The Feeder Effects of Cultured Rice Cells on the Early Development of Rice Zygotes

Abstract

Feeder cells and the synthetic auxin 2,4-dichlorophenoxyacetic acid (2,4-D) in a culture medium promote mitosis and cell division in cultured cells. These are also added to nutrient medium for the cultivation of highly active in mitosis and dividing zygotes, produced in vitro or isolated from pollinated ovaries. In the study, an in vitro fertilization (IVF) system was used to study the precise effects of feeder cells and 2,4-D on the growth and development of rice (Oryza sativa L.) zygote. The elimination of 2,4-D from the culture medium did not affect the early developmental profiles of the zygotes, but decreased the division rates of multicellular embryos. The omission of feeder cells resulted in defective karyogamy, fusion between male and female nuclei, and the subsequent first division of the cultured zygotes. The culture of zygotes in a conditioned medium corrected developmental disorders. Proteome analyses of the conditioned medium revealed the presence of abundant hydrolases possibly released from the feeder cells. Exogenously applied α-amylase ameliorated karyogamy and promoted zygote development. It is suggested that hydrolytic enzymes, including α-amylase, released from feeder cells may be involved in the progression of zygotic development.

Keywords: 2,4-D; amylase; cell wall; feeder cell; fertilization; hydrolytic enzyme; rice; zygote; zygotic development.

Figure 1

Developmental profiles of zygotes cultured in N6Z medium (A) and 2,4-D free N6Z medium (B,C). (A) Developmental profiles of the zygotes cultured in N6Z medium. After completion of karyogamy in zygote (Aa,Ab), the zygote developed into a two-celled embryo (Ac,Ad), multicellular embryo (Ae,Af), a globular-like embryo (Ag,Ah), and cell mass (Ai–An). (B) Developmental profiles of the zygotes cultured in 2,4-D free N6Z medium. After completion of karyogamy in zygote (Ba,Bb), the zygote developed into a two-celled embryo (Bc,Bd), multicellular embryo (Be,Bf), a globular-like embryo (Bg,Bh), and cell mass (Bi–Bn). (C) Developmentally arrested embryos cultured in 2,4-D free N6Z medium. Upper and lower panels are fluorescent and bright-field images, respectively. Bars, 20 µm.

Figure 2

Developmental profiles of zygotes cultured in feeder cell free N6Z medium. Zygotes cultured in feeder-cell free N6Z medium (A–C) and in feeder cell and 2,4-D free N6Z medium (D–F) were observed 1 day after fusion. Left and right panels are fluorescent and bright-field images, respectively. Bars, 20 µm.

Figure 3

Developmental profiles of the zygotes cultured in conditioned medium. (A) After completion of karyogamy in zygote (Aa–Ac), the zygote developed into a two-celled embryo (Ad–Af), multicellular embryo (Ag–Ai), a globular-like embryo (Aj–Ao), and cell mass (Ap–Ar). Bars, 20 µm (Aa–Ao), 100 µm (Ap–Ar). (B) The zygote arrested development. The zygote developed into a multicellular embryo (Ba–Bc) 3 days after fusion and arrested development thereafter (Bd–Bi). Upper, lower, and middle panels are fluorescent, bright-field, and merged images, respectively. Bars, 20 µm.

Figure 4

Developmental profiles of the zygotes cultured in conditioned medium without heat treatment (A–C) and in heat-treated conditioned medium (D). After completion of karyogamy in zygote cultured in non-treated conditioned medium (Aa–Ac), the zygote developed into a two-celled embryos (Bc–Bc) and multicellular embryos (Ca–Cc). In case of zygotes cultured in heat-treated conditioned medium, zygotes were degenerated after completion of karyogamy (Da–Dc). Left, right, and middle panels are fluorescent, bright-field, and merged images, respectively. Bars, 20 µm.

Figure 5

Effect of exogeneously applied α-amylase on the development of rice zygotes. Zygotes were cultured in 2,4-D free N6Z medium containing α-amylase at the concentration of 0.17 U/mL (A,B), 1.7 U/mL (C,D), and 17 U/mL (E,F). Left, right, and middle panels are fluorescent, bright-field, and merged images, respectively. Bars, 20 µm.