Anthocyanin accumulation enhanced in Lc-transgenic cotton under light and increased resistance to bollworm

Abstract

Breeding of naturally colored cotton fiber has been hampered by the limited germplasm, an alternative way is to use transgenic approach to create more germplasm for breeding. Here, we report our effort to engineer anthocyanin production in cotton. The maize Lc gene, under the control of the constitutive 35S promoter, was introduced into cotton through genetic transformation. Our data showed that the expression of the Lc gene alone is sufficient to trigger the accumulation of anthocyanin in a variety of cell types including fiber cells in cotton. However, the accumulation of colored anthocyanin in cotton fibers requires the participation of light signaling. These data indicate that it is feasible to engineer colored fibers through transgenic approach in cotton. Furthermore, we showed that the Lc-transgenic cotton plants are resistant to cotton bollworm. These transgenic plants are, therefore, potentially useful for cotton breeding against cotton bollworm.

Keywords: Anthocyanin; Cotton fiber; Gossypium hirsutum; Lc gene.

Fig. 1

Southern blot analysis of the Lc-transgenic cotton plants. Genomic DNA from T0 transgenic (a) and T1 transgenic plants (b) was digested by BamHI and fractionated using gel electrophoresis. The blot was probed with a DIG-labeled Lc probe. a Lanes 1–4 and 6–7 represent six independent transgenic lines as indicated. Lane 5 is a nontransgenic control (Wt). b Lanes 1–5 represent five T1 siblings from L143 showing Lc hybridization bands

Fig. 2

RT-PCR analysis of Lc expression patterns in transgenic cotton plants. a Upper panel Lc expression level in the indicated vegetative and reproductive tissues; lower panel Actin was used as internal control indicating no DNA contamination in the samples. b Lc expression in ovules and fibers at the indicated stages. Actin was used as an internal control

Fig. 3

Phenotypic characterization of Lc-transgenic cotton. a Red transgenic calluses and somatic embryos (bar 2.5 mm). b Non-transgenic calluses (bar 2.5 mm). c Transgenic purple colored leaf, indicating anthocyanin accumulation. d Non-transgenic leaf. e Anthocyanin accumulation on stem (red patches). f Transgenic flower bud with a red sepal. g Transgenic flower with a red anther. h T1 siblings showing the segregation of Lc-dependent anthocyanin accumulation in roots. r1 and r2 are positive seedlings with red roots; r3 and r4 are negative seedlings with white roots. i Transgenic immature ovules cultured under light for 1 week (bar 2 mm). Note red fiber cells. j Magnification of J showing a single red fiber cell (bar 10 μm). Note red oily liquid gathered in the central big vacuole. k Transgenic immature ovules cultured in completely dark for 1 week (bar 2 mm). l Transgenic leaf abaxial surface toward the sun; its freehand cross section with red palisade mesophyll cells. The epidermal cells are colorless and transparent (bar 1 mm). m Anthocyanin extract from transgenic dry leaves (e1), transgenic fresh leaves (e2), Wt leaves (e3), and empty control (e4)

Fig. 4

Anthocyanin content revealed by UV spectrometric analysis. Relative fractions of monomeric anthocyanin, polymeric anthocyanin (condensed tannins) and other compounds of the total anthocyanin calculated using the bisulfite leaching method. Values represent averages ± STEV, and every bar represents an average value from three repeated measurements

Fig. 5

Bioassay for cotton bollworm feeding on Lc and Wt leaves. a Leaf images after 2 days of feeding. b1 and c1 are Lc-transgenic leaves, a1 is a Wt leaf. The arrow points to worm (bar  2.2 cm). b Leaf images after 4 days of feeding. b2 and c2 are Lc-transgenic leaves, a2 is a Wt leaf (bar 2.2 cm). c Image showing larvae after feeding 5 days on Lc-transgenic red leaves. The arrows point to worms (bar 2 cm). d Image showing larvae after feeding 5 days on Wt leaves. The arrows point to worms (bar 1.8 cm)

Fig. 6

Statistical analysis of the larval weights after 3 days of rearing on Lc-transgenic and Wt leaves from Fig. 5b values are from 13 samples and represent averages ± STEV. Every bar represents the average weight repeated three times (n = 13)