阅读说明：本技术 一种提高子叶胚得出率的油菜小孢子培养基和培养方法 (Rape microspore culture medium and culture method for improving yield of cotyledon embryos ) 是由 万丽丽 王转茸 杨光圣 辛强 洪登峰 孙玉宏 高红霞 于 2019-10-12 设计创作，主要内容包括：本发明涉及植物组织培养技术领域,公开了一种提高子叶胚得出率的油菜小孢子培养基和培养方法,所述培养基为固液双层培养基,所述培养基上层为NLN13液体培养基,所述培养基下层为B5培养基改良的固体培养基。所述培养方法采用两步法培养油菜小孢子,第一步采用NLNM液体培养基培养,第二步采用固液双层培养基培养。本发明显著提高了子叶胚的得出率,而且保证了子叶胚的质量,进而提高小孢子培养获得双单倍体植株的效率。(The invention relates to the technical field of plant tissue culture, and discloses a rape microspore culture medium for improving the yield of cotyledon embryos and a culture method thereof, wherein the culture medium is a solid-liquid double-layer culture medium, the upper layer of the culture medium is an NLN13 liquid culture medium, and the lower layer of the culture medium is a solid culture medium improved by a B5 culture medium. The culture method adopts a two-step method to culture the microspores of the rape, the first step adopts NLNM liquid culture medium to culture, and the second step adopts solid-liquid double-layer culture medium to culture. The method obviously improves the yield of the cotyledon embryo, ensures the quality of the cotyledon embryo and further improves the efficiency of culturing the microspore to obtain the double haploid plant.)

1. A solid-liquid double-layer culture medium for culturing the microspores of the rape is characterized in that the upper layer of the culture medium is NLN13 liquid culture medium, and the lower layer of the culture medium is B5 culture medium improved solid culture medium;

the NLN13 liquid culture medium is as follows: each 1000mL of the solution contains 2.5g of KNO₃，2.5g MgSO₄·7H₂O，2.5g KH₂PO₄，10g Ca(NO₃)₂·4H₂O，0.556g FeSO₄·7H₂O，0.746g Na₂·EDTA，0.166g KI，1.24g H₃BO₃，3.474g MnSO₄.H₂O，2.118g ZnSO₄.7H₂O，0.05g Na₂MoO₄.2H₂O，0.005g CuSO₄.5H₂O，0.005gCoCl₂.6H₂O, 1g nicotinic acid, 0.4g glycine, 0.1g thiamine hydrochloride, 0.1g pyridoxine hydrochloride, 0.1g folic acid, 0.01g biotin, 130g sucrose, 0.1g inositol, 0.8g glutamine, 0.1g serine, 0.03g glutathione;

the solid culture medium is as follows: every 1000mL of the culture medium comprises 0.32g B5 basic culture medium powder, 0.1-0.75 g of activated carbon, 0.1-0.75 mg of 6-BA, 3g of sucrose and 0.55g of Phytagel.

2. The solid-liquid double-layer culture medium for culturing the rape microspores as claimed in claim 1, wherein the content of the activated carbon in the solid culture medium is 0.1 g/L.

3. The solid-liquid double-layer culture medium for culturing the cole microspores as claimed in claim 1, wherein the content of 6-BA in the solid culture medium is 0.25 mg/L.

4. A method for culturing microspores of rape for improving the yield of cotyledon embryos, which is characterized by adopting the solid-liquid double-layer culture medium of claim 1 and comprises the following steps:

s1, collecting and separating microspores: picking main inflorescence and branched flower buds, pretreating at low temperature, sterilizing, cleaning, adding microspore extraction buffer solution to extract microspores, filtering, centrifuging, collecting microspore cells, and suspending with NLNM liquid culture medium;

s2, microspore heat shock pretreatment: adjusting the number of microspore cells in the NLNM liquid culture medium in the step S1, putting the NLNM liquid culture medium into a culture dish, sealing the culture dish, and putting the NLNM liquid culture medium into an incubator at 31 +/-1 ℃ for dark culture for 48 hours;

s3, microspore cell culture: centrifuging and resuspending the microspore cells treated in the step S2, placing the microspore cells into a culture dish, sealing the culture dish, performing dark culture for a period of time, transferring the microspore suspension into the solid-liquid double-layer culture medium of claim 1, performing dark culture, supplementing NLN13 liquid culture medium every 1-2 weeks, and placing the culture dish containing the solid-liquid double-layer culture medium on a shaking bed, performing dark culture at 19-25 ℃ for 3-4 weeks when the size of embryos is visible with naked eyes.

5. The method for culturing the cole microspores capable of improving the yield of cotyledon embryos of claim 4, wherein the NLNM liquid culture medium comprises: each 1000mL of the solution contains 2.5g of KNO₃，2.5g MgSO₄·7H₂O，2.5g KH₂PO₄，10g Ca(NO₃)₂·4H₂O，0.556g FeSO₄·7H₂O，0.746g Na₂·EDTA，0.166g KI，1.24g H₃BO₃，3.474gMnSO₄.H₂O，2.118g ZnSO₄.7H₂O，0.05g Na₂MoO₄.2H₂O，0.005g CuSO₄.5H₂O，0.005gCoCl₂.6H₂O, 1g nicotinic acid, 0.4g glycine, 0.1g thiamine hydrochloride, 0.1g pyridoxine hydrochloride, 0.1g folic acid0.01g of biotin, 65g of sucrose, 0.1g of inositol, 0.8g of glutamine, 0.1g of serine, 0.03g of glutathione and 4g of mannitol.

6. The method for culturing Brassica campestris microspores with increased yield of cotyledon embryos of claim 4, wherein the culture temperature of the culture dish containing the solid-liquid double-layer medium on a shaker is 23 ℃.

Technical Field

The invention relates to the technical field of plant tissue culture, in particular to a rape microspore culture medium and a culture method for improving the yield of cotyledon embryos.

Background

Lichter from Germany in 1982 first reported that isolated microspores were successfully isolated from Brassica napus, and after induction of haploids by microspore culture followed by doubling to Doubled Haploids (DHs). The doubled haploids created by microspore culture are currently applied to the genetic map construction of important agronomic characters of rape, the positioning of important character QTLs and the doubled haploid breeding practice.

Although microspore culture technology systems with strong applicability have been established in brassica napus, there is a great difference in the ability to produce high quality cotyledon embryos during microspore culture for materials with different genotypes. Previous studies have shown that embryos obtained after microspore culture are mostly globular, heart-shaped or embryo-like structures (ELS), with only a few cotyledonary embryos. Cotyledon embryos can grow rapidly on a seedling culture medium to obtain plants with strong growth vigor and developed root systems, while embryos of other types and structures similar to the embryos are transferred to a regeneration culture medium and are difficult to grow into seedlings once, even are browned and die, so that the efficiency of obtaining double haploid plants by microspore culture is reduced to a great extent.

Disclosure of Invention

In view of the above, the invention obviously improves the yield of the cotyledon embryo in the culture of the rape microspore and ensures the quality of the cotyledon embryo by improving the culture medium and the culture method.

In order to achieve the above objects, one aspect of the present invention provides

A solid-liquid double-layer culture medium for culturing the microspores of rape, wherein the upper layer of the culture medium is NLN13 liquid culture medium, and the lower layer of the culture medium is B5 culture medium improved solid culture medium;

the NLN13 liquid culture medium is as follows: each 1000mL of the solution contains 2.5g of KNO₃，2.5g MgSO₄·7H₂O，2.5gKH₂PO₄，10g Ca(NO₃)₂·4H₂O，0.556g FeSO₄·7H₂O，0.746g Na₂·EDTA，0.166g KI，1.24gH₃BO₃，3.474g MnSO₄.H₂O，2.118g ZnSO₄.7H₂O，0.05g Na₂MoO₄.2H₂O，0.005g CuSO₄.5H₂O，0.005g CoCl₂.6H₂O, 1g nicotinic acid, 0.4g glycine, 0.1g thiamine hydrochloride, 0.1g pyridoxine hydrochloride, 0.1g folic acid, 0.01g biotin, 130g sucrose, 0.1g inositol, 0.8g glutamine, 0.1g serine, 0.03g glutathione;

the solid culture medium is as follows: every 1000mL of the culture medium contains B5 minimal medium, 0.1-0.75 g of activated carbon, 0.1-0.75 mg of 6-BA, 3g of sucrose and 0.55g of Phytagel;

preferably, the content of the activated carbon in the solid culture medium is 0.1 g/L.

Preferably, the content of 6-BA in the solid culture medium is 0.25 mg/L.

The invention provides a method for culturing microspores of rape for improving the yield of cotyledon embryos, which adopts the solid-liquid double-layer culture medium and specifically comprises the following steps:

s1, collecting and separating microspores: picking main inflorescence and branched flower buds, pretreating at low temperature, sterilizing, cleaning, adding microspore extraction buffer solution to extract microspores, filtering, centrifuging, collecting microspore cells, and suspending with NLNM liquid culture medium;

s2, microspore heat shock pretreatment: adjusting the number of microspore cells in the NLNM liquid culture medium in the step S1, putting the NLNM liquid culture medium into a culture dish, sealing the culture dish, and putting the NLNM liquid culture medium into an incubator at 31 +/-1 ℃ for dark culture for 48 hours;

s3, microspore cell culture: centrifuging and resuspending the microspore cells treated in the step S2, placing the microspore cells into a culture dish, sealing the culture dish, performing dark culture for a period of time, transferring the microspore suspension into the solid-liquid double-layer culture medium of claim 1, performing dark culture, supplementing NLN13 liquid culture medium every 1-2 weeks, and placing the culture dish containing the solid-liquid double-layer culture medium on a shaking bed for dark culture at 19-25 ℃ for 3-4 weeks when the size of embryos is visible with naked eyes.

Preferably, the NLNM liquid medium is: each 1000mL of the solution contains 2.5g of KNO₃，2.5g MgSO₄·7H₂O，2.5g KH₂PO₄，10g Ca(NO₃)₂·4H₂O，0.556g FeSO₄·7H₂O，0.746g Na₂·EDTA，0.166g KI，1.24g H₃BO₃，3.474g MnSO₄.H₂O，2.118g ZnSO₄.7H₂O，0.05g Na₂MoO₄.2H₂O，0.005gCuSO₄.5H₂O，0.005g CoCl₂.6H₂O, 1g of nicotinic acid, 0.4g of glycine, 0.1g of thiamine hydrochloride, 0.1g of pyridoxine hydrochloride, 0.1g of folic acid, 0.01g of biotin, 65g of sucrose, 0.1g of inositol, 0.8g of glutamine, 0.1g of serine, 0.03g of glutathione, 4g of mannitol.

Preferably, the culture temperature of the culture dish containing the solid-liquid double-layer culture medium on the shaking table is 23 ℃.

Compared with the prior art, the invention has the beneficial effects that: the method adopts a two-step method to culture the microspores of the rape, adopts an NLNM liquid culture medium to culture in the first step, adopts a solid-liquid double-layer culture medium to culture in the second step, obviously improves the yield of cotyledon embryos and ensures the high quality of the cotyledon embryos by designing the components of a lower solid culture medium and an upper liquid culture medium in the solid-liquid double-layer culture medium.

Drawings

FIG. 1 is a diagram showing the embryogenesis of microspores of vegetables on a liquid medium and a solid-liquid double-layer medium;

FIG. 2 is a statistical chart of the number of embryos produced on the liquid and solid-liquid double-layer culture medium of microspore cells of three lines;

FIG. 3 is a statistical chart of different oscillation times, types and numbers of embryos.

Detailed Description

In order that the invention may be better understood, it is further illustrated by the following specific examples, which are not to be construed as limiting the invention.

It should be noted that, as is known from the existing research, the content of each component in the culture medium has a great influence on the experimental result, so all the weight weighing in the examples is carried out by using a one-ten-thousandth balance.