CN101946704A - Method for regenerating Chinese rose plant by using immature seed as explant - Google Patents

Abstract

The invention belongs to the technical field of plant tissue and cell culture, and in particular relates to a plant regeneration method of rose somatic embryogenesis pathway. The method includes three culture stages: (1) Cultivate rose explants on the induction medium, the explants are immature zygotic embryos of roses, and directly induce somatic embryos from the explants; (2) inoculate somatic embryos Proliferate on the proliferation medium and promote its further differentiation; (3) Somatic embryos after proliferation and culture are inoculated on somatic embryo seedling formation medium to regenerate into complete plants. During the plant regeneration process of the somatic embryos cultured in vitro, roots and shoots occur simultaneously, and can form a complete plant without rooting culture. The method of the invention has the advantages of wide sources of explants, convenient material collection and the like. The obtained somatic embryo is a good recipient for the genetic transformation mediated by the agrobacterium, and the invention can also be used for the research of the genetic transformation of the rose.

Description

Method for regenerating rose plants using immature seeds as explants

technical field

The invention belongs to the technical field of plant tissue and cell culture, and specifically relates to a method for regenerating rose plants through somatic embryogenesis by using immature zygotic embryos of roses as explants.

Background technique

Rosa hybrida is a perennial evergreen woody plant belonging to the genus Rosa of the family Rosaceae. Rose has a long history of cultivation and is widely planted by countries all over the world. It is a very important ornamental flower with important commercial value and plays an important role in landscaping. Rose is known as the queen of flowers because of its graceful posture, rich and gorgeous flower colors, rich fragrance, long flowering period, strong adaptability, easy reproduction and convenient management. Roses can be used in a wide range of ways. For example, vine roses can be used to decorate corridors and arches, tree roses can be used to decorate main roads, shrub roses can be used to make hedges, polyflower roses can be used to decorate flower beds, tea roses can be used for exhibitions, and miniature roses can be used As an interior decoration, cut roses can be used in various places.

However, the vase life of rose flower is relatively short, and it is prone to powdery mildew and black spot, which have affected its ornamental value and economic value. In improving these traits, traditional breeding methods have great limitations. On the basis of keeping other traits of varieties relatively stable, plant genetic engineering technology can use exogenous genes to carry out directional improvement of the traits controlled by them, thus providing a new way for breeding new rose varieties. The establishment of rose regeneration system and genetic transformation system is an important basis for breeding with genetic engineering technology.

Plant somatic embryogenesis is a process in which plant somatic cells obtain new individuals through a developmental pathway similar to zygotic embryos under in vitro conditions. The establishment of the regeneration system of the somatic embryogenesis pathway is conducive to the rapid propagation of test-tube plantlets of roses, the production and cold storage of artificial seeds, and at the same time, embryogenic cells are the best recipients for genetic transformation of roses, and somatic embryos can be used as recipients Carry out research on genetic transformation of rose.

Different rose varieties and explants and experimental methods have been applied to the study of rose somatic embryogenesis, and the results show that it is difficult to find a general method for the induction of rose somatic embryos due to genotype limitations (Marchant et al., Somatic embryogenesis and plant regeneration in floribunda rose (Rosa hybrida L. cvs. Trumpeter and Glad Tidings). Plant Science, 1996, 120: 95-105). Embryogenic callus can be obtained by using the detached leaves or stem segments of Rosa hybrida cv. Carl Red and R. canina as explants (Visessuwan et al., Plant regeneration systems from leaf segment culture through embryogenic callus formation of Rosa hybrida and R. canina. Breed Sci, 1997, 47: 217-222). DeWit et al. (Somatic embryogenesis and regeneration of flowering plants in rose. Plant Cell Rep, 1990, 9: 456-458) also obtained leaves of R. hybrida cv. Domingo and R. hybrida cv. Vicky Brown as explants embryogenic callus. Rout et al. (Somatic embryogenesis in callus culture of Rosa hybrida L.cv.Landora.Plant Cell Tiss Org Cult, 1997, 27:65-69) in the study of somatic embryo induction in rose R.hybrida cv.Landora, the induction did not Somatic embryos can be obtained from mature leaf or stem segments. Marchant et al.(Somatic embryogenesis and plant regeneration in floribunda rose(Rosa hybrida L.cvs.Trumpeter and Glad Tidings).Plant Science, 1996,120:95-105) with rose R.hybrida cv.Trumpeter and R.hybrida cv .The petioles and roots of Glad Tidings were used as explants to induce somatic embryos. Somatic embryos were induced by immature seeds (Kim et al., Control of direct and indirect somatic embryogenesis by exogenous growth regulators in immature zygotic embryo cultures of rose. Plant Cell Tiss Org Cult, 2003, 74: 61-66; Kamo et al. al., Dispersal and size fractionation of embryogenic of callus increases the frequency of embryo maturation and conversion in hybrid tea roses. Plant Cell Rep, 2004, 22: 787-792). In addition, somatic embryos can be obtained by inducing rose petals (Arene et al., A comparison of the somaclonal variation level of Rosa hybrida L.cv. Meirutral plants regenerated from callus of direct induction from different vegetative and embryonic tissues. Euphytica, 1993 , 71:83-90). At present, there is no report on the induction of somatic embryos in rose 'Ziwu' at home and abroad.

Contents of the invention

The purpose of the present invention is to overcome the defects of the prior art, and provide a method for establishing a rose regeneration system by using immature zygotic embryos of rose 'Ziwu' as a material through somatic embryogenesis. At the same time, the somatic embryos obtained by the method of the invention can be subcultured for a long time and maintain their differentiation ability, and can be used as materials for genetic transformation of roses.

Technical scheme of the present invention and step are as follows:

A method for regenerating Chinese rose into a complete plant, comprising the following steps:

① Taking immature zygotic embryos of Chinese rose as explants, directly inducing somatic embryos from the explants in the induction medium;

② Inoculating the somatic embryos in step ① into a somatic embryo proliferation medium to proliferate and further differentiate them to obtain mature somatic embryos;

③ inoculating the mature somatic embryos obtained in step ② into the somatic embryo seedling growth medium to regenerate into complete plants;

in:

The composition of the induction medium is as follows: 1/2MS basic medium, 2,4-dichlorophenoxyacetic acid 0-3.0mg/L, 6-benzyl adenine 0-1.0mg/L, glucose 30g/L , vegetable gel 2.5g/L;

The composition of the somatic embryo proliferation medium is as follows: MS basic medium, 2,4-dichlorophenoxyacetic acid 1.0mg/L, 6-benzyl adenine 0.05mg/L, glucose 60g/L, phytocondensate Glue 2.5g/L;

The composition of the somatic embryo seedling growth medium is as follows: MS basic medium, 6-benzyl adenine 0-1.0mg/L, glucose 30g/L, plant gel 2.5g/L or MS basic medium, Benzodiazole diazide (TDZ) 1.0mg/L, 6-benzyl adenine 0.5mg/L, glucose 30g/L, plant gel 2.5g/L.

As a preferred scheme, the 2,4-dichlorophenoxyacetic acid in the induction medium is 2.0 mg/L, and the 6-benzyl adenine is 0.5 mg/L.

As a preferred solution, the composition of the medium for somatic embryonic cell seedling formation is as follows: MS basal medium, 6-benzyl adenine is 0.5 mg/L.

The invention uses immature zygotic embryos of the Chinese rose variety "Ziwu" as explants, directly induces somatic embryogenesis, and can maintain the characteristics of continuous proliferation and seedling formation of somatic embryos. These somatic embryos provide good recipients for the genetic transformation of roses mediated by Agrobacterium; at the same time, this regeneration system is also an ideal recipient for genetic transformations of roses and lays a certain technical foundation for the preparation of artificial seeds of new transgenic rose varieties .

The positive effect of the present invention is:

1. The source of explants used in the present invention is not limited by seasons, and the tissue and cell culture of rose 'Ziwu' can be carried out every year.

2. Rose somatic embryos cultured in vitro can proliferate continuously through secondary embryos, which has a good proliferation effect.

3. Rose somatic embryos cultured in vitro can be preserved for a long time and can maintain their ability to further differentiate into plantlets.

Description of drawings

Figure 1: Proliferation of somatic embryos of rose 'Ziwu' in the present invention.

Figure 2: Germination of somatic embryos of rose 'Ziwu' in the present invention.

Figure 3: A complete plant with roots and buds after germination of somatic embryos of rose 'Ziwu' in the present invention.

Figure 4: The regenerated plants of Chinese rose 'Ziwu' grown under field conditions in the present invention.

Detailed ways

Example 1

Test material selection, medium design and inoculation culture

1. The source and treatment of test materials:

The test material among the present invention is selected from and takes to grow robustly in the flower base of Huazhong Agricultural University, the excellent rose variety "Ziwu" without disease and insect pests (the original source variety is purchased from the flower nursery stock market of Hubei Academy of Agricultural Sciences, Wuchang, Wuhan City, Hubei Province. unripe fruit (8 weeks after free pollination). Fruit processing method: cut open the collected immature fruit of the Chinese rose 'Ziwu', take out the seeds inside, and select plump seeds that can sink in clear water. After rinsing with tap water for 30 minutes, put them into the mixed solution (concentrated sulfuric acid by volume: water=3:2) to soak the seeds for 30 minutes. Disinfect with 70% alcohol for 40 seconds on the ultra-clean workbench, then disinfect with 0.1% mercury liter for 10 minutes, and rinse with sterile water 3-4 times. The immature zygotic embryos were stripped as explants.

2. Culture medium design:

Table 1 is the composition and consumption amount of various media in the present invention.

The design of the in vitro culture medium of table 1 rose

Note: For the preparation of MS basic medium, refer to: Murashige T. and F. Skoog. Physiol. Plant, 1962, 15: 473-497; 1/2 MS basic medium is halved of the macronutrients and trace elements in MS basic medium Halved, Fe salt halved, organic elements unchanged.

The subculture medium for somatic embryos in Table 1 is not only used as a general subculture medium, but also the long-term subculture medium for somatic embryos referred to in the present invention.

The code names of the various components in the medium are as follows: 2,4-dichlorophenoxyacetic acid (2,4-D), 6-benzyl adenine (6-BA), and phytogel (GEL) can all be obtained from commercial Buy.

3. Culture conditions

The culture temperature in the culture room is 24±2°C, the light intensity is 1000-1500lx, and the light cycle is 14h; the dark culture temperature is 24±2°C.

4. Inoculation and cultivation:

Inoculate the immature zygotic embryos of Chinese rose ‘Ziwu’ on the medium induction medium (the medium is shown in Table 1) on the ultra-clean workbench, inoculate 10-12 explants in each petri dish, and culture in the dark . Two months later, somatic embryos were produced on immature zygotic embryos, and some explants turned brown and died, failing to induce somatic embryos.

The somatic embryos were inoculated on the somatic embryo proliferation medium (shown in Table 1), and cultured under light conditions. These somatic embryos can proliferate rapidly on the somatic embryo proliferation medium (see Figure 1), and the somatic embryos can be inoculated on the somatic embryo subculture medium (see Table 1) for long-term preservation. The mature somatic embryos were inoculated on the somatic embryo seedling-forming medium (shown in Table 1), and the somatic embryos matured and germinated (see Figure 2). The germinated somatic embryos were inoculated on fresh somatic embryo seedling formation medium, and the somatic embryos of rose 'Ziwu' could directly grow seedlings on the somatic embryo seedling formation medium due to their root-bud bipolar structure ( See Figure 3).

After 1 month, move the regenerated rose plants with well-developed root system to natural conditions with scattered light, harden the seedlings for a week, take out the rooted plantlets, wash the root culture medium, and then transplant them to the soil containing peat: perlite ( In a plastic cup with a volume ratio of 1:1), cultivate under low light for 3-4 days to make new roots grow, and then cultivate under strong light. When obvious new roots are seen in the plastic cup, it is transplanted into a pot with peat soil to make it grow healthily (see Figure 4).

Example 2

Effects of different concentration combinations of 2, 4-D and 6-BA on somatic embryogenesis in rose 'Ziwu'

The immature zygotic embryos of rose 'Ziwu' were used as explants to induce somatic embryogenesis. When the concentration of 2,4-D was 2mg/L, the highest induction rate of 'Ziwu' somatic embryos was 11.11%. Some immature zygotic embryos that did not generate somatic embryos were browned and died, and some produced non-embryogenic callus. The experimental results are shown in Table 2:

Table 2 Effects of different concentration combinations of 2, 4-D and 6-BA on somatic embryogenesis in rose 'Ziwu'

Example 3

Effects of Different Media on Germination of Somatic Embryos and Plantlet Regeneration of Rose 'Ziwu'

The effects of different media on the germination and plant regeneration of rose 'Ziwu' somatic embryos are shown in Table 3: In the somatic embryo germination test, there were significant differences in the effects of 9 media on the germination of rose somatic embryos . On MS+0.5mg/L 6-BA+3%Glucose+0.25%GEL medium, the germination rate of 'Ziwu' somatic embryos was the highest, reaching 86.67%, and the seedling rate per unit of somatic embryos was as high as 98.88%. 6-BA did not significantly promote the germination of somatic embryos and plant regeneration of rose 'Ziwu', and the effect of 6-BA was better than that of TDZ. During the plant regeneration process of somatic embryos of rose 'Ziwu', roots and buds occur simultaneously, and a complete plant with roots and buds can be formed without rooting culture.

Table 3 Effects of different seedling growth media on somatic embryo germination and plant regeneration of rose 'Ziwu'

Note: The data are shown as mean ± standard error, and different letters indicate significant differences at the P<0.05 level. Somatic embryo germination rate (%) = number of germinated somatic embryos / total number of inoculated somatic embryos × 100%; unit somatic embryo seedling rate (%) = total number of seedlings / total number of inoculated somatic embryos × 100% .

Claims (4)

1. one kind is regenerated as the method for whole plant with Chinese rose, and it comprises the following steps:

1. be explant with the Chinese rose immature zygotic embryos, described explant is directly induced somatic embryo in inducing culture;

2. the somatic embryo of step in 1. is inoculated in the somatic embryo proliferated culture medium propagation and makes its further differentiation, obtain mature somatic embryo;

3. the mature somatic embryo that 2. step is obtained is inoculated into somatic embryo and becomes in the seedling medium to be regenerated as whole plant;

Wherein

Described inducing culture composed as follows: 1/2MS minimal medium, 2,4 dichlorophenoxyacetic acid 0-3.0mg/L, 6-benzyladenine 0-1.0mg/L, glucose 30g/L, plant gel 2.5g/L;

Described somatic embryo proliferated culture medium composed as follows: MS minimal medium, 2,4 dichlorophenoxyacetic acid 1.0mg/L, 6-benzyladenine 0.05mg/L, glucose 60g/L, plant gel 2.5g/L;

Described somatic embryo becomes the composed as follows of seedling medium: the MS minimal medium, 6-benzyladenine 0-1.0mg/L, glucose 30g/L, plant gel 2.5g/L or MS minimal medium, azoles diazobenzene stem urea (TDZ) 1.0mg/L, 6-benzyladenine 0.5mg/L, glucose 30g/L, plant gel 2.5g/L.

2. method according to claim 1, the 2,4 dichlorophenoxyacetic acid in the wherein said inducing culture are 2.0mg/L, and 6-benzyladenine is 0.5mg/L.

3. method according to claim 1, it is 0.5mg/L that the thin blast embryo of wherein said body becomes the 6-benzyladenine in the seedling medium.

4. method according to claim 1, wherein the 2. described method of step also comprises described somatic embryo is inoculated on the somatic embryo subculture medium, makes its long-term subculture and keeps differentiation capability.

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