In vitro induction of tetraploids and their phenotypic and transcriptome analysis in Glehnia littoralis

Abstract

Background: Glehnia littoralis is a medicinal and edible plant species having commercial value and has several hundred years of cultivation history. Polyploid breeding is one of the most important and fastest ways to generate novel varieties. To obtain tetraploids of G. littoralis in vitro, colchicine treatment was given to the seeds and then were screened based on morphology, flow cytometry, and root tip pressing assays. Furthermore, transcriptome analysis was performed to identity the differentially expressed genes associated with phenotypic changes in tetraploid G. littoralis.

Results: The results showed that 0.05% (w/v) colchicine treatment for 48 h was effective in inducing tetraploids in G. littoralis. The tetraploid G. littoralis (2n = 4x = 44) was superior in leaf area, leaf thickness, petiole diameter, SPAD value (Chl SPAD), stomatal size, epidermal tissues thickness, palisade tissues thickness, and spongy tissues thickness to the diploid ones, while the stomatal density of tetraploids was significantly lower. Transcriptome sequencing revealed, a total of 1336 differentially expressed genes (DEGs) between tetraploids and diploids. Chromosome doubling may lead to DNA content change and gene dosage effect, which directly affects changes in quantitative traits, with changes such as increased chlorophyll content, larger stomata and thicker tissue of leaves. Several up-regulated DEGs were found related to growth and development in tetraploid G. littoralis such as CKI, PPDK, hisD and MDP1. KEGG pathway enrichment analyses showed that most of DEGs were enriched in metabolic pathways.

Conclusions: This is the first report of the successful induction of tetraploids in G. littoralis. The information presented in this study facilitate breeding programs and molecular breeding of G. littoralis varieties.

Keywords: Glehnia littoralis; Leaf; Phenotype; Tetraploid; Transcriptome.

Fig. 1

The induction of polyploidy G. littoralis from germinated seeds. (A) The survival rate of the seeds of G. littoralis on the proliferation medium. (B) Adventitious shoots regenerated from seeds after culture for 30 days (control). (C) Adventitious shoots regenerated from colchicine-treated seeds after culture for 30 days (T1). (D) Adventitious shoots regenerated from seeds after culture for 60 days (control). (E) Adventitious shoots regenerated from colchicine-treated seeds after culture for 60 days (T1). *p < 0.05, **p < 0.01. Note CK: control; T1: 0.05% (w/v) colchicine solution soaked seeds for 48 h; T2: 0.1% (w/v) colchicine solution soaked seeds for 24 h; T3: 0.15% (w/v) colchicine solution soaked seeds for 16 h

Fig. 2

Graded screening of tissue culture plantlets based on morphological characteristics. (A) diploid tissue culture plantlets. (B) first-grade tissue culture plantlets. (C) second-grade tissue culture plantlets. (D) third-grade tissue culture plantlets

Fig. 3

Determination of the ploidy of G. littoralis by flow cytometry. (A) diploid, (B) tetraploid, (C) chimera, and (D) chimera

Fig. 4

Shoots rooted after one month of culture on the rooting medium. (A) diploid, (B) tetraploid. (C) The number of roots after one month rooting culture. Verification of the ploidy of G. littoralis by root tip squash method. (D) diploid, (E) tetraploid. (F) The number of survival rate of plantlets transferred to the substrate after one month

Fig. 5

Comparison of leaf morphological traits between diploid and tetraploid. (A) diploid, (B) tetraploid. Growth of plantlets after a two-month transplantation. (C) diploid, (D) tetraploid. (E) comparison of the morphological characteristics of the leaves of diploid and tetraploid. *p < 0.05, **p < 0.01

Fig. 6

Stomata on leaf abaxial side recorded at 400x magnification. (A) diploid, (B) tetraploid. Guard cells in the lower epidermis of leaves and chloroplasts in guard cells. (C) diploid, (D) tetraploid. (E) comparison of the stomatal characteristics of the lower epidermis of diploid and tetraploid leaves. Sw: Stomatal width, Sl: Stomatal length, Pw: Pore width, Pl: Pore length. *p < 0.05, **p < 0.01

Fig. 7

Leaf anatomical characteristics of diploid and tetraploid G. littoralis. (A) diploid, (B) tetraploid. (C) comparison of anatomical characteristics of diploid and tetraploid leaves. *p < 0.05, **p < 0.01

Fig. 8

The number of gene annotation and differential expressed gene. (A) unigenes database statistics, (B) volcano map of differentially expressed genes in diploid (GL2) and tetraploid (GL4)

Fig. 9

GO and KEGG pathway enrichment analyses of DEGs between diploids and tetraploids. (A) GO enrichment analysis with the top 10 of each category. (B) KEGG enrichment analysis with the 30 most enriched KEGG pathways. Copyright permission has been granted for related KEGG images

Fig. 10

Expression analysis of the DEGs related to growth and development between diploid (GL2) and tetraploid (GL4) G. littoralis leaves

Fig. 11

qRT-PCR of 16 DEGs in diploid and tetraploid G. littoralis leaves. *p < 0.05, **p < 0.01