Generation method of hybrid liquidambar styraciflua sulphosine dependent nutritive organ origin somatic embryo

Abstract

The invention discloses a generation method of a hybrid sweetgum sulphonin-dependent nutritive organ-derived somatic embryo, which comprises the following steps: taking a mature nutritive organ as an explant, and carrying out callus induction, callus proliferation, embryogenic callus maturation, somatic embryo induction and somatic embryo development on a culture medium containing sulphonin, and performing somatic embryo germination on a culture medium which does not contain sulfonatin. According to the method disclosed by the invention, the induction of hybrid sweetgum leaf origin cotyledon embryos is realized in high-embryogenic genotypes and low-embryogenic genotypes by utilizing novel plant small peptide sulphogenin, and the sulphogenin is proved to be capable of remarkably improving the leaf callus induction rate, the cotyledon embryo formation rate and the germination rate, so that a stable and efficient system is provided for subsequent theoretical exploration, and the method has the advantages of being simple in process, low in cost and good in application prospect. And a solid foundation is laid for the development of hybrid sweetgum clonal forestry.

Description

Technical Field

[0001] The invention relates to a plant tissue culture method, in particular to a method for hybrid Liquidambar formosana leaf-derived somatic embryogenesis and obtaining regenerated plants, belonging to the technical field of plant asexual reproduction. Background Art

[0002] In recent years, due to the urgent need for large-scale reproduction or in-depth research of excellent varieties or clones in agriculture and forestry, somatic embryogenesis has become a research hotspot. This is because the regeneration of higher plants is mainly divided into the following three types: tissue repair, organogenesis and somatic embryogenesis (Xu Zhihong et al., 2019). Somatic embryogenesis, also known as somatic embryogenesis, has the characteristics of high efficiency, stable large-scale production, and single-cell origin. The polarity of its embryo allows it to develop directly into a plant without the rooting stage required for organogenesis (Zhang Tianze et al., 2022). Moreover, somatic embryogenesis is similar to the developmental stage of zygotic embryogenesis, which can provide a feasible reference model for studying the development of zygotic embryos, the earliest developmental event in the life cycle of higher plants (Guan et al., 2016). In addition, somatic embryogenesis is an important source of materials for genetic transformation and gene editing, and is also the best choice for producing haploids, somatic hybridization and artificial seeds.

[0003] The source of explants directly determines the ability of somatic embryogenesis. The process of explant to embryo regeneration first undergoes dedifferentiation to form omnipotent cells, and then redifferentiation to form embryos. Therefore, it is generally believed that the farther the explant is from the zygotic embryo stage, the more cell reprogramming processes are required to form embryonic callus tissue, and then induce somatic embryos (Xu Zhihong et al., 2019). At present, the source of explants for somatic embryogenesis of forest trees is mainly reproductive organs, including zygotic embryos, male flowers, etc., while the degree of differentiation of vegetative organs is relatively high, and it is difficult to establish a somatic embryo system. At present, it has only been successfully established in broad-leaved tree species such as poplar (Yu Zhishui et al., 2005) and eucalyptus (Corredoira et al., 2015), where somatic embryogenesis is relatively easy. However, vegetative organs can represent the genetic characteristics of the mother tree itself, and the materials are easy to obtain, which can be supplied throughout the year, and the supply is also very large. Therefore, it is very important to establish a somatic embryo system derived from vegetative organs to improve the production efficiency of excellent varieties.

[0004] Currently, the most commonly used method to induce somatic embryogenesis is to apply biological or abiotic stress to explants, and to change the levels of endogenous and exogenous plant growth regulators (PGRs) to trigger somatic embryogenesis. Currently, the most commonly used method is high-level plant hormone treatment, in addition to trauma, high temperature, chemical small molecules, osmotic pressure, etc. (Salaün et al., 2021). The most commonly used plant hormones are auxin and cytokinin. In addition, many studies have shown that abscisic acid (Chen et al., 2021; Thi et al., 2005; Rudus et al., 2009), ethylene (Roustan et al., 1989; Kepczynska et al., 2009; Zheng et al., 2013), salicylic acid (Hao et al., 2006; Hutchinson et al., 1996; Hong et al., 2008) and jasmonic acid (Rudus et al., 2005, 2009; Ahmadi et al., 2014) play a very important role in somatic embryogenesis. However, the biological process of somatic embryogenesis from vegetative organs is relatively complex, and traditional plant hormones cannot solve the problem of difficulty in establishing a somatic embryogenesis system for forest trees. With the further development of somatic embryogenesis research, the role of peptide hormones in plant growth and development has become an emerging hotspot, and it has also become a direction to solve the somatic embryogenesis from vegetative organs of forest trees.

[0005] Peptide hormones in plants are small peptides with several to dozens of amino acids. They are usually produced from precursor proteins containing N-terminal signal peptides, and are cleaved and modified to form mature peptides. So far, the modification of mature peptides can be divided into three main types: tyrosine sulfonation, proline hydroxylation, and arabinosylation. Small peptides are usually intercellular communication signals that coordinate plant defense and development processes in a non-cell-autonomous manner, including meristem maintenance, cell division, stomatal development, reproduction, and nodulation (Song et al., 2017). At present, several peptide hormones that are critical for plant regeneration have been discovered, including sulfopeptide (Phytosulfokine) (Li et al., 2024), CLAVATA3 (CLV3) / EMBRYO SURROUNDING REGION-related (CLE) (Yamaguchi et al., 2016), and PLANT PEPTIDE CONTAINING SULFATEDTYROSINE (PSY) (Ogawa-Ohnishi et al., 2022).

[0006] Sulfonates were first discovered as cell division factors during the proliferation culture of asparagus mesophyll cells (Matsubayashi et al., 1996). The process of somatic embryogenesis involves cell expansion and reprogramming. Sulfonates were found in carrots (Hanai et al., 2000) and hybrid tulip trees (Chen Jinhui et al., 2013) to promote somatic embryogenesis by accelerating embryonic cell proliferation, confirming the significant effect of sulfonates on promoting cell proliferation. In addition, studies have shown that sulfonates can promote the development of embryonic cells in Japanese larch (Umehara et al., 2005), wheat (Asif et al., 2014), and beans (Ochatt et al., 2018) to accelerate somatic embryogenesis. This phenomenon was subsequently explained in a study of Chinese fir. The study found that sulfonates can promote somatic embryogenesis by maintaining redox homeostasis and breaking away from genotype dependence. The study also showed that sulfonates also regulate Chinese fir somatic embryogenesis by participating in other biological pathways (Hao et al., 2023). The above studies indicate that sulfonyltransferase can be used as a potential key factor in promoting somatic embryogenesis, and this promoting effect is not only achieved by regulating cell proliferation, but also by other pathways, such as maintaining intracellular redox homeostasis.

[0007] At present, a method for obtaining regenerated plants by somatic embryogenesis of hybrid Liquidambar formosana petioles has been established, but the somatic embryo formation rate and germination rate are not high, and the system of obtaining regenerated plants by somatic embryogenesis using leaves as explants has not been conquered. Leaves are explants preferentially used in the field of asexual reproduction and are widely used in plant tissue culture and genetic engineering. Therefore, it is very necessary to break the bottleneck of leaf-derived somatic embryogenesis and then obtain regenerated plants. In addition, the functions of traditional plant hormones are currently clear, and some technologies cannot be solved by traditional plant hormones. Therefore, it is very necessary to explore some new plant hormones. The present invention uses a new type of plant small peptide sulfonyl peptide to break the bottleneck of leaf-derived somatic embryogenesis, and greatly improves the embryonic callus formation rate, somatic embryo formation rate and germination rate, which lays a solid foundation for the development of hybrid Liquidambar formosana clones, and also provides a reference scheme for the establishment of somatic embryo systems of other broad-leaved tree vegetative organs. Summary of the invention

[0008] The purpose of the present invention is to provide a novel method for obtaining hybrid Liquidambar somatic embryos in view of the technical problems such as low embryonic callus induction rate, low somatic embryo formation rate and low germination rate in the somatic embryogenesis process of petioles of hybrid Liquidambar, and the like. The method of the present invention uses functional leaves of hybrid Liquidambar as explants, establishes a somatic embryogenesis system through sulfopeptide, obtains somatic embryos of hybrid Liquidambar, significantly improves the breeding efficiency of hybrid Liquidambar, realizes large-scale asexual reproduction of excellent strains, and provides a reference approach for breakthroughs in somatic embryogenesis of vegetative organs of broad-leaved trees.

[0009] To achieve the purpose of the present invention, on one hand, the present invention provides a method for obtaining hybrid Liquidambar formosana somatic embryos, wherein mature organs of the hybrid Liquidambar formosana are used as explants and induced cultured on a callus induction medium, and the obtained callus is then induced cultured in a somatic embryo induction medium to obtain somatic embryos, wherein the somatic embryo induction medium contains sulfopeptide.

[0010] The callus induction medium is: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5-1.5mg / L + 6-BA 0.2-0.8mg / L + PSK-α (sulfopeptide) 0-0.2mg / L (preferably 0.1-0.2mg / L) + plant gel 1g / L, with a pH of 5.6-5.7.

[0011] In particular, the callus induction medium is: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5-1.5mg / L + 6-BA 0.2-0.8mg / L + PSK-α (sulfopeptide) 0.1-0.2mg / L + plant gel 1g / L.

[0012] In particular, the somatic embryo induction medium is: modified Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α 0-0.8mg / L (preferably 0.2-0.8mg / L) + plant gel 6g / L, with a pH of 5.6-5.7.

[0013] Particularly, the mature vegetative organs of hybrid Liquidambar formosana are leaves and petioles, preferably leaves.

[0014] In particular, leaves of hybrid Liquidambar seedlings that were 7 to 8 months old were selected for cultivation.

[0015] Another aspect of the present invention provides a method for hybrid maple leaf-derived somatic embryogenesis, comprising the following steps in order:

[0016] 1) Callus induction treatment

[0017] The sterile leaves of hybrid Liquidambar formosana were placed on callus induction medium for callus induction culture to obtain embryonic callus.

[0018] 2) Embryonic callus maturation treatment

[0019] Placing the embryonic callus in a liquid embryonic callus maturation medium and performing embryonic callus maturation culture under dark conditions;

[0020] 3) Somatic embryo induction

[0021] Placing the embryonic callus obtained by mature culture on a somatic embryo induction medium for somatic embryo induction culture;

[0022] 4) Somatic embryo development treatment

[0023] The tissue after somatic embryo induction culture is transferred to a somatic embryo development medium, and somatic embryo development culture is carried out under dark conditions to obtain cotyledon embryos;

[0024] 5) Somatic embryo germination treatment

[0025] The cotyledon embryos obtained by somatic embryo development culture are placed on a somatic embryo germination medium for somatic embryo germination culture to obtain hybrid Liquidambar formosana regenerated plants.

[0026] Wherein, in step 1), sterile leaves of 7-8 month old test tube seedlings of hybrid Liquidambar formosana are cut and placed on a callus induction medium to carry out the callus induction culture.

[0027] Particularly, the callus induction culture conditions are: dark culture, culture temperature is 25±2° C.; culture time: 25-35 days, preferably 30 days.

[0028] In particular, the callus induction medium is: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5-1.5mg / L + 6-BA 0.2-0.8mg / L + PSK-α (sulfopeptide) 0-0.2mg / L (preferably 0.1-0.2mg / L) + plant gel 1g / L, with a pH of 5.6-5.7.

[0029] In particular, the method further comprises step 1A): callus proliferation culture: transferring the embryonic callus obtained by the induction culture in step 1) to a callus proliferation culture medium, and performing callus proliferation culture under dark conditions.

[0030] Particularly, the temperature of the callus proliferation culture is 25±2°C.

[0031] In particular, the callus proliferation medium is: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5mg / L + 6-BA 0.2mg / L + PSK-α 0.1-0.2mg / L + plant gel 3g / L, with a pH of 5.6-5.7.

[0032] In particular, the callus proliferation medium is a modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5 + 6-BA 0.2mg / L + PSK-α 0.2mg / L + plant gel 3g / L, with a pH of 5.6-5.7.

[0033] Particularly, during the callus proliferation culture process, subculture is performed every 12-17 days (preferably 15 days) for 30 days.

[0034] In step 1A), after 15 days of callus proliferation culture, a more dispersed, granular secondary callus will grow. The secondary callus is considered to be able to induce somatic embryos. At this time, it is subcultured once and the proliferation process is cultured for 30 days to obtain a sufficient number of secondary embryonic callus.

[0035] In particular, the embryonic callus maturation culture conditions in step 2) are: dark culture; maturation culture temperature is 25±2°C; maturation culture time is 12-17 days, preferably 15 days; during the maturation culture process, suspension culture is performed, wherein the suspension culture rate is 120±20rpm.

[0036] Particularly, the embryonic callus maturation medium is: modified Blaydes basic medium + sucrose 40 g / L + PSK-α 0-0.8 mg / L (preferably 0.2-0.8 mg / L, more preferably 0.4 mg / L), with a pH of 5.6-5.7.

[0037] Particularly, during the maturation culture of embryonic callus, subculture is performed every 7 days; the maturation culture time is 12-17 days, preferably 15 days.

[0038] Step 2) The mesoembryonic callus is suspended and cultured for 15 days (usually 12-17 days), subcultured every 7 days, and mature culture reduces the content of exogenous plant hormones in the callus, which facilitates subsequent somatic embryo induction.

[0039] The callus maturation culture in step 2) is performed by suspension culture in a liquid culture medium, wherein every 0.2 g of callus is placed in 50 ml of liquid maturation culture medium.

[0040] In particular, the somatic embryo induction culture in step 3) is as follows: after filtering the embryonic callus after maturation, the mature embryonic callus together with the filter paper is placed on a solid somatic embryo induction medium for culture.

[0041] In particular, the culture conditions for the somatic embryo induction culture in step 3) are: dark culture; culture temperature is 25±2° C.; and culture time is 25-35 days, preferably 30 days.

[0042] In particular, the somatic embryo induction medium is: modified Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α0-0.8mg / L (preferably 0.2-0.8mg / L, more preferably 0.4mg / L) + plant gel 6g / L, with a pH of 5.6-5.7.

[0043] The purpose of somatic embryo induction culture is to absorb plant hormones in embryonic callus.

[0044] The purpose of step 3) is to further adsorb exogenous plant hormones in embryonic callus tissue through activated carbon and high-dose plant gel, provide a dry culture environment for somatic embryo induction, and improve the efficiency of somatic embryo induction. After 30 days (25-35 days) of culture, spherical embryos will be produced one after another.

[0045] In particular, the somatic embryo development culture conditions in step 4) are: dark culture; and a culture temperature of 25±2°C.

[0046] Particularly, the culture time for somatic embryo development is 55-65 days, preferably 60 days, and the culture is usually carried out until cotyledonary embryos are obtained.

[0047] In particular, during the somatic embryo development culture process, subculture is performed every 30 days. The fingerprint gel content in the somatic embryo development medium is lower than the plant gel content in the somatic embryo induction medium. The plant gel content in the medium is reduced, which reduces the osmotic pressure of the medium and is conducive to the development of globular embryos into heart-shaped embryos, torpedo-shaped embryos and cotyledon embryos.

[0048] In particular, the somatic embryo development medium is: modified Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α0-0.8mg / L (preferably 0.2-0.8mg / L, more preferably 0.4mg / L) + plant gel 3g / L, with a pH of 5.6-5.7.

[0049] In particular, the somatic embryo germination culture conditions in step 5) are: germination culture is carried out under light conditions; the culture temperature is 25±2° C.; the light intensity is 1000-1500 lx (preferably 1200 lx); and the light time is 14-18 h / d (preferably 16 h / d).

[0050] Particularly, the somatic embryo germination medium is: WPM basic medium + sucrose 30g / L + plant gel 3g / L, pH is 5.8-5.9.

[0051] In step 5), the somatic embryo germination culture time is 25-35 days, preferably 30 days; during the somatic embryo germination culture process, the culture conditions are changed from darkness to light and the regenerated seedlings are developed. The use of the basic culture medium in this step and the pH value of the culture medium are significantly different from other processes. After 15 days of culture, the cotyledon embryo will grow a complete root system and cotyledons will grow. After 30 days of culture, the cotyledon embryo will develop into a complete plant.

[0052] Compared with the prior art, the present invention has the following advantages:

[0053] 1. The present invention overcomes the technical difficulty of obtaining regenerated plants through somatic embryos using leaves as explants, which are more widely used in genetic engineering, production applications, etc. The existing technical system using petioles as explants is not convenient for use in other purposes because the petioles have a small surface area and are difficult to obtain.

[0054] 2. The present invention clearly distinguishes the process of hybrid Liquidambar formosana somatic embryogenesis into two processes: leaf dedifferentiation to induce callus and callus redifferentiation to form somatic embryos. It provides a suitable system for studying the molecular mechanism of plant tissue dedifferentiation and redifferentiation, and provides a stable and efficient technical system for genetic engineering breeding.

[0055] 3. Genotype differences have always been a key factor restricting somatic embryogenesis. The present invention is applicable to both high-embryonic genotypes and low-embryonic genotypes, and is of greater significance to production practice.

[0056] 4. The present invention adopts a novel small peptide hormone - sulfopeptide for embryonic callus induction culture, liquid maturation culture, somatic embryo induction culture, and somatic embryo development culture, which greatly improves the efficiency of somatic embryogenesis.

[0057] 5. The addition of sulfonyl sulfonate to the culture medium of the present invention improves the callus induction rate. An experiment was designed using three low-embryogenic genotypes ("Southeast-1-2", "HT-4", and "HT-10", which are seedlings) to explore the results. It was found that when 0.2 mg / L sulfonyl sulfonate was added to the callus induction medium, although there were differences in the embryonic callus induction rates of plants of different genotypes, they all reached more than 83%, and the overall callus induction rate was the highest.

[0058] 6. In the present invention, in the process of inducing somatic embryos, it was found in the low-embryonic genotype ("Southeast-1-2") that when sulfonyl peptidase was not added to the culture medium, the embryonic callus could not develop into somatic embryos. When 0.4 mg / L sulfonyl peptidase was added, the embryonic callus successfully induced somatic embryos and could normally develop into cotyledonary embryos. In the high-embryonic genotype ("SF15SH-5a", somatic embryo seedlings), it was found that when sulfonyl peptidase was not added to the culture medium, 38 cotyledonary embryos could be formed per 0.2 g of callus. When 0.4 mg / L sulfonyl peptidase was added, 82 cotyledonary embryos could be formed per 0.2 g of callus, and the cotyledonary embryo formation rate was significantly improved. In addition, the exogenous addition of sulfonyl peptidase did not affect the subsequent somatic embryo germination, and the somatic embryo germination rate could reach more than 50%, which is much better than the prior art. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 This is a diagram of callus induction from the leaves of hybrid Liquidambar formosana of genotype Southeast-1-2;

[0060] Figure 2This is a picture of callus induced from the leaves of hybrid Liquidambar formosana of genotype SF15SH-5a;

[0061] Figure 3 This is the embryonic callus diagram of the genotype Southeast-1-2;

[0062] Figure 4 This is the embryonic callus picture of genotype SF15SH-5a;

[0063] Figure 5 These are spherical embryos induced from leaves of the genotype SF15SH-5a;

[0064] Figure 6 This is a picture of the heart-shaped embryo induced from the leaves of the genotype SF15SH-5a;

[0065] Figure 7 This is a picture of torpedo-shaped embryos induced from leaves of genotype SF15SH-5a;

[0066] Figure 8 This is the cotyledon embryo image induced from leaves of genotype SF15SH-5a;

[0067] Fig. 9 The effect of sulfopeptide treatment on the cotyledon embryo formation rate during the process of embryogenic callus transformation into somatic embryo morphology of genotype SF15SH-5a;

[0068] Fig.10 The spherical embryos and heart-shaped embryos induced from leaves of the genotype Southeast-1-2 are shown;

[0069] Fig.11 This is a picture of the torpedo-shaped embryo induced from the leaves of the genotype Southeast-1-2;

[0070] Fig.12 This is the cotyledon embryo diagram induced from leaves of the genotype Southeast-1-2;

[0071] Fig.13 This is the picture of the cotyledon embryo germination and seedling formation of the genotype SF15SH-5a;

[0072] Fig.14 This is the effect of sulfonyl sulfonate treatment on the subsequent cotyledon embryo germination during the process of embryonic callus transformation into somatic embryo morphology in the genotype SF15SH-5a. DETAILED DESCRIPTION

[0073] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, these embodiments are exemplary only and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified and replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

[0074] Test materials

[0075] 1. Sterile leaves and petioles of hybrid Liquidambar formosana

[0076] The hybrid liquidambar formosana is a progeny obtained by hybridizing North American liquidambar formosana as the female parent and Chinese liquidambar formosana as the male parent. The present invention uses seedlings and somatic embryo seedlings.

[0077] The seedlings, that is, low-embryo seedlings, are based on the "Southeast-1" in the Liquidambar base in Yanling County, Xuchang City as the female parent, and the "Zhongshan No. 3" in Qingdao Zhongshan Park as the male parent. The three excellent offspring obtained after hybridization are: "Southeast-1-2", "HT-4" and "HT-10", among which "HT-4" and "HT-10" are excellent offspring bred through space mutagenesis.

[0078] Somatic embryo seedlings, also known as highly embryonic seedlings, are somatic embryo seedlings obtained by regeneration from the embryonic cell line SF15SH-5a derived from zygotic embryos.

[0079] In a sterile environment, young leaves and petioles of 7-8 month old test tube seedlings of hybrid Liquidambar formosana Southeast-1-2, HT-4, HT-10 and SF15SH-5a were collected and used as explant test materials of the present invention.

[0080] 2. Plant growth regulators

[0081] The plant growth regulators used in the present invention are 6-benzylaminopurine (6-BA) and 2,4-phenoxyacetic acid (2,4-D), which are purchased from Sigma, and sulfopeptide (PSK-α) is purchased from Nanjing Peptide Industry Biotechnology Co., Ltd.

[0082] 3. Culture medium

[0083] (1) The modified Blaydes basic medium formula is shown in Table 1:

[0084] Table 1 Formula of modified Blaydes medium

[0085]

[0086] (2) The formula of WPM basic culture medium is shown in Table 2:

[0087] Table 2 Formulation of WPM medium

[0088]

[0089] Modified Blaydes, WPM basic medium and modified WPM basic medium are basic medium. According to the volume of the prepared medium, the required macroelement mother solution components, trace element mother solution components and organic mixture mother solution components and weighed plant gel, sucrose, and hydrolyzed casein (part of the culture medium is added) are added to the deionized water in sequence, the pH is adjusted, and the medium is sterilized at a constant temperature of 121°C for 15 minutes.

[0090] The basic formula of modified Blaydes medium is as follows: Merkle SA, Neu KA, Battle PJ, Bailey RL. 1998. Somatic embryogenesis and plantlet regeneration from immature and mature tissues of sweetgum (Liquidambar styraciflua). Plant Science 132: 169-178.

[0091] (3) Callus induction medium:

[0092] Callus induction medium: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5-1.5mg / L + 6-BA 0.2-0.8mg / L + PSK-α 0-0.2mg / L (preferably 0.1-0.2mg / L, more preferably 0.2mg / L) + plant gel 1g / L, pH 5.6-5.7.

[0093] Preferably, it is: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5mg / L + 6-BA 0.2mg / L + PSK-α 0.2mg / L + plant gel 1g / L, with a pH of 5.6-5.7.

[0094] (4) Callus proliferation medium:

[0095] Modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5 + 6-BA 0.2mg / L + PSK-α 0.1-0.2mg / L (preferably 0.2mg / L) + plant gel 3g / L, pH 5.6-5.7.

[0096] Preferably, it is: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5 + 6-BA 0.2mg / L + PSK-α 0.2mg / L + plant gel 3g / L, with a pH of 5.6-5.7.

[0097] (5) Embryogenic callus maturation medium:

[0098] Modified Blaydes minimal medium + sucrose 40 g / L + PSK-α 0-0.8 mg / L (preferably 0.2-0.8 mg / L, more preferably 0.4 mg / L), pH 5.6-5.7.

[0099] Preferably, it is: modified Blaydes basic medium + sucrose 40 g / L + PSK-α 0.4 mg / L, pH 5.6-5.7.

[0100] (6) Somatic embryo induction medium:

[0101] Improved Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α 0-0.8mg / L (preferably 0.2-0.8mg / L, more preferably 0.4mg / L) + plant gel 6g / L, pH 5.6-5.7.

[0102] Preferably, it is: improved Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α 0.4mg / L + plant gel 6g / L, with a pH of 5.6-5.7.

[0103] (7) Somatic Embryo Development Medium:

[0104] Improved Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α0-0.8mg / L (preferably 0.2-0.8mg / L, more preferably 0.4mg / L) + plant gel 3g / L, pH 5.6-5.7.

[0105] Preferably, it is: modified Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α 0.4mg / L + plant gel 3g / L, with a pH of 5.6-5.7.

[0106] (8) Somatic embryo germination medium: WPM basic medium + sucrose 30 g / L + plant gel 3 g / L, pH 5.8-5.9.

[0107] 4. Culture conditions

[0108] The culture conditions for callus induction, callus proliferation, embryonic callus maturation, somatic embryo induction, and somatic embryo development were: dark culture at a temperature of 25±2°C.

[0109] The culture conditions for somatic embryo germination are: light culture, light intensity is 1200lx (usually 1000-1500lx), light time is 16h / d (usually 14-18h), and culture temperature is 25±2℃.

[0110] Genotype is a key influencing factor in the establishment of a somatic embryogenesis system, which seriously limits the development of various species to obtain regenerated plants through somatic embryogenesis. The exogenous sulfopeptide of the present invention breaks the limitations of hybrid Liquidambar formosana explants and genotypes. The present invention involves a total of 4 genotypes of hybrid Liquidambar formosana, and somatic embryos originating from 2 genotypes (Southeast-1-2, SF15SH-5a) are successfully induced through this system, and fully develop into cotyledon embryos. Among them, the hybrid Liquidambar formosana of the Southeast-1-2 genotype seedlings has a low somatic embryogenesis ability, and exogenous sulfopeptide needs to be added throughout the development process until cotyledon embryos are formed; the somatic embryogenesis ability of the somatic embryo seedling SF15SH-5a is higher, but the exogenous addition of different concentrations of sulfopeptide in the process of embryonic callus to somatic embryo morphology has a significant effect on the efficiency of cotyledon embryo formation.

[0111] Somatic embryogenesis from vegetative organs is mainly divided into two processes: process 1: dedifferentiation of mature vegetative organs to induce embryonic callus; process 2: differentiation and development of embryonic callus to form somatic embryos. In broad-leaved tree species, the bottleneck is mainly focused on the second process. Many embryonic tissues with structural states similar to embryonic callus cannot complete the maturation process and induce somatic embryos, and thus cannot form regenerated plants.

[0112] In the process of inducing callus from mature leaves of hybrid Liquidambar formosana, exogenous addition of sulfopeptide can improve the callus induction efficiency of leaves of three low-embryonic genotypes independently of genotype; and in the subsequent process of embryonic callus differentiation and development to form somatic embryos, embryonic callus of the Southeast-1-2 genotype without exogenous addition of sulfopeptide cannot form somatic embryos, while exogenous addition of sulfopeptide successfully enables Southeast-1-2 to complete the development of the complete stage of somatic embryos. The somatic embryo induction of Southeast-1-2 from seedlings is very critical and has more guiding significance for future application in production.

[0113] In addition, in the high-embryogenic genotype SF15SH-5a, it was found that the exogenous addition of sulfonylcholine to the process of embryonic callus tissue to somatic embryo morphology improved the efficiency of somatic embryo formation, and different concentrations of sulfonylcholine treatment had a significant effect on the cotyledon embryo formation rate.

[0114] Example 1 Callus induction from leaves of low-embryogenic genotypes

[0115] Under a sterile environment, young leaves of 7-8 month-old hybrid Liquidambar formosana test tube seedlings of three genotypes ("Southeast-1-2", "HT-4" and "HT-10") were cut as explants, cut into small pieces, and placed (i.e., inoculated) in the induction medium with the back of the leaves facing upward. The callus induction culture used genotypes (Southeast-1-2, HT-4, HT-10), 2,4-D, 6-BA and PSK-α as experimental factors, and each factor had three levels. An orthogonal experimental design was performed (as shown in Table 3). Three biological replicates were set for each experimental treatment, and 10 explants were inoculated per plate in each replicate, for a total of 30 explants. Callus induction was performed under dark conditions.

[0116] The inoculated leaves curled and shrank about 7 days after callus induction, callus appeared about 15 days later, and a large amount of primary callus appeared 30 days after induction. The primary callus had different states, including dense non-embryonic callus and loose spherical embryonic callus.

[0117] After 30 days of callus induction, more primary callus (such as Figure 1 ), the induced primary callus tissue was loose in texture, and the primary callus tissue induction rate was calculated according to formula (1). The statistical results are shown in Tables 3 and 4.

[0118] Callus induction rate = number of leaves that induced primary callus / number of leaves inoculated (1)

[0119] Table 3 Screening of callus induction conditions of hybrid Liquidambar formosana

[0120]

[0121] Note: Range = k (maximum value) - k (minimum value)

[0122] Table 4 Analysis of variance for screening of callus induction conditions of hybrid Liquidambar formosana

[0123]

[0124] Note: ** indicates extremely significant difference (p<0.01)

[0125] As shown in Tables 3 and 4, there are great differences in the primary callus induction rates of different genotypes. Sulfonate significantly affects the callus induction efficiency. The culture formula with the highest callus induction rate of different genotypes has an exogenous addition of 0.2 mg / l sulfonate. When 0.2 mg / l sulfonate induction medium is added, the callus induction rate of the three genotypes is above 83%. Sulfonate improves the callus induction rate of HT-4 and HT-10. Although the induction rate of Southeast-1-2 can reach 100% without the addition of exogenous sulfonate, the exogenous addition of sulfonate can make the tissue under the same induction time induce more callus, which also proves the key role of sulfonate in callus induction. In addition, the optimal culture medium for callus induction was screened out through range analysis: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5 + 6-BA 0.2mg / L + PSK-α0.2mg / L + plant gel 3g / L, pH 5.6-5.7.

[0126] Example 1A Callus induction from leaves of high embryogenic genotype SF15SH-5a

[0127] In a sterile environment, young leaves of SF15SH-5a genotype hybrid Liquidambar formosana test tube seedlings of 7 to 8 months old were cut as explants, and the leaves were cut into small pieces, and the leaves were placed (i.e., inoculated) in an induction medium with the back of the leaves facing upward. The induction medium used was the superior callus induction medium selected in Example 1. After 30 days (usually 25-35 days) of callus induction, primary callus (such as Figure 2 ).

[0128] Example 2 Low embryogenic gene callus proliferation culture

[0129] The primary calli of the three induced genotypes ("Southeast-1-2", "HT-4" and "HT-10") were separated from the explants and then inoculated on callus proliferation medium. After culturing for 15 days, it was clearly observed that the primary callus of Southeast-1-2 proliferated into new secondary embryonic callus. The secondary embryonic callus was looser and more granular than the primary callus (e.g. Figure 3 ), which is significantly different from the primary callus.

[0130] After 12-17 days (preferably 15 days) of callus proliferation culture, subculture is performed, and the proliferation culture is carried out for a total of 30 days to achieve doubling of the callus growth.

[0131] Example 2A Callus proliferation culture of high embryogenic genotype SF15SH-5a

[0132] The primary callus induced by the SF15SH-5a genotype was separated from the explants and inoculated into callus proliferation medium. Secondary embryonic callus appeared after 15 days of culture. After 30 days of proliferation culture, more secondary embryonic callus (such as Figure 4 ).

[0133] The transformation of embryonic callus into somatic embryo morphology is a key step of the present invention, which includes three parts: embryonic callus maturation, somatic embryo induction, and somatic embryo development culture. Both callus induction and proliferation stages require high levels of exogenous auxin and cytokinin stimulation, while the transformation of embryonic callus into somatic embryo morphology requires the removal of exogenous auxin and cytokinin, so the exogenous auxin and cytokinin content in the tissue is reduced during the embryonic callus maturation, somatic embryo induction, and development culture stages.

[0134] Example 3 Maturation culture of embryonic callus of high embryogenic genotype SF15SH-5a

[0135] In the process of somatic embryo morphogenesis of the present invention, sulfopeptidase is used as an experimental factor, a single factor experimental design is carried out, and 3 replicates are performed for each treatment. Embryonic callus maturation culture is to place embryonic callus in a liquid callus maturation medium for suspension culture to obtain mature cultured embryonic callus; 2,4-D and 6-BA are not added to the medium. Sulfopeptidase of the present invention may promote somatic embryo formation by regulating redox homeostasis.

[0136] The callus tissues (secondary embryonic callus tissues) obtained by callus tissue proliferation culture are inoculated into liquid embryonic callus tissue maturation medium containing different concentrations of sulfonyl peptide, and the callus tissues are subjected to liquid suspension maturation culture in the dark to obtain mature cultured embryonic callus tissues; wherein each 0.2 g of callus tissue is inoculated into 50 ml of liquid maturation medium; and the concentrations of sulfonyl peptide in the maturation medium are 0, 0.2, 0.4, and 0.8 mg / L, respectively; the suspension culture is carried out on a shaker at a rate of 120±20 rpm; the culture temperature is 25±2° C.; during the maturation process, the callus tissues are subcultured once every 7 days, and the suspension treatment is carried out for 15 days (usually 12-17 days).

[0137] Example 3A Somatic Embryo Induction Culture of High Embryogenic Genotype SF15SH-5a

[0138] The callus tissue after 15 days of liquid suspension mature culture was filtered out and placed with filter paper in somatic embryo induction medium. The somatic embryo induction medium was added with activated carbon and a high content of plant gel, which was conducive to the adsorption of exogenous auxin and cytokinin in the callus tissue and facilitated the formation of embryo morphology. No 2,4-D and 6-BA were added to the somatic embryo induction medium.

[0139] The culture system of liquid mature culture for 15 days is filtered to obtain mature culture embryonic callus, and the filtered embryonic callus together with the filtered filter paper are placed in a somatic embryo induction medium containing different concentrations of sulfonyl peptide, and somatic embryo induction culture is carried out under dark conditions, wherein the concentrations of sulfonyl peptide in the somatic embryo induction medium are 0, 0.2, 0.4, and 0.8 mg / L respectively; and each different treatment of embryonic callus is 0.2 g; somatic embryo induction culture is a critical period for the embryonic callus to develop into a somatic embryo morphology, and after somatic embryo induction culture for one month (30 days, usually 25-35 days), the callus develops successively, and some can grow spherical embryos.

[0140] Example 3B Development and culture of somatic embryos of the high-embryonic genotype SF15SH-5a

[0141] After culturing in a somatic embryo induction medium containing a high content of plant gel for about one month, the tissue after somatic embryo induction culture is transferred together with the filter paper to a somatic embryo development medium for somatic embryo development culture to obtain somatic embryos; the plant gel content in the somatic embryo development medium is reduced, because the embryonic callus and somatic embryos will be dehydrated and die due to long-term dry culture. Compared with the somatic embryo induction medium, the plant gel content in the development medium is lower than that in the somatic embryo induction medium. The low content of plant gel in the somatic embryo development medium will provide relatively moist and normal osmotic pressure, which is conducive to maintaining the moisture in the induced somatic embryos and facilitating growth. It is conducive to maintaining the moisture in the induced somatic embryos.

[0142] After 30 days of somatic embryo induction culture (usually 25-35 days), the somatic embryos were inoculated into somatic embryo development medium and cultured in the dark. The concentrations of sulfopeptide in the somatic embryo development medium were 0, 0.2, 0.4, and 0.8 mg / L, respectively. During the somatic embryo development culture, the somatic embryos completed the development of four morphologies: globular embryos, heart-shaped embryos, torpedo-shaped embryos, and cotyledon embryos ( Figure 5 , 6 , 7, 8).

[0143] The number of cotyledon embryos after somatic embryo development culture under different treatments was counted. The statistical results are as follows Fig. 9 In the process of embryonic callus tissue forming cotyledon embryo morphology, the exogenous addition of 0.4 mg / L sulfopeptide significantly increased the cotyledon embryo formation rate. When no sulfopeptide was added, 38 cotyledon embryos could be formed per 0.2 g callus tissue. When 0.4 mg / L sulfopeptide was added, 82 cotyledon embryos could be formed per 0.2 g callus tissue, which greatly increased the cotyledon embryo formation rate.

[0144] Embryogenic callus maturation, somatic embryo induction and somatic embryo development are three stages in the process of embryogenic callus developing into somatic embryo morphology. They are three essential stages that must be cultured in strict order. The three stages complete a biological process. Therefore, in the single-factor experimental design with sulfopeptide as the experimental factor in the three stages, the concentration of sulfopeptide was consistent in the three stages and corresponded to each other (i.e., the concentration of sulfopeptide was inherited and followed; for example, the concentration of sulfopeptide in the maturation medium was 0.4 mg / L, and the concentration of sulfopeptide in the somatic embryo induction medium and the somatic embryo reaction medium was 0.4 mg / L; other concentrations were the same). The three experimental designs together constituted an exploration of the effect of sulfopeptide on the embryo formation rate of hybrid Liquidambar formosana cotyledons, thereby screening out the optimal concentration of sulfopeptide in the process of hybrid Liquidambar formosana embryogenic callus differentiation into somatic embryos.

[0145] Example 3C Construction of embryonic callus of low-embryogenic genotype into somatic embryo morphology

[0146] The obtained embryonic callus of Southeast-1-2 was subjected to embryonic callus maturation culture, somatic embryo induction and somatic embryo development culture in sequence. The embryonic callus maturation medium, somatic embryo induction medium and somatic embryo development medium in the culture process were designed as two control experiments without adding sulfopeptide and with exogenous addition of 0.4 mg / L sulfopeptide, respectively. The other culture conditions were the same as those in Examples 3, 3A and 3B, respectively. After 30 days of somatic embryo induction, it was found that no globular embryos were formed in the treatment without adding sulfopeptide, and the embryonic callus did not develop; globular embryos were observed to be formed in the treatment with exogenous addition of 0.4 mg / L sulfopeptide, and the globular embryos were continuously transferred to the development medium treated with exogenous addition of 0.4 mg / L sulfopeptide, and the globular embryos completed the development of heart-shaped embryos and torpedo-shaped embryos until the cotyledonary embryos (such as Fig.10 , 11 , 12). This further proves that sulfonyltransferase plays a crucial role in the morphogenesis of somatic embryos.

[0147] The low-embryogenic genotypes HT-4 and HT-10 were also unable to successfully induce somatic embryos when treated with exogenous sulfonyltransferase, which shows that the genotype has a great influence on leaf-derived somatic embryogenesis, and it is difficult for a certain factor to be applicable to all genotypes.

[0148] Example 4 Somatic embryo germination culture of high embryogenic genotype SF15SH-5a

[0149] The above examples demonstrate that sulfopeptide significantly promotes the callus induction rate and the efficiency of cotyledon embryo formation. This example studies the effect of sulfopeptide on the quality of cotyledon embryo formation.

[0150] In a sterile environment, the cotyledon embryos produced by the four sulfonyltransferase treatments in Example 3B were inoculated onto the same germination medium without sulfonyltransferase added, and somatic embryo germination culture was performed under light conditions. Three plates were inoculated for each treatment, with 10 cotyledon embryos per plate, for a total of 30 cotyledon embryos.

[0151] Since the exogenous addition of 0.8 mg / L sulfopeptide inhibited the cotyledon embryo formation rate during the process of embryonic callus tissue to somatic embryo morphology, it was costly and had poor effect, so no experiment was set up in the germination culture.

[0152] After one week of somatic embryo germination culture under light conditions, the cotyledon embryo can be observed to change from white to green. After 15 days, green cotyledons and slender roots will grow together. After 30 days, the green plants will be fully developed. Fig.13 At this time, the germination rate of cotyledon embryos produced by each treatment in Example 3B was statistically calculated. The cotyledon embryo germination rate was calculated according to formula (2). The statistical results are as follows: Fig.14 shown.

[0153] Cotyledon embryo germination rate = number of cotyledon embryos germinated / number of cotyledon embryos inoculated (2)

[0154] Depend on Fig.14 It can be seen that adding sulfonyl sulfonate to the embryonic callus during the somatic embryo morphology process will not affect the developmental state of the corresponding cotyledon embryo, but will affect its subsequent germination. This proves that sulfonyl sulfonate has a significant promoting effect on somatic embryo formation and has no adverse effect on the acquisition of regenerated plants, and can be widely used.

[0155] The low-embryo genotype Southeast-1-2 produced a small number of cotyledon embryos, and the material was precious, so no germination test was conducted.

[0156] Example 5 Somatic embryogenesis culture using petioles as explants

[0157] 1. Callus induction culture

[0158] In a sterile environment, young petioles of 7-8 month old hybrid Liquidambar formosana test tube seedlings of four genotypes ("Southeast-1-2", "HT-4", "HT-10" and SF15SH-5a) were cut as explants, and the petioles were cut into pieces of about 1 cm and inoculated into induction medium. The rest was the same as in Examples 1 and 1A.

[0159] After 30 days of culture, more callus tissues were induced from the petioles, among which there were primary callus tissues with loose texture and better quality.

[0160] 2. Callus proliferation culture

[0161] The induced primary callus was separated from the explant, inoculated into a proliferation medium, and cultured for callus proliferation, which was the same as in Example 2 and 2A.

[0162] After 30 days of culture, some loose and granular embryonic calli were obtained from the two genotypes of Southeast-1-2 and SF15SH-5a, while the calli produced by HT-4 and HT-10 were relatively dense.

[0163] 3. Embryonic callus develops into somatic embryo morphology

[0164] This stage includes embryonic callus maturation culture, somatic embryo induction culture and somatic embryo development culture, and is the same as Example 3C except that the source of the material is embryonic callus obtained from petioles.

[0165] After 30 days of induction culture, the high-embryonic genotype SF15SH-5a could induce globular embryos and subsequently develop normally, but no somatic embryos were obtained from the three low-embryonic genotypes.

[0166] The invention discloses a novel plant small peptide sulfonate, which has a significant promoting effect on callus induction and somatic embryo morphogenesis, wherein the promoting effect of sulfonate on callus induction is attributed to a mitotic factor, which promotes cell division and callus formation; the promoting effect of sulfonate on somatic embryo morphogenesis is attributed to regulating the redox homeostasis in embryonic callus, thereby providing a suitable environment for somatic embryo morphogenesis and promoting the efficiency of cotyledon embryo formation.

[0167] The present invention successfully induced cotyledon embryos originating from leaves of seedlings using sulfopeptide, but there were differences in the effects of sulfopeptide on inducing somatic embryos from leaves and petioles, which requires further research.

[0168] The above embodiments of the present invention are only exemplary and do not constitute any limitation to the scope of the present invention. It should be understood by those skilled in the art that the details and forms of the technical solution of the present invention may be modified or replaced without departing from the spirit and scope of the present invention, but these modifications and replacements all fall within the scope of protection of the present invention.

Claims

1. A method for generating hybrid liquidambar formosana-dependent vegetative organ-derived somatic embryos, characterized in that: The mature vegetative organs of hybrid Liquidambar formosana are used as explants and induced cultured on a callus induction medium. The obtained callus is then induced cultured in a somatic embryo induction medium to obtain somatic embryos, wherein the somatic embryo induction medium contains sulfopeptide.

2. The method of generating as claimed in claim 1, characterized in that: The mature vegetative organs of hybrid Liquidambar formosana are leaves and petioles, preferably leaves.

3. A method for obtaining hybrid Liquidambar formosana regenerated plants, characterized in that: The steps include the following steps in order: 1) Callus induction The sterile leaves of hybrid Liquidambar formosana were placed on callus induction medium to induce callus and obtain embryonic callus. 2) Embryonic callus maturation The embryonic callus is placed in a liquid maturation medium and the embryonic callus is cultured for maturation under dark conditions; 3) Somatic embryo induction placing the mature cultured embryonic callus on a somatic embryo induction medium for somatic embryo induction culture; 4) Somatic embryo development After 25-35 days of somatic embryo induction culture, the tissue is transferred to a somatic embryo development medium, and somatic embryo development culture is carried out under dark conditions to obtain cotyledon embryos; 5) Somatic embryo germination The cotyledon embryos obtained by somatic embryo development culture are placed on a somatic embryo germination medium for somatic embryo germination culture to obtain hybrid Liquidambar formosana regenerated plants.

4. The method according to claim 3, characterized in that: The callus induction medium in step 1) is: modified Blaydes basic medium + hydrolyzed casein 1g / L + sucrose 40g / L + 2,4-D 0.5-1.5mg / L + 6-BA 0.2-0.8mg / L + PSK-α 0-0.2mg / L + plant gel 1g / L, with a pH of 5.6-5.

7.

5. The method according to claim 3 or 4, characterized in that: The method further comprises step 1A): callus proliferation culture: transferring the embryonic callus obtained by the induction culture in step 1) to a callus proliferation culture medium, and performing callus proliferation culture in the dark.

6. The method according to claim 3 or 4, characterized in that: The maturation medium in step 2) is: modified Blaydes basic medium + sucrose 40 g / L + PSK-α 0-0.8 mg / L (preferably 0.2-0.8 mg / L), pH 5.6-5.

7.

7. The method according to claim 3 or 4, characterized in that: The somatic embryo induction medium in step 3) is: modified Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α 0-0.8mg / L (preferably 0.2-0.8mg / L) + plant gel 6g / L, with a pH of 5.6-5.

7.

8. The method according to claim 3 or 4, characterized in that: The somatic embryo development medium in step 4) is: modified Blaydes basic medium + activated carbon 1g / L + sucrose 40g / L + PSK-α 0-0.8mg / L (preferably 0.2-0.8mg / L) + plant gel 3g / L, with a pH of 5.6-5.

7.

9. The method according to claim 3 or 4, characterized in that: The somatic embryo germination medium in step 5) is: WPM basic medium + sucrose 30g / L + plant gel 3g / L, pH 5.8-5.9.

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