Induction and phenotype analysis of autotetraploids of Morus Mongolica

Abstract

Background: Morus mongolica holds significant value in medicinal applications, livestock fodder production, and ecological restoration. Although polyploidization is well-documented in plants for enhancing stress tolerance and altering nutrient composition, the specific beneficial traits conferred by genome duplication in M. mongolica have not yet been fully characterized.

Results: Our results demonstrated that a 72 h preculture followed by 72 h of colchicine treatment (30 mg·L^-¹), yielded a tetraploid rate of 25%. Significant differences were observed between diploid and tetraploid plants in plant height, basal diameter, leaf area, and chlorophyll content. Furthermore, tetraploids exhibited significantly higher levels of total sugars, Ca, Se, total phenols, and flavonoids, compared to diploid M. mongolica. Also, tetraploid plants are better equipped to endure extended periods of drought stress, ultimately resulting in improved survival rates, through earlier drought perception and more finely tuned physiological regulation.

Conclusion: This study established an effective tetraploid induction protocol for M. mongolica, characterized the beneficial traits of the induced tetraploids, and thereby lays the foundation for tetraploid induction and breeding.

Keywords: Drought tolerance; Morphological characteristics; Morus mongolica; Nutrient composition; Tetraploid.

Fig. 1

The complete process of the regeneration system from stem of M. mongolica in vitro. (A) Selection and treatment of M. mongolica explants; (B) Primary culture of explants; (C) Axillary buds elongation and callus differentiation; (D) Axillary buds proliferation; (E) Adventitious roots induction; (F) Rooted plantlets. Bar = 0.5 cm

Fig. 2

The ploidy levels assessments based on flow cytometry and chromosome counting of colchicine-treated and non-treated M. mongolica plantlets. Flow cytometry analysis of tetraploid (A) and diploid (B) plantlets; Chromosome counting slides of tetraploid (C, 2n = 4x = 56) and diploid (D, 2n = 2x = 28). Bar = 5㎛

Fig. 3

The comparison between tetraploid (T) and diploid (D) on the seedlings morphologies grown in the greenhouse (A), the morphology of the first unfolded leaves (B), the morphology of the second (C), the morphology of the third leaf (D), plant hight (E), stem diameter (F), lesf area (G) and chlorophyll content (H) of M. mongolica

Fig. 4

Analysis of nutrient composition in diploid and tetraploid M. mongolica leaves. (A) Crude protein content (CP). (B) Crude fiber content (CF). (C) Total sugar content (TS). (D) Total phenol content (TP). (E) Total flavonoid content (TF). (F) Calcium content (Ca). (G) Selenium content (Se)

Fig. 5

Morphological changes of seedlings (A) and roots (B) in diploid and autotetraploid M. mongolica under drought-stress. Bar = 1.0 cm

Fig. 6

Morphological differences of plant height (A), fresh weight per-plant (B), shoot fresh weight (C), root fresh weight (D) and seeding mortality rate (E) in diploid and tetraploid M. mongolica plants under control and drought conditions. Significant difference analysis used two-way ANOVA followed by Duncan’s multiple range test. The vertical bars show the standard error

Fig. 7

Physiological differences of relative electrical conductivity (REC) (A), malondialdehyde (MDA) (B), proline (Pro) (C), soluble sugar (SS) (D), superoxidase (SOD) (E), catalase (CAT) (F), superoxide anion (O₂⁻) (G), and hydrogen peroxide (H₂O₂) (H) in diploid and tetraploid M. mongolica plants under control and drought conditions