CN111492979B

CN111492979B - Primula forbesii somatic embryo induction method

Info

Publication number: CN111492979B
Application number: CN202010451248.5A
Authority: CN
Inventors: 贾茵; 黄远祥; 李西; 周鹏; 江明艳; 姜贝贝
Original assignee: Sichuan Tianyi Ecological Garden Group Co ltd; Sichuan Agricultural University
Current assignee: Sichuan Tianyi Ecological Garden Group Co ltd; Sichuan Agricultural University
Priority date: 2020-05-25
Filing date: 2020-05-25
Publication date: 2022-03-22
Anticipated expiration: 2040-05-25
Also published as: CN111492979A

Abstract

The invention discloses a primula forbesii somatic embryo induction method, which comprises the following steps: 1) aseptic seeding to obtain primula forbesii aseptic seedling; 2) inoculating the sterile seedling leaf obtained in the step 1) to an embryonic callus induction culture medium to induce embryonic callus; 3) inducing somatic embryos from the embryogenic callus induced in the step 2); 4) the somatic embryo germinates and roots. The invention establishes the primula forbesii somatic embryo induction method for the first time, can quickly obtain primula forbesii somatic cell seedlings, and has high induction efficiency and short seedling period. In addition, compared with the traditional approaches of inducing adventitious buds by using leaf callus and directly inducing adventitious buds by using leaves, the method can obviously improve the genetic transformation efficiency mediated by agrobacterium tumefaciens and lays a foundation for primula forbesii transgenic breeding.

Description

Primula forbesii somatic embryo induction method

Technical Field

The invention belongs to the technical field of plant biology, and particularly relates to a primula forbesii somatic embryo induction method.

Background

Plant of Primulaceae family, genus 74 in total, wherein Primulina belongs to (PrimulaLinn.) is the largest genus in primulinaceae, approximately 500 species are in the world, most of the plants are located in northern hemisphere, the plants of the genus are mainly distributed in two areas, one is himalayas, and the other is Yunnan, Tibet and Sichuan areas of China. Primula is a famous ornamental flower in China, and the planting history at home and abroad has been for hundreds of years (Zhang Yan Li, Xiao Primula: (Primula forbesii) Preliminary study of biological Properties [ D]Beijing university of forestry, 2003.]Its advantages are short flowering time, long flowering period, rich colours, unique flower type and high ornamental value, and its combination with azalea and gentian is named as flower of three mountains in the world and flower of three natural famous flowers in China (Huqing, seed and plant family) [ M]Beijing scientific Press, 1999.]. China is vast and has a great variety of primrose, and there are 300 kinds of primula in China (China academy of sciences, China plant Commission, China plant (volume 59, volume 2) [ M ]]Beijing scientific Press, 1990]The resources are widely distributed, but the resources are located in remote mountain areas in the southwest area of China, the connection with the outside is not smooth, the resources are in a wild state, the acquisition and research are difficult, and many types of the resources are yet to be developed and utilized. In addition, natural factors such as earthquakes and debris flows and artificial factors such as tourism development, excessive grazing and excavation cause the wild primrose resources to be seriously damaged, particularly rare primrose varieties, the number of the wild primrose varieties is greatly reduced, the living environment is also damaged to a certain degree, and practical and effective protection measures are urgently needed. In the process of garden application of primula sikkmensis hook, the great potential of the plant resource is discovered abroad, a great deal of research is carried out on the plant resource, the process of horticultural application is promoted, and cultivated horticulture has a large variety and is widely planted at home and abroad. Compared with the foreign countries, the garden application research aspect of primula is far behind in China, and many primula resources are in the wild state for a long time and have not been shown in relevant research and report, so many resources cannot be obtainedCan be effectively utilized and can not be converted into commodities. Particularly, in the introduction aspect, the method is still in the initial exploration stage, and the garden application potential of a plurality of wild primula is huge, so that the development and utilization are urgently needed, and the advantages of abundant primula resources in China are not brought into play.

In order to further make new progress on the breeding work of primula, abundant germplasm resources in China are fully utilized, and the development and utilization strength of certain germplasm resources specific to China is increased. Primula forbesii (A. B)Primula forbesii) The primula forbesii is also called primula forbesii, belongs to primula genus of primula family, is a special species in China, is a biennial herb flower distributed in Yunnan and Sichuan areas, and has weak and thin rhizome and a plurality of deficient roots. The leaf base is in a lotus socket shape, usually in an oval shape, and is mostly fine and soft; 1 to a plurality of scapes, extracted from the leaf cluster, the inflorescence is pulverized, 1-2 or more than 2 circles of umbelliform inflorescences are provided, 5-15 flowers are arranged in each circle, 5 petals are arranged, the corolla is light red or light purple, the tip of the lobe is deeply sunken, and the diameter is about 1cm (old and beautiful, 1990). The primula plant is a few of primula plants with strong fragrance, has high ornamental value, strong vitality, easy survival of seeding and propagation, frequent and large-area growth in the field and excellent ornamental effect. And the two primula sikkmensis flowers are distributed in areas with lower altitude, so that introduction and domestication are relatively easy, and the primula sikkmensis flower and sweet flower ground cover plants have great potential in garden development. Therefore, the research on the regeneration system of primula forbesii is actively developed, the introduction and domestication progress is accelerated, the wild resources are fully utilized, the primula forbesii becomes a cultivated species with commercial value, and the method has very important significance for creating new species with independent intellectual property rights in China.

The primula plant is mainly propagated by seeds, is greatly influenced by environmental conditions, has low seed activity and difficult artificial cultivation, is fine in seeds and short in service life, requires newly harvested seeds to be sown in time, otherwise has extremely low emergence amount and low efficiency, and is not beneficial to large-scale application in gardens. Under the continuous development of biotechnology, aiming at the problem of low plant propagation efficiency, tissue culture is an important way for rapidly and efficiently solving the problem, is widely applied to breeding new varieties of garden plants, and has the characteristics of low cost, high production efficiency, high propagation speed, capability of producing virus-free seedlings and the like. Xiaoxianbao et al, tissue culture and plant regeneration of primula forbesii, plant physiology communication, Vol.41, No. 6, month 12 2005, discloses tissue culture and plant regeneration of primula forbesii, and specifically discloses inducing and proliferating clumpy buds by using young leaves of primula forbesii as explants. The primula forbesii tender leaves can firstly induce callus on different culture media, the callus can be differentiated into adventitious buds, and the adventitious buds can also be directly differentiated from the leaves without callus process.

At present, primula plants applied to gardens in China mainly comprise Tibetan primula: (A)P. sinensis) Primula sikkimensis (four seasons) ((P. obconica) Primula europaea (b), primula europaea (b)P. vulgaris) The flower type is various, the color is gorgeous, but the flower fragrance character is generally lacked. The unique strong flower fragrance of primula forbesii also makes the primula forbesii an ideal material for improving the flower fragrance character of the plant. The primula species are various, the genetic background is complex, the characteristics of self incompatibility, abnormal style flower columns, large variation of interspecific chromosome base number and the like exist, and the variety with rich flower fragrance is not many, so that the cultivation of the aromatic primula by the conventional breeding method is often difficult to realize. With the continuous updating of unconventional breeding means, molecular breeding can directionally modify a certain target character or certain target characters of the primula by introducing exogenous genes, and further realize the precise breeding of primula molecular design. In future, the improvement of the floral character of the primula by utilizing the plant genetic transformation technology becomes an important target of primula breeding. The agrobacterium infection method is a common method in genetic transformation of plants. However, in primula forbesii genetic transformation, after agrobacterium infection is found, transformed seedlings cannot be effectively obtained by either direct budding or adventitious callus budding. After agrobacterium infection, the resistant callus can not differentiate adventitious buds or directly realize direct sprouting from leaves, so that the development of a primula forbesii differentiation method capable of effectively improving the transformation rate is urgently needed.

Disclosure of Invention

In order to solve the above problems, the present invention provides a method for inducing primula forbesii somatic embryos.

1. A primula forbesii somatic embryo induction method comprises the following steps:

1) aseptic seeding to obtain primula forbesii aseptic seedling;

2) inoculating the sterile seedling leaf obtained in the step 1) to an embryonic callus induction culture medium to induce embryonic callus;

3) inducing somatic embryos from the embryogenic callus induced in the step 2);

4) the somatic embryo germinates and roots.

Wherein the embryogenic callus induction medium is MS medium added with 0.1-0.5 mg/L2, 4-D and 0.5-1.0 mg/L6-BA, preferably, the embryogenic callus induction medium is also added with 200-400 mg/L hydrolyzed casein.

In one embodiment of the present invention, the somatic embryo is induced by inoculating the embryogenic callus into a somatic embryo differentiation medium, wherein the somatic embryo differentiation medium is MS medium supplemented with 0.05-0.2mg/L of 2,4-D and 1.0-3.0mg/L of 6-BA, and preferably, the somatic embryo differentiation medium is further supplemented with 200-400 mg/L of hydrolyzed casein.

In another embodiment of the invention, the induced embryoid is inoculated into a rooting medium for germination and rooting, wherein the rooting medium is 1/2MS medium added with 0.01-0.05mg/L IBA.

Wherein the aseptic seeding comprises the following specific steps:

selecting seeds of the artificially cultured plants harvested in the same year, and storing the seeds in a refrigerator at 4 ℃ in the dark; sterilizing with 75% alcohol for 30S before inoculation, washing with sterile water for 5-6 times, soaking with 1% sodium hypochlorite solution for 1min, washing with sterile water for 5-6 times, sucking off excessive water with sterile filter paper, and inoculating in MS culture medium.

Wherein, when inoculating the leaf, the outer edge of the leaf is cut off and divided into 0.5-1cm²With the leaves facing down, in contact with the medium.

In another embodiment of the present invention, the method further comprises the step of proliferating embryogenic callus in the MS medium supplemented with 0.1-0.3 mg/L2, 4-D, 0.2-0.8 mg/L6-BA, 200-400 mg/L casein hydrolysate.

The invention establishes the primula forbesii somatic embryo induction method for the first time, can quickly obtain primula forbesii somatic cell seedlings, and has high induction efficiency and short seedling period. In addition, compared with the traditional approach of adventitious buds of leaf callus and direct adventitious buds of leaves, the method can obviously improve the genetic transformation efficiency mediated by agrobacterium tumefaciens and lays a foundation for primula forbesii transgenic breeding.

Detailed Description

The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.

EXAMPLE 1 preparation of sterile seedlings

Primula forbesii seeds used in the test are picked in a greenhouse of Sichuan agricultural university in 2019, 4 months and 25 days. The seeds are air-dried in time after being picked, wrapped by a sulfuric acid paper bag and refrigerated in a refrigerator at 4 ℃. Mature and plump seeds are selected as explant materials and tested in the laboratories of the institute of landscape, garden and forestry, Sichuan university.

Sterilizing primula forbesii seeds with 75% ethanol for 30s, washing with sterile water for 5-6 times, sterilizing with 1% sodium hypochlorite for 1min, washing with sterile water for 5-6 times, inoculating the seeds into MS culture medium for culture, inoculating 2 seeds per bottle, and adding 6 g.L into the culture medium^-1Agar was solidified and 30 g.L was added^-1Sucrose, pH adjusted to 5.8 before autoclaving. The culture temperature of the seeds is (25 +/-2) DEG C, the illumination intensity is 1500 lx-2000 lx, and the illumination time is 12 h/d. When the seedling grows to 6-8 cm high and the number of the dark green leaves reaches 3-6, the callus of the leaves can be induced.

EXAMPLE 2 Induction of embryogenic callus

Taking young leaf of aseptic seedling in super clean bench, cutting into 0.5-1cm²The leaf of (5) was inoculated into the callus induction medium in a horizontal manner with the leaf back facing upward and the culture medium being contacted with the leaf on the paraxial surface.

Selecting cytokinin as 6-BA, auxin as 2,4-D, and culture medium with 0.0 mg.L of 2,4-D respectively^-1、0.1mg•L^-1、0.3mg•L^-1、0.5mg•L^-16-BA 0.5 mg.L each^-1、1.0mg•L^-1A total of 8 treatment combinations were designed. Each treatment was inoculated in 10 flasks with 3 explants per flask, and repeated 3 times. MS is taken as a basic culture medium, and 6 g.L of MS is added into all the culture media^-1Agar was solidified and 30 g.L was added^-1Sucrose, pH adjusted to 5.8 before autoclaving. Dark culture is carried out 7 days before inoculation, then a fluorescent lamp is used as a culture light source, the illumination time is 12h/d, the illumination intensity is 1500 lx-2000 lx, and the temperature of a culture room is (25 +/-2) DEG C. The induction of the callus was observed and counted daily.

The hormone proportioning concentration has more obvious influence on the formation of the embryogenic callus of the primula forbesii leaf explant. In each culture medium added with different hormones, the leaf blade in the culture medium can generate callus, and about 7-12 days of leaf blade explants are firstly generated at the main vein lacerated wound part.

As can be seen from Table 1, the hormone concentration has certain influence on primula forbesii leaf induction callus, the over-high and under-low 2,4-D concentrations can inhibit the generation of callus and influence the shape and texture of callus, and when the 6-BA concentration is added for a certain time, the callus induction rate tends to increase and then decrease along with the increase of the 2,4-D concentration. In the medium without 2,4-D, the number of calli was small and the texture was dense green. And with the increase of the concentration of 2,4-D, the induction rate of the callus is improved, and the callus becomes light green callus with loose particle morphology, and the callus with the morphology is embryonic callus. However, when the concentration of 2,4-D is 0.5mg/L and the concentration of 6-BA is 1.0mg/L, the induction rate and the quantity of the callus are both high, but the shape of the callus is changed into a grey loose semitransparent water stain shape.

TABLE 1 callus induction rate of primula forbesii aseptic seedling leaf explants at different hormone concentration ratios

Note: lower case letters indicate differences at a 5% significance level, the same below. More + means more callus and vice versa, less.

Example 3 Induction of embryoid bodies

Three kinds of induced callus, namely compact green callus, granular loose light green callus and gray loose translucent water-soaked callus are inoculated into MS culture medium added with 0.05, 0.1, 0.15 and 0.2 mg/L2, 4-D and 1.0, 2.0 and 3.0 mg/L6-BA, and other conditions of the culture medium are the same as the above.

The results show that compact green callus and loose translucent water-soaked callus can not effectively induce embryoid in 12 kinds of culture medium or the induction rate is very low. Adventitious buds are induced from individual compact green callus blocks. Granular loose light green callus can induce embryoid in different culture mediums, and the result is shown in Table 2.

TABLE 2 Effect of different hormone combination media on primula forbesii embryogenic callus induced embryoid

As shown in Table 2, the granular loose pale green callus can induce somatic embryos in all the culture media, but the influence of different culture media on the induction rate is more obvious. When the concentration of 6-BA is constant, the differentiation rate tends to increase first and then decrease as the concentration of 2,4-D is increased. When the 2,4-D concentration is added for a certain time, the differentiation rate of the somatic embryo is reduced under the condition of high concentration of 6-BA. As can be seen from Table 2, the induction rates of the somatic embryos in culture were the highest for 0.1mg/L of 2,4-D and 1-2mg/L of 6-BA.

EXAMPLE 4 proliferation of embryogenic callus

The induced embryogenic callus was inoculated into MS medium supplemented with 0.1-0.3 mg/L2, 4-D, 0.2-0.8 mg/L6-BA (Table 3), and the effect of different media on the proliferation of embryogenic callus was observed. The proliferation coefficients indicate the increase in the volume of the callus, and + indicates 1-2 fold, + indicates 3-4, and + + indicates 5 fold or more, and the results are shown in Table 3.

TABLE 3 Effect of different hormone combination media on primula forbesii embryogenic callus proliferation

As can be seen from Table 3, the different callus growth media had little effect on callus growth, each of the media was able to grow callus efficiently, and the different media did not change the shape and texture of callus.

EXAMPLE 5 Effect of exogenous additives on embryogenic callus and embryoid body Induction

Taking young leaf of aseptic seedling in super clean bench, cutting into 0.5-1cm²The leaf was placed with the back side facing up and contacted with the medium paraxially, and inoculated horizontally to MS +2, 4-D0.3 mg.L supplemented with hydrolyzed casein at various concentrations^-1+6-BA1.0mg•L^-1Embryogenic callus induction rates for medium, different media are shown in Table 4.

TABLE 4 Effect of different concentrations of hydrolyzed Casein on the embryogenic callus induction rate of primula forbesii

Treatment of	Hydrolyzed casein concentration (mg.L)^-1）	Embryogenic callus induction rate (%)
			1	0	76.88±9.14b
2	200	80.22±9.11ab
			3	400	85.89±10.69a
4	600	72.88±6.34c
			5	800	65.59±3.64c

As can be seen from Table 4, the addition of hydrolyzed casein contributes to the improvement of the induction rate of embryogenic callus. However, when the concentration of hydrolyzed casein exceeds 600mg/L, the induction of callus is rather inhibited.

Inoculating the induced embryogenic callus to MS +2, 4-D0.1 mg.L supplemented with 0, 200, 400, 600, 800mg/L hydrolyzed casein^-1+6-BA1.0mg•L^-1Similar results were obtained when embryoid bodies were induced in the medium, i.e., addition of 200mg/L or 400mg/L of hydrolyzed casein contributed to the induction of somatic embryos, whereas the somatic embryo induction rate was rather decreased in the high-concentration hydrolyzed casein medium.

Example 6 Effect of different morphoietic formation modes of primula forbesii on Agrobacterium-mediated transformation efficiency

Thawing the pBI121 agrobacterium liquid preserved at-80 ℃ on crushed ice, dipping a small amount of the liquid by using an inoculating needle, streaking on a solid plate culture medium added with 50mg/L Kan and 80mg/L Rif, and performing inversion dark culture in a constant-temperature incubator at 28 ℃ for 2 d. Picking single colony with toothpick into liquid LB culture medium containing 50mg/L Kan and 80mg/L Rif, shaking and culturing for 16-24 h to logarithmic phase, thenThen according to the following steps of 1: inoculating 100 percent of the culture medium into a fresh liquid culture medium without antibiotics for continuous shaking culture for 4-6 h to OD₆₀₀Up to about 0.6. And (3) taking fresh bacteria liquid under an aseptic condition, centrifuging the bacteria liquid in a 50ml centrifuge tube at the normal temperature of 8000rpm for 10min, removing supernatant, and carrying out basic suspension precipitation by using 1/2MS liquid culture medium for infection transformation.

Taking young leaf of aseptic seedling in super clean bench, cutting into 0.5-1cm²Placing the left and right small blocks (with partial vein) in the prepared bacterial liquid, infecting for 8min, and intermittently shaking to make the bacterial liquid and plant material fully contact; transferring the infected plant material to sterile filter paper, sucking off the redundant bacteria liquid, transferring to a MS co-culture medium, and co-culturing in the dark at 25 ℃ for 2 d. After co-culture, transfer to the embryogenic callus medium of MS +2, 4-D0.3 mg/L +6-BA1.0mg/L + kanamycin 15mg/L + carbenicillin 300 mg/L.

After about 15 days, loose resistant embryogenic callus with greenish granules was induced at the incision. After 1 month, the embryogenic callus proliferated to about 0.5 cm in diameter, the untransformed part of the explant and the untransformed control underwent browning death, and the induction rate of the resistant embryogenic callus was 15.23. + -. 4.23% (number of leaves from which the resistant embryogenic callus was induced/total number of leaves).

The induced embryogenic callus was inoculated to MS +2, 4-D0.1 mg/L +6-BA1.0mg/L + kanamycin 25mg/L + carbenicillin 200mg/L medium, and 1 half month later, embryoid was induced from the resistant embryogenic callus at an induction rate of 68.77 + -3.45% (number of embryogenic callus clusters/total cluster). The induced embryoid is inoculated in a culture medium of 1/2MS, 0.02mg/L IBA, 25mg/L kanamycin and 200mg/L carbenicillin, and the embryoid can normally germinate and root.

Since pBI121 carries GUS reporter gene, the resistant seedlings were detected by conventional GUS histochemical staining method, and 11 lines of 15 tested lines can display blue plaques, which indicates that the GUS gene has been successfully transformed.

Comparative example 1

The bacterial suspension and primula forbesii material were the same as in example 6 except that the culture medium was MS +6-BA1.0+ NAA0.1 (refer to Xiagua Kishinouye et al, tissue culture and plant regeneration in primula forbesii, physiological communication of plants, Vol. 41, No. 6, 2005, Medium (7)), and the morphogenesis was achieved by callus-induced adventitious bud pathway.

About 30 days later, a small amount of green compact callus can be induced at the incision, and the induction rate of the resistant callus is only 2.48 +/-1.86%. In addition, the callus proliferation rate is slow, and after transfer, resistant shoots cannot be effectively proliferated or induced.

Comparative example 2

The bacterial suspension and primula forbesii material were the same as in example 6 except that the culture medium was MS +6-BA 1+ NAA 0.05 (refer to Xiagua kusnezoffii et al, tissue culture and plant regeneration of primula forbesii, physiological communication of plants, Vol. 41, No. 6, 2005, Medium (3)), and the morphogenesis was carried out by directly inducing adventitious bud pathway in leaf.

After the leaves infected by the agrobacterium are transferred to the culture medium, resistant buds cannot be successfully induced, and the leaves die due to browning.

As can be seen from the above comparative examples, different morphogenetic patterns have a significant effect on primula forbesii genetic transformation. The embryoid approach can significantly improve primula forbesii genetic transformation rate.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the technical principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A primula forbesii somatic embryo induction method is characterized by comprising the following steps:

1) aseptic seeding to obtain primula forbesii aseptic seedling;

2) inoculating the sterile seedling leaf obtained in the step 1) to an embryonic callus induction culture medium to induce embryonic callus; the embryogenic callus induction culture medium is MS +2, 4-D0.3 mg.L^-1+6-BA 0.5mg•L^-1Or MS +2, 4-D0.5 mg.L^-1+6-BA 0.5mg•L^-1Or MS +2, 4-D0.3 mg.L^-1+6-BA 1.0mg•L^-1；

3) Inoculating the embryogenic callus induced in the step 2) into a somatic embryo differentiation medium to induce a somatic embryo, wherein the somatic embryo differentiation medium is MS +2, 4-D0.1 mg.L^-1+6-BA1.0mg•L^-1Or MS +2, 4-D0.1 mg.L^-1+6-BA2.0mg•L^-1；

4) The somatic embryo germinates and roots.

2. The method for inducing primula forbesii somatic embryos of claim 1, wherein 200-400 mg/L hydrolyzed casein is further added to the embryogenic callus induction medium.

3. The method for inducing primula forbesii somatic embryos of claim 1, wherein 200-400 mg/L hydrolyzed casein is further added to the somatic embryo differentiation medium.

4. The method for inducing primula forbesii somatic embryos of claim 1, wherein the embryoid bodies induced in the step 3) are inoculated into a rooting medium for germination and rooting, wherein the rooting medium is 1/2MS medium added with 0.01-0.05mg/L IBA.

5. The primula forbesii somatic embryo induction method according to claim 1, characterized in that the specific steps of aseptic seeding are:

selecting seeds of the artificially cultured plants harvested in the same year, and storing the seeds in a refrigerator at 4 ℃ in the dark; sterilizing with 75% ethanol for 30s before inoculation, washing with sterile water for 5-6 times, soaking with 1% sodium hypochlorite solution for 1min, washing with sterile water for 5-6 times, sucking off excessive water with sterile filter paper, and inoculating in MS culture medium.

6. The method for inducing primula forbesii somatic embryos of claim 1, wherein the outer edges of the leaves are cut off and divided into 0.5-1cm at the time of inoculation of the leaves²With the leaves facing down, in contact with the medium.

7. The method for inducing primula forbesii somatic embryos of any one of claims 1 to 6, further comprising the step of proliferating embryogenic callus in the MS medium supplemented with 0.1 to 0.3mg/L of 2,4-D, 0.2 to 0.8mg/L of 6-BA, 200-400 mg/L of casein hydrolysate.