) alternating culture for 5 weeks. After the end of this step 4): the germination rate of zygotic embryos reached 88.9%, bipolar zygotic embryos (zygotic embryos that produce both plumules and radicles) accounted for 42.5%, unipolar zygotic embryos (zygotic embryos that produce only plumules) accounted for 20%, and zygotic embryos that formed callus accounted for 26.4%.

[0189] Note: Generally, after dark culture, the isolated embryos are transferred to light / dark alternating culture for 5-7 days and begin to turn green and the cotyledons elongate. Then, light / dark alternating culture continues and the radicle elongates. After 5 weeks of light / dark (12 / 12) alternating culture, the radicle of the resulting bipolar zygote (a zygote that has both a plumule and a radicle) can elongate to ≥2cm, and 2-3 adventitious roots will emerge from the base of the hypocotyl, each of which can grow to ≥1cm (generally 1-1.5cm).

[0190] In step 5): the rooted seedling culture medium is B5 medium + BA 0.2 mg / l + NAA 0.5 mg / l. The bipolar zygotic embryo (a zygotic embryo that grows both plumule and radicle) is first cultured in the dark for 3 days and then transferred to light / dark alternating culture. After 5 weeks of light / dark alternating culture, the plumule of the bipolar zygotic embryo continues to elongate, and the radicle and adventitious roots grow further (the length of the radicle can reach ≥3 cm), and the average height of the plant can reach 3.6 cm.

[0191] In step 6), zygotic embryos with only plumules and zygotic embryos with callus formation were cut into 5mm*5mm pieces and transferred to callus induction and proliferation medium. They were first cultured in the dark for 14 days, then transferred to alternating light / dark culture for 30 days to obtain callus. The medium consisted of MS medium + BA 1mg / L + NAA 0.3mg / L. The callus induction rate of the small pieces reached 92%, and the callus was yellow-green and granular.

[0192] In step 7), the callus tissue was transferred to differentiation medium and cultured in the dark for 6 days, then transferred to light / dark alternating culture for 35 days. The differentiation medium was B5 medium + BA 1 mg / L + 2,4-D 0.5 mg / L. After 5 weeks of culture, the induction rate of adventitious shoots reached 77%, and the average height of adventitious shoots reached 3.4 cm.

[0193] In step 8), the adventitious shoots generated on the callus tissue are transferred to the adventitious shoot proliferation culture medium. The conditions for proliferation culture are: first, dark culture for 6 days, and then light / dark alternating culture; when the height of the adventitious shoots reaches 2.5-3 cm, they are transferred to the rooting culture medium for rooting.

[0194] The adventitious shoot proliferation medium consisted of B5 medium + BA 0.2 mg / L + KT 1 mg / L + IBA 0.2 mg / L. New adventitious shoots emerged from the base of intact adventitious shoots or from the cut surfaces of cut adventitious shoots, with the number of shoots ranging from 2 to 3. A single adventitious shoot longitudinally cut in half typically yielded 4 to 6 new adventitious shoots during culture; a single adventitious shoot longitudinally cut into quarters typically yielded 8 to 12 new adventitious shoots. Longitudinally cutting large adventitious shoots through the center significantly increased the propagation rate.

[0195] In step 8), the adventitious bud proliferation culture process can generally be carried out once every 5 weeks, and the proliferation culture can be carried out continuously.

[0196] In step 10), the adventitious shoots obtained in step 8) are transferred to rooting medium: 1 / 2 B5 + NAA 0.5 mg + sucrose 25 g / L + agar 6.6 g / L + activated charcoal 0.5 g / L, pH 5.8. The rooting culture conditions are as follows: first, dark culture for 7 days, then transfer to light / dark alternating culture; the rooting culture is ended when 3 to 5 roots with a length ≥3 cm are produced at the base of the adventitious shoots; the resulting plants are named rooted plants.

[0197] Generally, roots will develop in 4 to 5 weeks, with a rooting rate of 100%, and the average plant height will be 4.5 cm.

[0198] Step 11) After two months of hardening off, transplant the seedlings to the understory; the survival rate can reach 82%.

[0199] The survival rate is the number of transplanted plants that survived divided by the total number of transplanted plants.

[0200] Experiment 2 follows the method described in Example 1 above, except that the culture medium is different, as follows:

[0201] Step 3) Use the "ex vivo embryo peeling method" of Method 1:

[0202] Seed embryo germination medium (medium for germination of incompletely zygotic embryos): 1 / 2 MS + BA 0.5 mg / L + sucrose 25 g / L + agar 7.4 g / L, pH 5.8;

[0203] Step 4) After the culture is completed: the germination rate of zygotic embryos reaches 82%, bipolar zygotic embryos account for 30%, unipolar zygotic embryos account for 24%, and zygotic embryos with callus growth account for 28%.

[0204] In step 5), the rooted seedling culture medium was B5 medium + BA 0.2 mg / l + IBA 0.5 mg / l, and the average height of the resulting plants reached 3.4 cm.

[0205] In step 6), the induction and proliferation medium was MS medium + BA 0.1 mg / L + NAA 0.6 mg / L + IAA 0.4 mg / L, and the callus induction rate reached 85%. The callus was yellowish-green with nodular protrusions.

[0206] In step 7), the differentiation medium was B5 medium + TDZ 1 mg / l. After 5 weeks of culture, the induction rate of adventitious shoots reached 73%, and the average height of adventitious shoots reached 3.0 cm.

[0207] In step 8), the culture medium for adventitious shoot proliferation is B5 medium + BA 0.2 mg / L + KT 1 mg / L + NAA 0.2 mg / L. New adventitious shoots grow from the base of intact adventitious shoots or from the cut surface of cut adventitious shoots, with the number of adventitious shoots ranging from 1 to 2. If an adventitious shoot is longitudinally cut in half, 2 to 4 adventitious shoots will appear during culture; if an adventitious shoot is longitudinally cut into quarters, 4 to 8 adventitious shoots will appear during culture. Longitudinal cutting of large adventitious shoots through the center can greatly increase the propagation rate.

[0208] In step 10), the rooting medium is 1 / 2 B5 + IBA 0.5 mg / L + sucrose 25 g / L + agar 6.6 g / L + activated charcoal 0.5 g / L, pH 5.8. Generally, roots will form in 4-5 weeks, with a rooting rate of 92% and an average plant height of 4.0 cm.

[0209] Step 11) After two months of hardening off, transplant the seedlings to the understory; the survival rate can reach 74%.

[0210] Experiment 3: Following the method described in Example 1 above, the specific conditions are set as follows:

[0211] Step 3) Using the "intact seed inoculation method" of Method 2: For incompletely developed zygote embryos, without separating the embryo, insert the embryo-containing end of the intact seed into the seed germination medium. The intact seed germination medium is: 1 / 2 MS + CPPU 0.2 mg / L + sucrose 25 g / L + fresh coconut water 100 ml / L + agar 7.0 g / L.

[0212] First, the seeds were cultured in the dark. After 2 months, the seeds began to germinate, with a germination rate of 53.8%. Then, the seeds were cultured in alternating light and dark (12h / 12h) conditions for 5 weeks. Clusters of somatic embryos (with cotyledon morphology) appeared at the base of the plant, with an average of 7.8 somatic embryos.

[0213] Step 9): After cutting the somatic embryos into 5mm × 5mm pieces, transfer them to somatic embryo proliferation medium: B5 medium + BA 0.1 mg / L + KT 0.5 mg / L + NAA 0.1 mg / L. Culture conditions: first in the dark for 6 days, then transfer to light / dark alternating culture; end this step when the length of the somatic embryos reaches 2.5–3 cm; the required culture time for this step is approximately 35 days. The percentage of bipolar plants formed was 42%, with an average height of 2.8 cm; the percentage of unipolar plants (only a plumule, no root at the base) was 36%; and the percentage of somatic embryos that browned and died was 22%.

[0214] Step 10) Transfer the unipolar plantlets produced from the above somatic embryos to a rooting medium for rooting culture. The rooting medium is 1 / 2 B5 + NAA 0.5 mg / L + sucrose 25 g / L + agar 6.6 g / L + activated carbon 0.5 g / L, pH 5.8. The rooting culture conditions are as follows: first, culture in the dark for 7 days, then transfer to light / dark alternating culture; the rooting culture is ended when 3-5 roots with a length ≥3 cm are produced at the base of the adventitious buds; the resulting plants are named rooted plantlets. Generally, rooting occurs in 4-5 weeks, with a rooting rate of 90%, and the average plant height is 3.4 cm.

[0215] Step 11) After two months of hardening off, transplant the seedlings to the understory; the survival rate can reach 66.7%.

[0216] Of the three experiments mentioned above, Experiment 1 is generally preferred because it has a high germination rate of in vitro embryos, a short germination time, and a high yield of bipolar zygote embryos. Subsequent embryonic regeneration occurs through the callus pathway, which has a high callus induction rate (92%), a high proportion of adventitious buds differentiated from the callus (77%), and a large number of adventitious buds (up to 8-12).

[0217] It should be noted that the above three experiments used fresh capsules that were not stored in a refrigerator. The results obtained by operating the capsules stored at 4°C for one month according to the above experimental method were not significantly different from the corresponding experimental results.

[0218] Comparative Example 1-1: The culture medium for the germination of the incomplete zygote embryo in Experiment 1 was changed to the conventional 1 / 2 MS medium (without hormones), while the rest was the same as in Experiment 1. Due to the lack of hormones, the results obtained were as follows: Step 4) showed that the zygote could not expand and slowly withered and died.

[0219] Comparative Examples 1-2: The bipolar zygotic embryos (zygotic embryos with both plumules and radicles) from Experiment 1 were not transferred to the rooted seedling medium prepared by adding hormones to the B5 medium described in step 5) of Experiment 1. Instead, they continued to use the medium of "1 / 2 MS + KT 0.5 mg / L + sucrose 25 g / L + agar 7.4 g / L, pH 5.8". The results showed that, as in step 5), although the plumules and adventitious roots of the plants could grow further, after 5 weeks of alternating light and dark culture, the average height of the plants reached 2.8 cm. The length of the radicles and adventitious roots at the base of the hypocotyl increased slowly. Although hardening and transplanting were possible, the growth of the plants and the survival rate after transplanting were far inferior to those in Experiment 1.

[0220] Compared to Experiment 1, in Comparative Examples 1-3: if callus tissue was transferred to MS medium containing 1 mg / L BA, 0.5 mg / L 2,4-D, 30 g / L sucrose, and 7.2 g / L agar (pH 5.8), after 5 weeks of culture, the plant base easily turned brown, the adventitious bud induction rate reached 45%, and the average height of the adventitious buds reached 3.0 cm. Experiment 1, on the other hand, showed an adventitious bud induction rate of 77% and an average height of 3.4 cm.

[0221] Comparative Examples 1-4: The adventitious bud proliferation medium used after cutting the adventitious buds in Experiment 1 was changed to "MS medium + BA 0.2 + KT 1 mg / l + IBA 0.2 mg / l", that is, "B5 medium" was changed to "MS medium", while the hormone concentration remained unchanged. The results showed that the area near the cut of the adventitious bud was easily browned, which affected the regeneration of the adventitious bud, and the number of adventitious buds was only 1 to 2.

[0222] Comparative Examples 1-5: Adventitious buds (medium and large adventitious buds) with a base diameter greater than 3 mm from Experiment 1 were directly inoculated into the proliferation medium, with the rest being the same as in Experiment 1. Only 2-3 adventitious buds appeared at the base of the adventitious buds. However, after longitudinal cutting and culture in Experiment 1, 2-3 adventitious buds appeared in each cut piece.

[0223] Comparative Examples 1-6: Adventitious buds were transferred to a rooting medium consisting only of 1 / 2 B5 medium (i.e., 1 / 2 B5 blank medium without added auxin), with the rest being the same as in Experiment 1. The results showed that, as in step 10), roots could be formed in 4-5 weeks, with a rooting rate of only 30%, and the average plant height was 3.4 cm.

[0224] After two months of hardening off, the seedlings are transplanted into the forest floor, where the survival rate can reach 50%.

[0225] Comparative Example 2-1: The culture medium for the germination of incompletely developed zygotic embryos in Experiment 2 was changed to conventional MS medium, while the rest remained the same as in Experiment 2. The results showed that the germination rate of zygotic embryos reached 65%, with bipolar zygotic embryos accounting for 18%, unipolar zygotic embryos accounting for 26%, and zygotic embryos with callus formation accounting for 21%. The corresponding results for Experiment 2 were: a germination rate of zygotic embryos of 82%, with bipolar zygotic embryos accounting for 30%, unipolar zygotic embryos accounting for 24%, and zygotic embryos with callus formation accounting for 28%.

[0226] Comparative Example 2-2: The induction and proliferation medium was changed to "MS medium + BA 0.1 mg / L + 2,4-D 0.6 mg / L".

[0227] +IAA 0.4mg / l” ​​The rest is the same as Experiment 2; the results show that the callus induction rate reached 88%, but the callus was loose and water-soaked, which is not conducive to the subsequent differentiation of adventitious buds. Therefore, its use is not recommended.

[0228] Comparative Examples 2-3: The differentiation medium was changed to B5 medium + TDZ 0.5 mg / L, with the rest remaining the same as in Experiment 2. The results showed that after 5 weeks of culture, the induction rate of adventitious shoots reached 56%, and the average height of adventitious shoots reached 3.1 cm. The corresponding results for Experiment 2 were: the induction rate of adventitious shoots reached 73%, and the average height of adventitious shoots reached 3.0 cm.

[0229] Comparative Examples 2-4: The adventitious bud proliferation medium was changed to B5 medium + BA 1 mg / l + KT 0.2 mg / l + NAA 0.2 mg / l. The results showed in step 8) that due to the high concentration of BA, the adventitious buds were prone to vitrification; therefore, it is not recommended to use it.

[0230] Comparative Example 3-1: The "components corresponding to 1 / 2 MS medium" in the culture medium for germinating intact seed embryos were changed to "components corresponding to VW medium," while the rest remained the same as in Experiment 3. The results showed that seeds germinated successively after 3.5 months, with a germination rate of 42.9%. Granular somatic embryos appeared at the base of the embryo, with an average of 4.5 somatic embryos in clusters. In contrast, Experiment 3 showed that seeds germinated successively after 2 months, with a germination rate of 53.8%, and an average of 7.8 somatic embryos in clusters.

[0231] Comparative Example 3-2 was modified by cutting somatic embryos into small pieces and transferring them to the following somatic embryo proliferation medium: MS medium + BA 0.1 mg / L + KT 0.5 mg / L + NAA 0.1 mg / L, with the rest remaining the same as in Experiment 3. The somatic embryo cuts were prone to browning, with a 30% rate of bipolar plant formation and an average height of 2.5 cm; a 45% rate of unipolar plant formation (only a plumule, no root at the base); and a 25% rate of somatic embryo browning and death. The corresponding results for Experiment 2 were: a 42% rate of bipolar plant formation and an average height of 2.8 cm; a 36% rate of unipolar plant formation (only a plumule, no root at the base); and a 22% rate of somatic embryo browning and death.

[0232] Comparative Example 3-3: In Experiment 3, the "1 / 2 B5" in the rooting medium was replaced with "1 / 2 MS," while the rest remained the same as in Experiment 3. The rooting rate reached 75%, and the average plant height was 3.0 cm. After two months of hardening-off, the seedlings were transplanted into the forest, and the survival rate reached 60%. Experiment 3 yielded the following results: a rooting rate of 90%, an average plant height of 3.4 cm, and a survival rate of 66.7% after two months of hardening-off.

[0233] Finally, it should be noted that the above examples are merely some specific embodiments of the present invention. Obviously, the present invention is not limited to the above embodiments and many variations are possible. All variations that can be directly derived or conceived by those skilled in the art from the disclosure of the present invention should be considered within the scope of protection of the present invention.

Claims

1. A method for rapid propagation of *Lilium buckwheat-leaf* through the culture of incompletely developed zygote embryos, characterized by: The seeds of *Lilium buckwheat* were used as explants, and the seeds contained underdeveloped zygotic embryos.

2. The method for rapid propagation of *Lilium buckwheat-leaf* through culturing incompletely developed zygotic embryos according to claim 1, comprising the following steps: 1) Material collection: Collect buckwheat leaves and large lily capsules; 2) Seed surface sterilization: The seeds from the buckwheat leaf lily capsules are removed and sterilized to obtain sterilized seeds; 3) Isolation and inoculation of underdeveloped embryos: This includes the following two methods: Method 1: In vitro embryo separation method: The seed embryos were removed from the sterilized seeds and named as isolated embryos; the isolated embryos were then inoculated horizontally onto the seed embryo germination medium. Method 2: Whole Seed Inoculation Method Insert the embryonic end of the sterilized seed obliquely into the germination medium of intact seeds; 4) Embryo germination: ① The isolated embryos inoculated onto the seed embryo germination medium according to Method 1 are first induced and cultured. The induction and culture conditions are: dark culture for 7-10 days; then transferred to light / dark alternating culture for 35±1 days; at least one of the following is obtained accordingly: bipolar zygote, unipolar zygote, or zygote forming callus tissue; ② After sterilization, the seeds inoculated into the complete seed germination medium according to method 2 are first cultured in the dark until the embryo germinates, and the resulting embryo is named seed embryo; then transferred to light / dark alternating culture, and cultured for another 35±1 days, the resulting embryo is named somatic embryo. 5) Cultivation of rooted seedlings: The bipolar zygote obtained in step 4) is transferred to a rooted seedling culture medium for growth culture; the growth culture is as follows: first, it is cultured in the dark for 2-3 days, and then transferred to light / dark alternating culture for 35±1 days. The resulting seedling is named a rooted seedling. 6) Induction and proliferation culture of callus tissue: The unipolar zygotes and callus-forming zygotes obtained in step 4) were cut into pieces and transferred to an induction and proliferation culture medium for culture. They were first cultured in the dark for 10-14 days, and then transferred to light / dark alternating culture for 30±1 days to obtain callus tissue. 7) Differentiation of adventitious buds in callus tissue: The callus tissue obtained in step 6) was transferred to differentiation culture medium, wherein the differentiation culture was performed as follows: dark culture for 5-6 days, followed by light / dark alternating culture for 35±1 days. 8) Proliferation culture of adventitious buds: Adventitious shoots differentiated from callus tissue were transferred to adventitious shoot proliferation medium for culture. The conditions for the proliferation culture are as follows: first, dark culture for 5-6 days, followed by light / dark alternating culture; when the height of the adventitious buds reaches 2.5-3 cm, they are transferred to rooting medium for rooting. 9) Proliferation of somatic embryos: The somatic embryos obtained in step 4) are directly transferred or cut into pieces and then transferred to somatic embryo proliferation medium for culture. The culture conditions are as follows: first, culture in the dark for 5-6 days, then transfer to light / dark alternating culture; when the length of the somatic embryo reaches 2.5-3 cm, this step is ended; thus, plants with roots and shoots, as well as adventitious buds, are obtained accordingly. 10) Rooting: The adventitious buds obtained in step 8) and step 9) are transferred to rooting medium for further culture. The conditions for rooting culture are as follows: first, culture in the dark for 5-7 days, then transfer to light / dark alternating culture; the rooting culture is ended when 3-5 roots with a length ≥3 cm are produced at the base of the adventitious buds; the resulting plants are named rooted plants. 11) Transplanting: Transplant the rooted seedlings obtained in step 5), the plants with roots and buds obtained in step 9), and the rooted plants obtained in step 10.

3. The method for rapid propagation of *Lilium buckwheat-leaf* through the culture of incompletely developed zygotes according to claim 2, characterized in that: The temperature for dark incubation is 22~24℃; The light / dark alternating culture was performed as follows: during light culture, the light intensity was 35~40 µmol·m⁻¹. -2 .s -1 The light and dark incubation periods are 12 hours at a temperature of 24–26°C; the dark incubation period is 12 hours at a temperature of 22–24°C; the above light and dark incubation periods are alternated.

4. The method for rapid propagation of *Lilium buckwheat-leaf* by culturing incompletely developed zygotes according to claim 2 or 3, characterized in that: The seed embryo germination medium in step 3) of method one is: 1 / 2 MS + KT 0~0.5 mg / l + BA 0~0.5 mg / l + sucrose 25 g / l + agar 7.3~7.4 g / l, pH 5.8~6.0; The complete seed germination medium in step 3) of method two is: 1 / 2 MS + CPPU 0~0.2 mg / L + sucrose 25 g / L + fresh coconut water 100 ml / L + agar 6.9~7.0 g / L; The rooted seedling culture medium in step 5) is: B5 medium + BA 0~0.2 mg / l + NAA 0~0.5 mg / l + IBA 0~0.5 mg / l; The induction and proliferation medium in step 6) is: MS medium + BA 0~1 mg / l + NAA 0~0.6 mg / l + IAA 0~0.4 mg / l; The differentiation medium in step 7) is: B5 medium + BA 0~1 mg / l + 2,4-D 0~0.5 mg / l + TDZ 0~1 mg / l; The adventitious bud proliferation medium in step 8) is: B5 medium + BA 0~0.2 mg / l + KT 0~1 mg / l + IBA 0~0.5 mg / l + NAA 0~0.5 mg / l; The somatic embryo proliferation medium for step 9) is: B5 medium + BA 0~0.1 mg / L + KT 0~0.5 mg / L + NAA 0~0.1 mg / L; The rooting medium for step 10) is any of the following: 1 / 2 MS + 25 g / L sucrose + 7.3~7.4 g / L agar + 0.5 g / L activated charcoal + 0~0.5 mg / L IBA + 0~0.5 mg / L NAA; pH 5.8~6.0; 1 / 2 B5 + sucrose 25 g / l + agar 6.6~6.7 g / l + activated carbon 0.5 g / l + IBA 0~0.5 mg / l + NAA 0~0.5 mg / l, the pH of the culture medium is 5.8~6.

0.

5. The method for rapid propagation of *Lilium buckwheat-leaf* through the culture of incompletely developed zygotes according to claim 4, characterized in that... As a preferred option: Seed embryo germination medium is any of the following: 1 / 2 MS + KT 0.5 mg / L + sucrose 25 g / L + agar 7.4 g / L, pH 5.8; 1 / 2 MS + BA 0.5 mg / L + sucrose 25 g / L + agar 7.4 g / L, pH 5.8; The complete seed germination medium was: 1 / 2 MS + CPPU 0.2 mg / L + sucrose 25 g / L + fresh coconut water 100 ml / L + agar 7.0 g / L; The culture medium for rooted seedlings is any of the following: B5 medium + BA 0.2 mg / L + NAA 0.5 mg / L; B5 medium + BA 0.2 mg / L + IBA 0.5 mg / L; The induction and proliferation culture medium is any one of the following: MS medium + BA 1 mg / L + NAA 0.3 mg / L; MS medium + BA 0.1 mg / L + NAA 0.6 mg / L + IAA 0.4 mg / L; The differentiation medium is any of the following: B5 medium + BA 1 mg / L + 2,4-D 0.5 mg / L; B5 medium + TDZ 1 mg / L; The adventitious bud proliferation medium is any one of the following: B5 medium + BA 0.2 mg / L + KT 1 mg / L + IBA 0.2 mg / L; B5 medium + BA 0.2 mg / L + KT 1 mg / L + NAA 0.2 mg / L; Somatic embryo proliferation medium: B5 medium + BA 0.1 mg / L + KT 0.5 mg / L + NAA 0.1 mg / L; The rooting medium is any of the following: 1 / 2 B5 + NAA 0.5 mg / L + sucrose 25 g / L + agar 6.6 g / L + activated charcoal 0.5 g / L, pH 5.

8. 1 / 2 B5+ IBA 0.5 mg / L + sucrose 25 g / L + agar 6.6 g / L + activated charcoal 0.5 g / L, pH 5.

8.

6. The method for rapid propagation of *Lilium buckwheat-leaf* by culturing incompletely developed zygotes according to any one of claims 2 to 5, characterized in that: Step 8) is as follows: When the adventitious buds differentiated from the callus tissue obtained in step 7) are small adventitious buds with a base of <3 mm, the cut small adventitious buds are directly transferred to the adventitious bud proliferation culture medium. When the adventitious buds differentiated from the callus tissue obtained in step 7) are medium-sized adventitious buds with a base of 3 mm to 10 mm, the medium-sized adventitious buds are cut in half longitudinally from the center and then transferred to the adventitious bud proliferation medium. When the adventitious buds differentiated from the callus tissue obtained in step 7) are large adventitious buds with a base >10 mm, the large adventitious buds are cut into four longitudinal sections from the center and then transferred to the adventitious bud proliferation culture medium.

7. The method for rapid propagation of *Lilium buckwheat-leaf* by culturing incompletely developed zygote embryos according to claim 6, characterized in that... Step 11): Transplanting was carried out on a mixed substrate of peat moss: perlite: garden soil = 2:1:

1.

8. The method for rapid propagation of *Lilium buckwheatense* by culturing incompletely developed zygotes according to claim 7, characterized in that: Step 2) is as follows: the capsule is first washed with water, then the pericarp is wiped with alcohol cotton, the pericarp is cut open, the seeds are taken out, the seeds are first soaked in 75% alcohol for 20-30 seconds, rinsed with sterile water, then sterilized with sodium hypochlorite solution containing 0.5% available chlorine for 6-8 minutes, and rinsed with sterile water; sterilized seeds are obtained.

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