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Tanbaguro: a new model genotype of soybean for tissue culture study

Tomoko Hatanaka1, Shinya Yoshihara, Seiko Imoto, Naotsugu Uchida and Hyoe Tsugawa

Faculty of Agriculture, Kobe University, 1-1 Rokko, nada, Kobe, 657-8501, Japan Email thata@kobe-u.ac.jp

Abstract

Tanbaguro, a local variety of Japanese soybean [Glycine max (L.) Merrill] with a black seed coat, is very popular in Japan. In spite of its high price, its uses and demands are increasing. The objectives of this study were to establish a micropropagation system for black seed coat soybeans to obtain many uniform plants. A standard procedure of embryogenic culture was applied to two strains of Tanbaguro, “Hyokeikuro-3” and “Shin-tanbaguro”. Two regular cultivars, “Jack” and “Tamahomare” were used as controls. Our results showed that “Hyokeikuro-3” had a higher embryogenic capacity than Jack, a model cultivar for tissue culture study.

Media summary

One popular Japanese variety of soybean with a black seed coat has a higher embryogenic capacity than a model cultivar Jack.

Key Words

Hyokeikuro-3, black soybean, embryogenic capacity

Introduction

A local soybean variety that is commonly called “Tanbaguro” originated in western Japan. Tanbaguro seeds are characterized by a black seed coat, huge size, and rounded shape compared to regular soybean seeds (Fig. 1, Table 1). In general, black seed coat soybeans are bigger than regular yellow soybeans and Tanbaguro would be the biggest soybean in the world. It is thought that temperature and day-length in western Japan provides conditions to make the seeds huge.

Figure 1. Comparison between Tanbaguro (left) and Jack (right). The bar indicates 1 cm.

Table 1. Mean seed weights of several soybean cultivars and strains

Cultivar/Strain

Seed weight (mg/grain)

Seed skin color

Origin

Jack

216.6

Yellow

USA

Chapman

251.4

Yellow

USA

Sachiyutaka

270.7

Yellow

Japan

Tamahomare

363.6

Yellow

Japan

Chusei-Hikariguro

440.6

Black

Japan

Tokachikuro

442.9

Black

Japan

Tanbaguro

729.4

Black

Japan

Black seed coats contain anthocyanin, which is an effective antioxidant, in addition to soybean’s primary functional substance, isoflavone. Anthocyanin makes black soybean more valuable than regular soybean. Among many soybean varieties with black seed coats, Tanbaguro is also known for its excellent taste. These advantages make it highly valuable for food products, especially nimame (cooked beans) and edamame (boiled immature beans). While soybean is cultivated mainly as a forage or an oil crop in the world, it is an important food crop in Japan. Therefore the quality of the taste and the shape of the beans affects the price significantly. The market price of Tanbaguro seeds is three to six times higher than that of regular soybean seeds (Table 2).

Table 2. Market price of soybeans in Japan

 

Regular soybean

Tanbaguro

Price (Yen/250g)

200 ~ 500

600 ~ 1,200

Within several years, edamame of Tanbaguro has become popular in Japan although the price is almost ten times higher than regular edamame. The usage of this crop is expanding to breads, cakes, kinako (soybean powder), and beverages. They are usually more expensive than regular ones but are still getting more popular in Japan. Because of these properties, this soybean is expected as a good profitable crop in future.

Tanbaguro has been selected to bear bigger seeds by local breeders, however it has not improved with respect to adaptation to agricultural environments. It has many disadvantages, such as virus susceptibility, wide spreading plant habit, susceptibility to lodging, etc., so that a large amount of labor is required to cultivate this crop. Tanbaguro is considered to contain high genetic diversity because local farmers had maintained seeds by themselves traditionally until a few decades ago. Thus, there are several strains in a variety generally called “Tanbaguro”. In other words, this crop will be a good genetic resource. On the other hand, we will need genetically uniform plants for a systematic study of the physiology of Tanbaguro.

Soybean tissue culture or transformation is relatively difficult compared to other plants. Plant genotype of soybean is one of factors which have influenced plant regeneration (Komatsuda and Ohyama 1988; Parrott et al. 1989a; Bailey et al. 1993). Among hundreds of soybean cultivars, only several cultivars have been used for these experiments. “Jack” is one of the exemplary cultivars used for tissue culture and transformation studies (Stewart et al. 1996; Santarem et al. 1998; Yan et al. 2000). To our knowledge, there is no reports about successful tissue culture and regeneration of black seed coat soybeans. We report here that Tanbaguro can be a useful source for the study of soybean tissue culture.

Methods

Plant materials

Four varieties of soybean [Glycine max (L.) Merrill] were used to test the embryogenic capacity. “Hyokeikuro-3” and “Shin-tanbaguro” are strains of the black seed coat soybean, Tanbaguro, and two regular yellow cultivars, “Jack” and “Tamahomare”, were used as controls. Jack is a well-known model cultivar for tissue culture study and Tamahomare is a common commercial cultivar in Japan.

Culture conditions

Embryogenic cultures were initiated as described in Samoylov et al. (1998a). Immature zygotic embryos between 4 and 12 mm long were collected and were classified by length. The cotyledons were excised and the ends with the embryonic axes were removed. They were cultured on an induction medium (MSD40) at 25C, a light intensity of 10 μmolm-2s-1, and a 23-h photoperiod to induce embryogenesis.

Table 3. Media compositions

Medium

Composition

Induction
(MSD40)

MS macro salts, MS micro salts (Murashige and Skoog 1962),
B5 vitamins (Gamborg et al. 1968), 40 mg/L 2,4-D, 3% sucrose, 0.25% Gellan gum, pH 7.0

Proliferation
(MSD20)

MS macro salts, MS micro salts, B5 vitamins, 20 mg/L 2,4-D,
3% sucrose, 0.25% Gellan gum, pH 5.8

Maturation
(FNL0S3S3SGM)

FN Lite macro salts (Samoylov et al. 1998a), MS micro salts, B5 vitamins,
30mM glutamine, 2mM methionine, 3% sucrose, 3% sorbitol, pH 5.8

Conversion
(MS0)

MS macro salts, MS micro salts, B5 vitamins, 3% sucrose,
0.25% Gellan gum, pH 5.8

Explants that formed somatic embryos were counted after 4 weeks of culture. The somatic embryos were proliferated on a MSD20 medium for 3 weeks followed by maturation culture in a liquid FNL0S3S3GM medium on a rotary shaker (125 rpm) at 25C, a 16-h photoperiod, with a 2-week subculture period (Samoylov et al. 1998b). The maturated embryos were desiccated in an empty Petri dish for 3 – 5 days and transferred onto a conversion medium (MS0). The compositions of MSD40, MSD20, FNL0S3S3GM, and MS0 media are described in Table 3 (Parrott 2000).

Results and Discussion

Table 4 shows the regeneration rates of each cultivar/strain related to the length of the major axis of cotyledons. Rates of embryogenesis were calculated by the formula, (explants formed somatic embryos) / (total explants) x 100.

Table 4. Regeneration rates of each soybean cultivar/strain related to its cotyledon size.

Cultivar or Strain

Rate of embryogenesis (%)

Cotyledon length of the major axis (mm)

≤5

6

7

8

≥9

Jack

63.3

47.6

36.7

15.1

6.4

Tamahomare

3.3

10.7

11.4

12.5

6.9

Shin-tanbaguro

7.6

7.8

7.1

5.4

8.3

Hyokeikuro-3

70.3

59.6

59.8

32.9

9.1

We could not obtain efficient embryogenesis in Tamahomare. It is a typical Japanese cultivated soybean but does not appeared to be an appropriate material for tissue culture study. As expected, Jack showed a high embryogenic capacity especially at 5 mm and less of the cotyledon length. However, the best result was obtained from Hyokeikuro-3. The rates of embryogenesis were higher than Jack in all cotyledon lengths. Interestingly, Hyokeikuro-3 had the best embryogenesis at 5 mm and less of the cotyledon length like Jack.

In our experiments, the best rate of embryogenesis of Hyokeikuro-3 was 8.5 times higher than that of Shin-tanbaguro while there is no obvious difference between Shin-tanbaguro and Hyokeikuro-3 in their seed and plant appearances. We attempted to distinguish several strains of Tanbaguro by an SSR analysis using 34 microsatellite markers, but we have not found any differences between Shin-tanbaguro and Hyokeikuro (our unpublished data). Therefore we do not yet have appropriate explanations for the difference in their embryogenic capacities.

The formed somatic embryos of Hyokeikuro-3 were matured in a maturation medium to a cotyledonary stage. Currently, the cotyledonary embryos formed only roots after a desiccation process, and there is difficulty in converting them to adult plants (Figure 3). We are planning to modify the procedure to solve this problem.

Figure 3. Cotyledonary embryos of Hyokeikuro-3 after the desiccation process.

The bar indicates 1 cm.

Conclusion

“Hyokeikuro-3” is not only a tasty black soybean, but also could become a model genotype for tissue culture and transformation studies. Our micropropagation system can produce a huge amount of genetically uniform clones and will help to investigate the physiology of Tanbaguro.

References

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