Establishment of embryogenic Pinus thunbergii Parl. suspension cultures: growth parameters, dynamic analysis, and plant regenerative capacities

Abstract

Background: Pinus thunbergii is an economically important conifer species that plays a fundamental role in forest ecosystems. However, the population has declined dramatically in recent years as a result of the pine wilt disease outbreak. Thus, developing pine wilt-resistant P. thunbergii is an effective strategy for combating this epidemic.

Results: The somatic embryogenesis of nematode-resistant P. thunbergii was previously reported by our group. The current study looked into the potential commercialization of suspension cultures as a means of large-scale production of nematode-resistant P. thunbergii seedlings. According to our findings, P. thunbergii suspension cultures were suitable for an initial inoculum of embryogenic tissue (2 g) and a subculture inoculum (6.7% (v/v)). Suspension cultures were cultivated for 8-10 days in a 30 mL liquid medium (Gupta and Durzan medium, DCR medium) to facilitate their maturation. The suspension cultures produced a large number of high-quality somatic embryos, which were then used to regenerate the plants and move them into the field. A more accurate assessment of the quality of suspension cultures for somatic embryogenesis could come from the suspension's dynamics. The results showed that the medium's phosphate, ammonium, nitrate, and carbohydrates were quickly eaten from day 0 to day 10. In terms of the absorption of nitrogen sources, the ammonium (NH₄⁺) was absorbed prior to nitrate (NO₃^-). Additionally, the activity of mitochondrial succinate dehydrogenase and superoxide dismutase was directly related to cell growth.

Conclusions: This study presents an approach for selecting appropriate suspension cultures for efficient somatic maturation of P. thunbergii that can also be applied to other conifers. Furthermore, it is possible to commercialize nematode-resistant P. thunbergii seedlings using bioreactors, according to the suspension culture system we describe. To the best of our knowledge, this is the first work to describe a P. thunbergii suspension culture.

Keywords: Pinus thunbergii; Enzyme activity; Mass propagation; Nutrient consumption; Somatic embryogenesis.

Fig. 1

Suspension cells proliferation of P. thunbergii. The performance of suspension cells (1539-1) cultured for seven days with an inoculum content of 3.3% (A), 6.7% (B), 10% (C) and 16.7% (D). The performance of proliferation rate in genotypes (1539-1, 1536-1 and 2237-1) and subculture inoculum (E). Data represent the mean ± SD of nine replicates. Capital and lowercase letters represent the significant differences (P < 0.05) of genotype and inoculum amount on the proliferation rate by Duncan’s test, respectively

Fig. 2

Proliferation (A) and activities (B) of P. thunbergii suspension cultures (cell line 1539-1). Various uppercase and lowercase letters indicate significant differences (P < 0.05) between treatments over time based on Duncan’s test. Data represent the mean ± SD of three replicates. FW indicates fresh weight. DW indicates dry weight

Fig. 3

Structures of suspension cultures in lag phase (A) exponential phase (B), and aging phase (C) of P. thunbergii. Bars A-C 200 μm

Fig. 4

Dynamics of P. thunbergii suspensions (cell line 1539-1). Consumption of carbohydrate (A) ammonium and nitrate (B) and phosphate (C). pH changes (D). Various uppercase and lowercase letters indicate significant differences (P < 0.05) between treatments over time based on Duncan’s test. Data represent the mean ± SD of three replicates. I-VI indicates significant differences (P < 0.05) between treatments (fructose) over time based on Duncan’s test

Fig. 5

Changes of intracellular enzyme activities in cell line 1539-1. (A) Activities of CAT and POD. (B) Activities of SOD. (C) The correlation analysis between SOD and phosphate. Various uppercase and lowercase letters indicate significant differences (P < 0.05) between treatments over time based on Duncan’s test. Data represent the mean ± SD of three replicates

Fig. 6

The evaluation indications for other genotypes in the established suspension culture system. A-H indicate cell line1536-1; I-P indicate cell line 2237-1. (A, I) The proliferation or growth of embryogenic cells in the suspension culture system. FW represents fresh weight. The acronym DW stands for dry weight. (B, J) Cell activity varies in a suspension culture system. (C, K) Variations in the intracellular CAT and POD enzyme activity. (D, L) Variations in intracellular SOD enzyme activity. (E, M) Ammonium and nitrate consumption. (F, N) The consumption of phosphate. (G, O) Consumption of carbohydrates. And (H, P) pH varies in liquid medium. The data show the mean ± SD

Fig. 7

Somatic embryos (SE) maturation and plant regeneration of nematode-resistant P. thunbergii. Variations in the genotype of mature somatic embryos produced by cell mass and suspension cells (A). Maturation of somatic embryos from suspension cells (B) and cell mass (C). Germinated somatic embryos on semisolid medium (D and E). Enhancement of regenerated plants in sterile culture chamber (F). The regenerated plants were transplanted to the field (G) and the tolerance of individuals were screened for PWN (H, I). Data represent the mean ± SD. * indicates P < 0.05, *** indicates P < 0.001, **** indicates P < 0.0001

Fig. 8

Mass propagation of nematode-resistant P. thunbergii plantlets by suspension culture