KR100736207B1 - Method for regenerating Sesamum indicum L. - Google Patents

Abstract

The present invention removes pears from mature cotyledon immediately before germination of sesame seeds, induces malformation and converts them into news bodies, and then grows them into complete plants to produce seeds for in-situ plant disease, disaster resistance and high-quality sesame breeding. It relates to a regeneration method.

Sesame seeds (Sesamum indicum L.), biotechnological methods, embryonic matured cotyledons, in-flight regeneration, molecular breeding

Description

Method for regenerating in-vehicle plants in sesame seeds {Method for regenerating Sesamum indicum L.}

1 is a photograph of plants regenerated through induction of germination in mature cotyledon immediately before germination from which sesame seeds were removed.

A, cotyledons of sesame seeds with embryos and embryos immediately before germination

B, initial callus formation at the cut plane after 3 days of culture

C, malformation after 2 weeks of culture

D-F: malformation-3 wt% sucrose (D), 6 wt% sucrose (E) 9 wt% sucrose (F).

Root Formation in MS Medium Containing G, NAA (0.5mg / l)

H, Purification process in a mixture of sand and horticultural clay (1: 1, v / v)

I, mature plants in the greenhouse

J, harvest seeds

Figure 2 is a graph showing the rate of malformation at each developmental stage of the explant. Vertical bars represent mean ± S.E.

Figure 3 is a graph showing the rate of malaria formation by breed. Vertical bars represent mean ± S.E.

The present invention removes pears from mature cotyledon immediately before germination of sesame seeds, induces malformation, converts them to news bodies, and grows them into complete plants to produce seeds for in-situ plant disease, disaster resistance and high quality sesame breeding It relates to a regeneration method.

Sesame seeds ( Sesamum indicum L.) is an important paid crop, but growers are suffering from difficulties due to their weakness in cultivation stability due to weakness in diseases and natural disasters. Also, due to the nature of sesame oil, it is a crop that has much room for improvement. Breeders have tried to cultivate sesame varieties that are highly resistant to diseases and disasters, and contain high-quality fatty acids and antioxidants in order to overcome these shortcomings, but promising varieties have not been developed yet.

Recently, gene conversion technology has been developed in various crops, and molecular breeding systems have been established using them, and new crops having high disease resistance, disaster resistance, and quality characteristics are being developed one after another. In order to use these new molecular breeding techniques for sesame breeding, in-flight regeneration technology, which is a prior art of gene conversion technology, is essential. However, the available technology has not yet been developed in sesame seeds, which has become an obstacle to molecular breeding.

Existing in-flight regeneration techniques in sesame seeds (Jeya Mary and Jayabalan 1997) and regeneration techniques using cotyledons, roots, and upper hypocotyls in young seedlings (Xu et al. 1997) have been reported. This was as low as 12 to 13%, and there was a problem of poor reproducibility. There are also reports of multiple transit in shoot-tips of young seedlings (Rao and Vidyanath 1997) and interaxed ligaments (Gangopadhyay et al. 1998), but these organs are low as pro-meristematic organs. Because of the rate of transformation, the genetic transformation is ineffective. Successful gene conversion is known to be effective in regenerating plants through the formation of malformed germs from meristematic-free organs. No such regeneration techniques have been reported in sesame seeds.

Therefore, the present inventors established a technique for inducing high-fidelity sprouts from the cotyledons from which the embryos of the mature seeds, which are the non-dividing organs of sesame seeds, were removed, and regenerating sesame seeds in the cabin. It was found that it can be produced to complete the present invention.

Accordingly, it is an object of the present invention to provide a method for inducing malaria with high efficiency in sesame mature cotyledon immediately before germination.

It is also an object of the present invention to provide a method for deriving a newsletter and a complete plant from the indeterminate child and a method for obtaining seeds therefrom.

In order to achieve the above object, the present invention provides an in-vehicle regeneration method that can obtain seeds by removing embryos from mature cotyledon immediately before germination of sesame seeds and inducing germination and growing them into complete plants.

The in-vehicle regeneration method according to the present invention will be described in more detail.

In the sesame seeds, completely mature seeds were absorbed until just before germination, and then the cotyledons removed from the seeds and embryos were separated by 0.01-2.0 mg / l of IAA (Indole-3-acetic acid) and 0.01-benzyl-aminopurine (BA) 0.01 Callus and malaria were incubated for 2-3 weeks in MS medium containing -7.0 mg / l, ABA (Abscisic acid) 0.01-2.0 mg / l, AgNO ₃ 0.01-7 mg / l and sucrose (1-9% by weight). MS medium containing IAA 0.01-2.0 mg / l, BA 0.01-7.0 mg / l, ABA 0.01-2.0 mg / l, AgNO ₃ 0.01-7 mg / l and sucrose (1-9% by weight) Induce multiple shoots and subculture two to three times every two weeks to multiply.

By cutting the multi-diameter, the medium containing only half of the MS medium containing 0.01-0.5mg / L NAA (a-naphthalene acetic acid) (hereinafter referred to as '1/2 MS medium') to the full MS medium (hereinafter '1' / 1 MS medium ') to induce rooting. Rooted planters are transplanted into a mixed soil mixed with sand and horticultural soils in a 1: 1 volume ratio to flower and produce seeds.

When cultivated cotyledons of mature seeds immediately before germination were cultured in MS medium + IAA 1.0 / l + BA 5.0 mg / l with 3% by weight sucrose, callus was formed on the cut surface after 3 to 5 days. After a week or two, the calus developed a malformed child. ABA and AgNO ₃ treatments were also more effective in inducing infertility. In the medium without ABA and AgNO ₃ , the rate of malformation was 17%, but when ABA and AgNO ₃ were combined with BA and IAA, respectively, it was doubled to 34% and increased to 50% when combined. In other words, ABA and AgNO ₃ were found to be more effective when used simultaneously than when used individually.

The developmental period of the explants used as a material is very important for the induction of malaria in sesame seeds. The differentiation ability to produce malaria is only observed in mature cotyledon just before germination.

Cotyledon explants were cultured in various concentrations of sucrose (3, 6, and 9% by weight) medium, and in 9% sucrose medium, the sections browned and died after 6 weeks. However, transfer of 3% medium to 9% sucrose medium after 2 weeks of pre-cultivation resulted in a very high rate of 64-70% dendrite formation.

The rooting rate was 98% in complete MS medium containing 0.5 mg / l NAA. Plant roots after rooting had a 95% purity in sand and horticultural mixtures (1: 1, v / v), and plants continued to grow and bloom to produce seeds.

Hereinafter, an Example and a test example are given and this invention is demonstrated in detail. Such examples or test examples are intended to describe the present invention in detail, but the scope of the present invention is not limited to these examples or test examples.

Example 1

After absorbing water from the sesame seeds until immediately before germination, the cotyledons removed from the seeds and embryos were treated with IAA 1.0 mg / l, BA 5.0 mg / l, ABA 1.0 mg / l, AgNO ₃ 5.0 mg / l and Two weeks of incubation in MS medium containing 3% by weight sucrose induced calus and malaria. After 3 to 5 days, callus was formed on the cut surface (Fig. 1B), and after 1 to 2 weeks, a callus emerged from the callus (Fig. 1C). After 4-6 weeks, negative children also appeared on the basal side of the explant (FIGS. 1D-F).

Again, multiple doses were induced by transferring to MS medium containing 1.0 mg / l of IAA, 5.0 mg / l of BA, 1.0 mg / l of ABA, 5.0 mg / l of AgNO ₃ and 3% by weight of sucrose. Multiple passages were proliferated by 2-3 passages.

The multiple diameters were cut and transferred to 1 / 2-1 / 1 MS medium containing 0.3 mg / L NAA to induce rooting (FIG. 1G). The rooted newsletters were transplanted into a mixed soil mixed with sand and horticultural clay in a 1: 1 weight ratio to bloom (FIG. 1H) and to produce seeds (FIGS. 1I and J).

[Test Example 1]

Table 1 shows the results of incubating cotyledons removed from the seed and embryos from mature sesame seeds immediately before germination with 3% by weight sucrose and various concentrations of IAA and BA based on MS medium. As a result, it was confirmed that the highest adventitious bud formation was obtained in the combination of IAA 1.0 mg / L and BA 5.0 mg / L. Kinetin and Zetin were not effective in the formation of malaria.

IAA (mg / L) BA (mg / L) Negative Childbirth Rate (%) Germination / Intersection Number 0 2.5 3 ± 0.2 1.1 ± 0.12 0 5.0 6 ± 0.5 2.3 ± 0.16 0 10.0 0 0 0.5 2.5 7 ± 0.3 2.3 ± 0.34 0.5 5.0 12 ± 0.7 4.3 ± 0.52 0.5 10.0 6 ± 0.5 3.0 ± 0.43 1.0 2.5 9 ± 0.5 2.0 ± 0.19 1.0 5.0 17 ± 1.2 5.3 ± 0.64 1.0 10.0 10 ± 1.1 4.7 ± 0.37 2.0 2.5 5 ± 0.3 3.3 ± 0.26 2.0 5.0 11 ± 0.9 3.7 ± 0.45 2.0 10.0 7 ± 0.4 2.3 ± 0.32

The data represent the mean ± S.E. Of three independent experiments.

[Test Example 2]

Induction of malaria in sesame seeds is very important because the developmental period of the material is very important in order to determine the most suitable time of development of the dendritic formation of the fragments, the results are shown in FIG. Referring to the result of Figure 2, the differentiation ability to produce a malaria appeared only in the mature cotyledon immediately before germination with embryos removed. In the cotyledons removed from the 40-day-old immature seeds, the germinal formation rate was less than 3%, and in the cotyledon explants 1-2 weeks after germination. Even when the hypocotyls and roots were germinated 1 to 2 weeks after germination, callus was formed at high frequency, but no malaria was differentiated at all.

In addition, the cotyledon explants were cultured in various concentrations of sucrose (3, 6, and 9% by weight) medium, and in 9% sucrose medium, the sections were browned and killed after 6 weeks. However, after 2 weeks of pre-culture in 9% sucrose medium and transfer to 3% medium, the rate of malformation was very high. However, when precultured in 12% sucrose medium, the rate of malformation was zero. These results suggest that the developmental period (mature seed) of the explants is important for the induction of malaria, which is essential for organogenesis in sesame seeds, and the temporary (2 weeks) high concentration of sucrose (9% by weight) treatment results in malformation. It has been found to promote.

[Test Example 3]

In order to determine the effect of ABA and AgNO ₃ treatment on induction of malaria, the rate of formation of malaria according to the content of each component shown in Table 2 was examined. In the medium without ABA and AgNO ₃ , the rate of malformation was 17%, but when ABA and AgNO ₃ were combined with BA and IAA respectively, it was doubled to 34%, and increased to 50% when all were combined (Table 2). ).

Treatment group % Abnormal child formation rate IAA (mg / L) BA (mg / L) ABA (mg / L) AgNO ₃ (mg / L) 1.0 2.5 0 0 12 ± 1.4 1.0 5.0 0 0 17 ± 2.1 1.0 10.0 0 0 11 ± 0.9 1.0 2.5 One 0 33 ± 4.2 1.0 5.0 One 0 34 ± 3.7 1.0 10.0 One 0 26 ± 5.3 1.0 2.5 One 5 47 ± 5.3 1.0 5.0 One 5 50 ± 6.7 1.0 10.0 One 5 29 ± 4.5

The data represent the mean ± S.E. Of three independent experiments.

ABA is a plant hormone that responds to stress and is known to promote seed maturation and inhibit germination. In the present invention, ABA has been found to be effective in inducing and propagating malaria in sesame seeds. AgNO ₃ has also been reported to enhance plant regeneration as an ethylene inhibitor, which also improves the frequency of growth in sesame seeds. It was confirmed that ABA and AgNO ₃ were more effective when used at the same time than when using embryonic cotyledons with germinated cotyledons.

[Test Example 4]

5 mg / ℓ BA, 1 mg / ℓ IAA from cotyledons of mature seeds from which pears were removed from nine sesame varieties (anam, dasaek, baekbaek, mangeum, namsan, osan, pungnam, tainnan, and yonghyuk). , Incubated for 2 weeks in different concentrations of sucrose medium (3, 6 and 9% by weight) containing 1 mg / l ABA and 5 mg / l AgNO ₃ and then subcultured in 3% sucrose medium with the same hormones added. After each of the varieties were examined for the rate of formation, the results are shown in FIG. In the results of FIG. 3, nine varieties precultured at 3% sucrose had 29-50% denaturation rate, but precultured at 9% sucrose were very high at 64-70%, indicating high sucrose treatment. It was confirmed that it promotes formation.

[Test Example 5]

After growth of 2 to 3 cm in size, the embryos were cut and transferred to 1/2 MS medium without hormone (Murashige & Skoog medium), complete MS medium containing 0.5 mg / L NAA, and induced rooting. 1G). The rooting rate was 98% of complete MS medium containing 0.5 mg / L NAA, 90% of 1/2 MS medium and 85% of complete MS medium, but the rate of differentiation into complete plantlets was 0.5 mg / L NAA. The total MS medium contained was 57%, slightly lower than half MS medium 58% and complete MS medium 62% (Table 3).

Culture medium Rooting rate (%) Differentiation rate (%) 1/2 MS 90 ± 12.3 58 ± 5.4 MS 85 ± 13.5 62 ± 8.2 MS + 0.5 mg / l NAA 98 ± 22.1 57 ± 7.6

The data represent the mean ± S.E. Of three independent experiments.

[Test Example 6]

In order to determine the most suitable soil for transplanting rooted plantlets, grafts about 10 cm in size were transplanted into loess, sand, and sand and horticultural mixtures (1: 1, v / v), respectively. Was put on the vinyl and then bloomed for 2 months to find out the rate of purification, the results are shown in Table 4 below. As a result, the purifying rate of ocher, sand, and sand and horticultural clay mixture was 20%, 55% and 95%, respectively, and sand and horticultural clay mixture (1: 1) showed the best purifying ratio. Purified plants continued to grow and bloom to produce seeds.

Soil type Purification rate (%) ocher 20 ± 4.2 sand 55 ± 5.7 Mixture of sand and gardening clay 95 ± 11.5

The data represent the mean ± S.E. Of three independent experiments.

Through the above test examples in the present invention using sesame seeds from the cotyledons of which the embryos of mature seeds were removed immediately before germination, sesame seeds were regenerated in the cabin at high efficiency, and after being converted into the news material, a mixture of horticulture clay and sand (1: 1) ) Were able to grow well into complete plants and successfully bloom to produce seeds. During this process, high concentrations of sucrose, BA, IAA, AgNO ₃ and ABA played an important role in the induction of malaria based on MS medium.

As described above, in the present invention, by inducing hormonal and sucrose treatment with mature cotyledons removed immediately from germ from sesame seeds, the induction of high quality in vitro and cultivation of sequential and complete plants in sequence A seed harvesting technique was provided, which will enable molecular breeding in sesame as an essential technology for genetic transformation technology that enables disease resistance, hazard resistance and high quality breeding in sesame seeds.

In addition, the present invention targets sesame seeds ( in In vitro , high-efficiency induction of indeterminate embryos and sequential growth of news and complete plants to obtain seeds, which can be used in genetic transformation technology for molecular breeding and functional genomics in sesame seeds.

Claims (4)

After absorbing water from the sesame seeds to the end of germination, the cotyledons from which the seeds and embryos were removed were 0.01-2.0 mg / l of IAA (Indole-3-acetic acid) and 6-benzyl-aminopurine (BA). Incubate for 2-3 weeks in MS medium containing 0.01-7.0mg / l, ABA (Abscisic acid) 0.01-2.0mg / l, AgNO ₃ 0.01-7mg / l and sucrose (1-9% by weight) MS medium containing IAA 0.01-2.0mg / l, BA 0.01-7.0mg / l, ABA 0.01-2.0mg / l, AgNO ₃ 0.01-7mg / l and sucrose (1-9% by weight) Method of regenerating in-flight plants from sesame through the process of inducing multiple shoots and subculture two to three times every two weeks to multiply multiply. The method according to claim 1, wherein the produced multi-diameters are cut and 1/2 (MS medium containing only half of the medium) -1 / 1MS medium containing all 0.01-0.5 mg / L a-naphthalene acetic acid (NAA). Medium) to further induce rooting. 3. The method according to claim 2, wherein the rooted plantlets are transplanted into a mixed soil in which sand and horticultural clay are mixed in a 1: 1 volume ratio to flower and produce seeds. delete

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