阅读说明：本技术 一种促进枫香属植物体胚成熟的液体悬浮培养方法 (Liquid suspension culture method for promoting maturation of somatic embryos of liquidambar plants ) 是由 孔立生 张金凤 赵健 齐帅征 于 2019-08-22 设计创作，主要内容包括：本发明公开了一种促进枫香属植物体细胞胚胎(体胚)成熟的液体悬浮培养方法,属于林木组培技术领域,包括对枫香属植物的胚性愈伤组织进行体胚发生培养后接种于体胚液体成熟培养基中进行体胚的液体悬浮成熟培养。采用本发明的枫香属树种的体胚成熟悬浮培养方法促使枫香属植物体胚在液体培养基中正常成熟,在短时间内生产大量的枫香成熟体胚,成熟体胚经萌发,成苗效率高。本发明方法简便,再生周期短,繁殖系数高,移栽成活率高,出苗整齐一致,便于养护管理,节约人力及土地资源,生产成本低,不受季节限制持续获得再生植株,特别适用于枫香苗木产业化生产。(The invention discloses a liquid suspension culture method for promoting maturation of somatic embryos (somatic embryos) of liquidambar plants, belongs to the technical field of forest tissue culture, and comprises the steps of performing somatic embryogenic culture on embryogenic calluses of the liquidambar plants, and then inoculating the embryogenic calluses into a somatic embryo liquid maturation culture medium for performing liquid suspension maturation culture on somatic embryos. By adopting the liquidambar seed somatic embryo maturation suspension culture method, the somatic embryos of liquidambar plants are promoted to normally mature in a liquid culture medium, a large number of liquidambar mature somatic embryos are produced in a short time, and the mature somatic embryos are germinated to have high seedling formation efficiency. The method is simple and convenient, short in regeneration period, high in propagation coefficient, high in transplanting survival rate, uniform in seedling emergence, convenient to maintain and manage, low in production cost, free of seasonal restrictions, and capable of continuously obtaining regenerated plants, and is particularly suitable for industrialized production of liquidambar formosana seedlings.)

1. A liquid suspension culture method for promoting maturation of somatic embryos of liquidambar plants is characterized by comprising the steps of performing somatic embryogenesis culture on embryonic calluses of the liquidambar plants, and then inoculating the embryonic calluses into a somatic embryo liquid maturation culture medium for liquid suspension maturation culture of somatic embryos.

2. The propagation method as claimed in claim 1, wherein the liquid maturation medium of somatic embryos is modified Blaydes minimal medium + sucrose 40 g/L.

3. The propagation method as claimed in claim 1, wherein the solid medium used for the somatic embryogenesis culture is modified Blaydes minimal medium + sucrose 40g/L + phytogel 3-6 g/L.

4. A liquid suspension culture method for promoting maturation of somatic embryos of liquidambar plants is characterized by comprising the following steps:

1) inoculating the liquidambar plant explant to an induction culture medium, and performing embryonic tissue induction culture to obtain an embryonic callus;

2) inoculating the embryogenic callus to a somatic embryogenesis culture medium, and performing somatic embryogenesis culture to obtain immature somatic embryos;

3) inoculating the callus with immature somatic embryos into a somatic embryo liquid maturation culture medium, and performing somatic embryo liquid maturation culture to obtain mature somatic embryos.

5. The method as claimed in claim 4, further comprising step 3A) of passing the mixture after the liquid maturation culture of the embryos through a sieve having a pore size of more than 1mm, wherein embryos which do not pass through the sieve are said mature embryos.

6. The method as claimed in claim 4 or 5, wherein the liquid medium for somatic embryo maturation in step 3) is modified Blaydes medium + sucrose 40 g/L.

7. The method according to claim 4 or 5, wherein the somatic embryogenesis medium in step 2) is modified Blaydes minimal medium + sucrose 40g/L + phytogel 3-6 g/L.

8. The method according to claim 4 or 5, further comprising the step 1A) of inoculating the embryogenic callus obtained by the culture in the step 1) to a multiplication medium, performing the multiplication culture of the embryogenic callus, obtaining the multiplied embryogenic callus, and then inoculating the multiplied embryogenic callus to a somatic embryogenesis medium, and performing the somatic embryogenesis culture.

9. The method according to claim 8, wherein the embryogenic callus proliferation culture in step 1A) is performed by inoculating embryogenic callus to an embryogenic callus solid proliferation medium; or inoculating the embryogenic callus into the embryogenic callus liquid multiplication culture medium for embryogenic callus liquid multiplication culture.

10. The method for breeding hybrid sweetgum is characterized by comprising the steps of performing somatic embryogenesis culture on embryogenic callus of the hybrid sweetgum and then performing liquid suspension maturation culture.

Technical Field

The invention relates to a plant tissue culture method, in particular to a liquidambar formosana somatic embryo mature liquid suspension culture method, and belongs to the technical field of cell engineering breeding in forestry industry.

Background

The Liquidambar spp tree species is an important forestry resource worldwide, particularly Liquidambar formosana and Liquidambar styraciflua (L.styraciflua), which have high economic value, ornamental value and ecological value, and hybrid Liquidambar styraciflua obtained by the hybridization of the Liquidambar styraciflua and the Liquidambar styraciflua has hybrid vigor.

The liquidambar formosana can be propagated sexually or asexually, and the asexual propagation efficiency of the liquidambar formosana is greatly improved along with the continuous progress of a cuttage technology and a tissue culture rapid propagation technology in recent years.

Tissue culture is an efficient rapid propagation technique for forest trees, and is widely applied to various tree species, such as poplar, eucalyptus, fir and the like. Besides the purpose of in vitro rapid propagation, the organ in vitro regeneration technology also provides technical support for developing forest genetic transformation and in vitro chromosome doubling research. At present, tissue culture and rapid propagation researches carried out by liquidambar styraciflua in liquidambar are deeper, and tissue culture researchers of liquidambar styraciflua tree species successively establish tissue culture regeneration or somatic embryogenesis systems by utilizing liquidambar styraciflua and liquidambar styraciflua embryonic axis, cotyledon, inflorescence axis, leaf blade, petiole and other organs. For example, Sommer and Brown used North America sweetgum embryonic axis cutting for somatic embryo induction, and then the America university Merkle team successfully established North America sweetgum and hybrid sweetgum somatic embryogenesis systems with North America sweetgum and hybrid sweetgum immature embryos and inflorescence axes, respectively, completed suspension culture of embryogenic callus, allowed embryogenic tissue proliferation, and performed somatic embryo synchronization studies (Merkle, Neu et al 1998; Venndrame, Holliday et al 2001; Merkle, Battle et al 2003). Also, as reported by Brand and Lineberger (1988), leaf blades and petioles of liquidambar styraciflua are differentiated to obtain regenerated plants. Kim, Sommer et al (1997) established a regeneration system of Liquidambar styraciflua adventitious buds from in vitro hypocotyls as research materials. The Shijisen team of Nanjing forestry university makes a relatively comprehensive study on tissue culture in-vitro rapid propagation of sweetgum, and the team (2003) develops a set of sweetgum tissue culture technology by respectively using stem tips, leaves, leaf stalks, hypocotyls, male inflorescence axes and immature embryos as explants. The xulin (2008) takes the isolated leaves as the material, and establishes a liquidambar formosana adventitious bud regeneration system. Gongzheng, Wanghong and so on (2012) induced adventitious buds by using tender buds and explored the conditions for rooting such as IBA and NAA.

The research on the regeneration and propagation method of the isolated organ of liquidambar formosana is numerous. In various asexual in vitro propagation methods, the somatic embryo technology has the characteristics of thorough rejuvenation, high propagation efficiency and the like, but because the embryogenic tissue of the liquidambar plant is directly transferred into a liquid mature culture medium, plant cells are difficult to directly transfer from a multiplication state to a somatic embryo development mature state, so that the somatic embryo of the liquidambar plant cannot mature in the liquid culture medium. Therefore, the existing technology for promoting the maturation of somatic embryos of liquidambar plants must use a solid maturation medium all the time. However, when somatic embryos of liquidambar are matured on a solid medium, only embryos on the surface of embryogenic tissue can be matured. Due to the different degrees of exposure to the medium, the synchronization of the embryo development maturation is poor. Mature somatic embryos on solid medium are typically basal-associated with embryogenic tissue. Because the plant tissues are dry, a plurality of somatic embryos are difficult to separate, the method is not beneficial to industrial production, and the method is also not beneficial to synchronous screening and subsequent germination.

Disclosure of Invention

The invention aims to solve the technical defects that the somatic embryos of liquidambar cannot normally mature in a liquid culture medium and the liquid culture cannot be reflected in the mature stage of the somatic embryos in the conventional tissue culture and rapid propagation method of liquidambar seeds, and the liquidambar seedling production efficiency is low and the production cost is high in the prior art, and provides a liquid suspension culture method for promoting the maturation of the somatic embryos (somatic embryos) of the liquidambar; performing synchronous management on somatic embryo development; improve the germination consistency of the follow-up somatic embryos.

In order to achieve the purpose, the invention provides a liquid suspension culture method for promoting maturation of somatic embryos of liquidambar, which comprises the steps of performing somatic embryogenic culture on embryogenic callus of the liquidambar, and then inoculating the embryogenic callus into a somatic embryo liquid maturation culture medium to perform liquid suspension maturation culture of the somatic embryos.

Wherein the liquidambar genus plant is liquidambar formosana, liquidambar styraciflua or hybrid liquidambar formosana.

In particular, the hybrid liquidambar formosana is (liquidambar formosana x liquidambar formosana).

Wherein the somatic embryo liquid maturation culture medium is an improved Blaydes minimal medium plus sucrose of 40 g/L.

In particular, the culture conditions for liquid suspension culture of the somatic embryo are as follows: culturing on a shaking table at 25 + -2 deg.C in dark.

Wherein the culture medium used for the somatic embryogenesis culture is an improved Blaydes minimal medium, 40g/L sucrose and 3-6g/L plant gel.

In particular, the culture conditions for the somatic embryogenesis culture are: culturing at 25 + -2 deg.C in dark.

The invention also provides a liquid suspension culture method for promoting maturation of somatic embryos of liquidambar, which comprises the following steps of:

1) inoculating the liquidambar plant explant to an induction culture medium, and performing embryonic tissue induction culture to obtain an embryonic callus;

2) inoculating the embryogenic callus to a somatic embryogenesis culture medium, and performing somatic embryogenesis culture to obtain immature somatic embryos;

3) inoculating the callus with immature somatic embryos into a somatic embryo liquid maturation culture medium, and performing somatic embryo liquid maturation culture to obtain mature somatic embryos.

Wherein, the explant in the step 1) is one or more of immature seed embryo, mature seed embryo and rachis, preferably immature seed embryo.

The explants of the Liquidambar genus Plant of the present invention are sterilized and disinfected according to the method described in the literature (W.A. Vendra.C.P.Holliday.S.A.Merkle et al, clone propagation of sweet potato (Liquidambar styracifolia. times. L.formalana) by systematic embryo production, Plant Cell Rep (2001)20: 691-.

In particular, the induction medium in step 1) is: modified Blaydes minimal medium, hydrolyzed casein 1g/L, sucrose 40g/L, plant gel 2.5-3g/L +2, 4-D1-4 mg/L +6-BA0-2 mg/L.

In particular, the culture conditions of the embryogenic tissue induction culture are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; the culture time is 3-4 weeks.

In particular, it also includes the subculture of embryogenic callus every 3-4 weeks.

In particular, the embryonic tissue subculture medium is: improved Blaydes minimal medium, 1g/L hydrolyzed casein, 40g/L sucrose, 2.5-3g/L plant gel, 2, 4-D0.5-2 mg/L and 6-BA0-1 mg/L.

In particular, the culture conditions for the subculture were: under dark conditions, the culture temperature (25 +/-2) DEG C; the culture time is 3-4 weeks.

Particularly, the method also comprises a step 1A) of inoculating the embryogenic callus obtained by the culture in the step 1) to a multiplication culture medium, carrying out multiplication culture on the embryogenic callus, obtaining the embryogenic callus after the multiplication culture, and then carrying out somatic embryogenesis culture.

Wherein, the embryogenic callus proliferation culture in the step 1A) is to inoculate the embryogenic callus to an embryogenic callus solid proliferation culture medium for the embryogenic callus solid proliferation culture; or inoculating the embryogenic callus to the embryogenic callus liquid multiplication culture medium for embryogenic callus liquid multiplication culture.

In particular, the embryogenic callus solid multiplication culture medium is as follows: modified Blaydes minimal medium + 1g/L hydrolyzed casein + 40g/L sucrose + 2.5-3g/L plant gel +2, 4-D0.5-2 mg/L +6-BA0-1 mg/L, preferably modified Blaydes minimal medium + 1g/L hydrolyzed casein + 40g/L sucrose + 2.5-3g/L plant gel +2, 4-D0.5-2 mg/L +6-BA0.25-1 mg/L. Adjusting pH to 5.8, and sterilizing at 121 deg.C for 15 min.

In particular, the culture conditions of the solid propagation culture of the embryogenic callus are as follows: under dark conditions, the culture temperature (25 +/-2) DEG C; the culture time is 3-4 weeks.

In particular, the liquid multiplication culture medium for the embryogenic callus comprises: modified Blaydes minimal medium + hydrolyzed casein 1g/L + sucrose 40g/L +2, 4-D0.5-2 mg/L +6-BA0-1.0mg/L, preferably modified Blaydes minimal medium + hydrolyzed casein 1g/L + sucrose 40g/L +2, 4-D0.5-2 mg/L +6-BA0.25-1 mg/L. Adjusting pH to 5.8, and sterilizing at 121 deg.C for 15 min.

In particular, the culture conditions of the liquid multiplication culture of the embryogenic callus are as follows: under dark conditions, the culture temperature (25 +/-2) DEG C; shaking table 100-; the culture time is 3-4 weeks.

Wherein the somatic embryogenesis medium in the step 2) is an improved Blaydes minimal medium, sucrose 40g/L and plant gel 3-6 g/L.

In particular, the culture conditions for the somatic embryogenesis culture are: under dark conditions, the culture temperature (25 +/-2) DEG C; the culture time is 1-4 months, preferably 1-2 months.

In particular, the immature somatic embryo is a preliminarily developed somatic embryo,

in particular, the immature somatic embryos include somatic embryos at any developmental stage from globular embryos to immature cotyledonary embryos.

In particular, the immature somatic embryo is a globular embryo or/and a heart-shaped embryo.

In particular, the immature somatic embryo is obtained by inoculating the proliferating embryogenic callus onto a somatic embryogenesis medium and culturing at (25 +/-2) DEG C in the dark for 1-4 months.

Wherein, the liquid mature culture medium of the somatic embryos in the step 3) is modified Blaydes culture medium plus sucrose 40 g/L.

In particular, the culture conditions of the liquid mature culture of the somatic embryo are as follows: under dark conditions, the culture temperature is 25 +/-2 ℃; shaking table 100-; the culture time is 4-8 weeks.

In particular, the process of somatic embryo maturation culture also comprises the step of subculturing the callus with the initial somatic embryos, wherein the subculturing is carried out every 3-4 weeks.

Particularly, the method also comprises a step 3A) of passing the mixture subjected to liquid maturation culture of the somatic embryos through a sieve with the aperture larger than 1mm, and obtaining the somatic embryos which do not pass through the sieve holes, namely mature somatic embryos.

In particular, the sieve has a pore size of 1 to 5mm, preferably 2 to 4 mm.

The invention provides a propagation method of hybrid liquidambar formosana, which comprises the following steps of performing somatic embryogenesis culture on embryogenic callus of the hybrid liquidambar formosana and then performing liquid suspension maturation culture.

The invention also provides a propagation method of hybrid liquidambar formosana, which further comprises the following steps:

A) inoculating the sterilized explant of the hybrid sweetgum to an induction culture medium for embryonic tissue induction culture;

B) inoculating the embryogenic callus obtained by induction culture to a proliferation culture medium for embryogenic callus proliferation culture;

C) inoculating the embryogenic callus after propagation culture to a somatic embryogenesis culture medium, and performing somatic embryogenesis culture to obtain an immature somatic embryo;

D) inoculating the callus with immature somatic embryos into a somatic embryo liquid maturation culture medium, and performing somatic embryo liquid maturation culture to obtain mature somatic embryos;

E) inoculating the mature somatic embryos to a germination culture medium, and performing somatic embryo germination culture to obtain somatic embryo seedlings;

F) hardening seedlings, culturing in a greenhouse and transplanting the somatic embryo seedlings.

When the somatic embryos of the liquidambar plants are matured on the solid culture medium, only embryos on the surfaces of embryonic tissues can be matured, the somatic embryo development synchronism is poor, and the bases of the matured embryos are connected with the embryonic tissues and are not easy to separate; in addition, because the plant tissues are dry, a plurality of somatic embryos are mixed together and are difficult to separate, so that the method is not beneficial to industrial production and synchronous screening and subsequent germination; moreover, the somatic embryos of the liquidambar cannot normally mature in a liquid culture medium, and mature somatic embryos cannot be obtained.

Compared with the prior tissue culture propagation method of liquidambar, the invention has the following advantages:

1. the method overcomes the technical defect that the callus of the liquidambar plant can not normally develop into mature somatic embryos in a liquid culture medium, the method carries out mature culture on the somatic embryos in two stages in the process of culturing the somatic embryos, promotes the somatic embryos of the liquidambar plant to normally mature in the liquid culture medium and convert into somatic embryo seedlings in the subsequent culture stage, and the production efficiency of the mature somatic embryo seedlings is high.

The process of the somatic embryo culture technology of the invention is divided into two stages: the primary role of the first stage is to initiate the process of somatic embryo maturation on solid maturation medium (i.e., somatic embryogenesis medium) where embryogenic tissue is transferred from callus proliferation to somatic embryo development. The primary function of the second stage is to promote further growth of the somatic embryos in liquid maturation medium until complete maturation.

The first stage is as follows: the embryogenic callus initiates the maturation process of the somatic embryo on the solid somatic embryogenesis medium, and if the embryogenic tissue is directly cultured in a liquid maturation medium, it is difficult to promote the maturation of the somatic embryo in the liquid. The method can ensure the process of switching the embryogenic tissue from the multiplication state to the somatic embryo development; secondly, before the somatic embryo maturation culture is started, the embryonic callus with the initial small somatic embryos (spherical embryos and heart-shaped embryos) is selected to enter liquid maturation culture by differentiating the developmental morphology of the somatic embryos, so that the quality of plant tissues and the high efficiency of the production of the somatic embryos are ensured.

And a second stage: promoting the further growth of somatic embryo until the somatic embryo is completely mature. Mature embryos are called cotyledonary embryos, have a volume which is several times to tens of times that of spherical embryos and heart-shaped embryos, and have two well-developed cotyledons. In the method, the micro early-stage somatic embryos cultured in the first-stage somatic embryogenesis can contact with a culture medium in all directions in liquid maturation culture so as to grow rapidly; the liquid culture cost is low, the efficiency is high, and the method is particularly suitable for large-scale production; and the embryos matured in the liquid culture medium are independent and dispersed, which is beneficial to screening and later-stage treatment.

2. The method can efficiently produce mature somatic embryos by liquid culture, namely more mature somatic embryos are produced in a shorter time, and the mature somatic embryos are germinated and cultured to obtain more somatic embryo seedlings, so that more liquidambar formosana seedlings for afforestation are provided;

3. the method of the invention obviously reduces the production cost of the liquidambar plant propagation;

4. by adopting the liquid maturation culture, the somatic embryo development of the liquidambar plants can be synchronized, so that the somatic embryo development can be more synchronized, and the subsequent culture and management are facilitated.

5. In the method, the liquid culture medium is adopted for mature culture of the somatic embryos, the mature somatic embryos are independent and dispersed in the liquid culture medium, and the mature somatic embryos are screened by a sieve to obtain a large number of somatic embryos which are independent from each other, mature and consistent or similar in development degree, so that the defect that the somatic embryos formed by mature culture by adopting the solid culture medium are not easy to separate is overcome.

6. In the method, qualified mature embryos with the same or basically the same development degree are obtained by screening by using a sieve, and other tissues are removed. Whereas techniques for maturing embryos in liquid make screening possible. The mature somatic embryos in the liquid culture medium are screened by a sieve, the operation is convenient and easy, and the efficiency is high. In the method, the mature embryos cultured in liquid and the liquid culture medium are sieved together under the aseptic condition, the embryos which are left in the sieve and can not pass through the sieve holes are taken as the mature embryos, and the mature culture of the embryos is continued until the mature embryos pass through the sieve holes.

Drawings

FIG. 1 shows the embryogenic callus of hybrid sweetgum;

FIG. 2 shows the liquidambar formosana embryonic tissue (yellow) and the somatic embryo at the early development stage (white) in somatic embryogenesis culture;

FIG. 3 is an enlarged view of a portion of the immature embryos of FIG. 2;

FIG. 4 is a mature somatic embryo obtained by liquid maturation culture of somatic embryos;

FIG. 5 is an enlarged view of a mature somatic embryo obtained by liquid maturation culture of the somatic embryo: left) mature cotyledon embryos; right) germinating somatic embryos;

FIG. 6A shows a single mature somatic embryo inoculated into a germination medium to germinate and transform into a somatic embryo seedling with actively growing ends;

FIG. 6B is a graph of the in-flask growth of germination transformed somatic embryos in germination medium;

FIG. 7 shows hybrid liquidambar formosana somatic embryo seedlings in greenhouse after hardening treatment.

Detailed Description

The invention will be further described with reference to specific embodiments, and the advantages and features of the invention will become apparent as the description proceeds. These examples are illustrative only and do not limit the scope of the present invention in any way. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention, and that such changes and modifications may be made without departing from the spirit and scope of the invention.