Over-Expression of the Cell-Cycle Gene LaCDKB1;2 Promotes Cell Proliferation and the Formation of Normal Cotyledonary Embryos during Larix kaempferi Somatic Embryogenesis

Abstract

Somatic embryogenesis is an effective tool for the production of forest tree seedlings with desirable characteristics; however, the low initiation frequency and productivity of high-quality mature somatic embryos are still limiting factors for Larix kaempferi (Japanese larch). Here, we analyzed the expression pattern of L. kaempferi cyclin-dependent kinase B 1;2 (LaCDKB1;2) during somatic embryogenesis in L. kaempferi and its relationship with the cell proliferation rate. We also analyzed the effect of LaCDKB1;2 over-expression on somatic embryo quality. The results revealed a positive correlation between LaCDKB1;2 expression and the cell proliferation rate during the proliferation stage. After LaCDKB1;2 over-expression, the proliferation rate of cultures increased, and the number of somatic embryos in transgenic cultures was 2.69 times that in non-transformed cultures. Notably, the number of normal cotyledonary embryos in transgenic cultures was 3 times that in non-transformed cultures, indicating that LaCDKB1;2 not only increases the proliferation of cultures and the number of somatic embryos but also improves the quality of somatic embryos. These results provide insight into the regulatory mechanisms of somatic embryogenesis as well as new Larix breeding material.

Keywords: 2; LaCDKB1; Larix kaempferi; cell-cycle gene; somatic embryogenesis.

Figure 1

Structures and conserved domains of the LaCDKB1;2 protein. e: α-helixes; h: β-strands (extended strands); c: random coils. Red line: ATP-binding region signature [V(10)GEGTYGKV(18)]; blue line: PPTALRE motif; green line: serine/threonine protein kinase acting-site signature [V(131)MHRDLKPHNLLV(143)]. AA: amino-acid sequence; Pred: prediction of secondary structures.

Figure 2

Phylogenetic analysis of LaCDKB1;2. The tree was constructed with 17 amino acid sequences of CDKs using the neighbor-joining method with MEGA6.0. Values at each node are bootstrap support percentages from 1000 replications. Species and accession numbers: L. keampferi (LaCDKB1;1, MW132640; LaCDKB1;2, MW132641; LaCDKB1;3, MW132642; LaCDKA1, MW132639), A. thaliana (AtCDKB1;1, AT3g54180; AtCDKB1;2, AT2g38620; AtCDKB2;1, AT1g76540; AtCDKB2;2, AT1g20930; AtCDKA1, AT3g48750; AtCDKE1, AT5g63610; AtCDKC2, AT5g64960; AtCDKC1, AT5g10270; AtCDKD2, AT1g66750; AtCDKD3, AT1g18040; AtCDKD1, AT1g73690; AtCDKF1, AT4g28980), and P. trichocarpa (PtCDKB1;2, U5FL09).

Figure 3

Subcellular localization of the LaCDKB1;2 protein. GFP: the distribution pattern of the LaCDKB1;2-GFP fusion protein; RFP: the distribution pattern of the AtPYR1-RFP fusion protein used as a positive marker; Bright: bright-field; Merged: merged image of LaCDKB1;2-GFP and bright-field. Scale bar: 10 µm.

Figure 4

Expression pattern of LaCDKB1;2 during L. kaempferi somatic embryogenesis. Culture line S287, grown in a proliferation medium for 2 or 15 days and in a maturation medium for 4, 7, 14, 21, 28, 35, or 42 days, was harvested for qRT-PCR, with LaEF1A1 used as the internal control. Data are presented as the mean ± SE.

Figure 5

Expression patterns of LaCDKB1;2 during ABA treatment of L. kaempferi cultures. Cultures with (+ABA) or without (−ABA) ABA treatment were harvested at 0, 3, 6, 9, 12, or 48 h for qRT-PCR, with LaEF1A1 used as the internal control. Data are presented as the mean ± SE.

Figure 6

Proliferation rates of six L. kaempferi culture lines and expression pattern of LaCDKB1;2. (A) Proliferation rates of six culture lines (H20, W13, X46, H10, S287, and X58), which are equal to the weights of increased cultures grown on a solid proliferation medium for 20 days divided by the weight on day 1. (B) Relative expression level of LaCDKB1;2 assayed by qRT-PCR with LaEF1A1 as the internal control. Data are presented as the mean ± SE.

Figure 7

Generation and characterization of L. kaempferi transgenic (LaCDKB1;2 over-expression) cultures. (A) Several clumps of hygromycin-resistant S287 cultures (indicated by circles) were obtained in a solid proliferation medium with 400 mg/L cefotaxime and 5 mg/L hygromycin. (B) One clump of hygromycin-resistant S287 culture (indicated by a five-pointed star) survived and was proliferated in a solid proliferation medium with 5 mg/L hygromycin. (C) PCR amplification of the DNA fragment of GFP. M: 2000 DNA marker; ck+: positive control; ck–: negative control. (D) Expression of GFP assayed by Western blot, with actin as the internal control. (E) Expression of LaCDKB1;2 assayed by qRT-PCR, with LaEF1A1 as the internal control. (F) Cell proliferation rate in non-transformed and transgenic cultures. Data are presented as the mean ± SE. p-values for the expression of LaCDKB1;2 and the proliferation rate were generated to compare non-transformed and transgenic S287 cultures.

Figure 8

Characterization of L. kaempferi transgenic (LaCDKB1;2 over-expression) cultures. (A) Non-transformed and LaCDKB1;2 overexpression S287 cultures of the same weight (0.5 g) were cultured on a solid maturation medium in a plate for 42 days and then used to analyze the morphology (B) and number (C) of somatic embryos. Data are presented as the total number of somatic embryos generated from 10 plates of culture.

Figure 9

The statistical analysis of somatic embryos derived from L. kaempferi transgenic (LaCDKB1;2 over-expression) cultures. (A) The numbers and photographs of four types of somatic embryo. (B) Statistical analysis of four types of somatic embryo. Non-transformed and LaCDKB1;2 over-expression S287 cultures of the same weight (0.5 g) were cultured on a solid maturation medium for 42 days in a plate and then used to analyze the morphology and number of somatic embryos. Data are presented as the total numbers of the four types of somatic embryo generated from 10 plates of culture. N: number of cotyledons.