‘Pink cotton candy’—A new dye‐free cotton

Abstract

White cotton is the dominant natural fibre, accounting for a $USD 36 billion share of the $USD 1.5 trillion global textile industry and, for many decades, has been dyed post-production. Modern cotton ginning and spinning processes require longer and stronger fibres, favouring superior white cotton varieties, which are more amenable to post-harvest dyeing (Gong et al., 2018; Vreeland, 1999). However, large quantities of synthetic dyes from textile dyeing released into the environment/waterways are harming the health of humans and other organisms (Manzoor and Sharma, 2020). Eco-friendly alternatives are urgently needed to reduce pollution and save water; coloured dye-free cotton could be a solution. While naturally coloured cotton has been known for more than 5000 years and occurs in all four species of cultivated cotton, that is Gossypium (G.) hirsutum, G. barbadense, G. herbaceum and G. arboretum (Murthy, 2001; Vreeland, 1999), these coloured varieties generally have low yield, poor fibre quality and variable and unstable colours (Rathinamoorthy and Parthiban, 2019; Vreeland, 1999). Although conventional breeding has improved the properties of some coloured cotton, quality and yield remain low compared with white cotton and colour range is limited (Murthy, 2001; Rathinamoorthy and Parthiban, 2019). Betalains are tyrosine-derived pigments found naturally in the order Caryophyllales of flowering plants, and fungi and bacteria (Timoneda et al., 2019). Betalains comprise two classes of compounds, that is yellow-orange betaxanthins and red-violet betacyanins. These compounds are synthesized through a series of enzymatic steps including hydroxylases, dioxygenases and glucosyl transferases that produce visible colours. As the Malvaceae typically do not produce betalains, we embarked on genetically engineering the betalain pathway in G. hirsutum, the world's largest plant-based fibre commodity. We designed constructs that included the coding sequences (CDS) of BvDODA1 (Beta vulgaris, GeneBank ID HQ656027.1), BvCYP76AD1 (HQ656023.1) and MjcDOPA5GT (Mirabilis jalapa, AB182643.1; Polturak et al., 2017; Timoneda et al., 2019; Figure 1a,b). The CDSs were optimized for Arabidopsis and synthesized by GeneArt (Table S1). Constructs (pAGM4723 vector) were assembled via Golden Gate cloning with a 2 × 35 S-driven kanamycin resistance gene. The betalain genes were driven by either a 2 × 35 S constitutive promoter or a ltp3/8K12 (LTP) mid-late-stage cotton fibre-specific promoter (Figure 1b, Table S1). Transgenic plants were generated through tissue culture transformation (Murray et al., 1999; Figure 1c). Coker 315-11 was used as the recipient of transformation by infection with Agrobacterium tumefaciens strain AGL1 containing different constructs. Transgenic and wild-type (controls) cotton plants were grown in a greenhouse at 28°C/20°C (day/night) with natural light. The pattern of betalain production differed strongly between these two promoters. Under the fibre-specific promoter, betalain pigments were successfully produced in cotton fibres and resulted in visibly pink fibres (Figure 1d–f). Plants were either green or partially purple (leaky expression in old tissues) and flowers resembled those of control plants at 0 days postanthesis (DPA; Figure S1). By contrast, the constitutive promoter-driven construct generated purple/violet plants with purple leaves, stems, boll coats and bracts (Figure S1). The flowers were pink at opening (0 DPA) while wild type had green leaves with creamy yellow flowers (Figure S1). However, constitutive promoter-driven constructs generated white fibres throughout development, similar to wild type, although ovules and mature seed interior were pink/purple (Figure S1). Despite strong betalain accumulation during fibre development in the fibre-specific lines, the colour faded to light brown/pink in the final days of boll maturation, when the bolls dried and opened (transgenic lines: 55–60 DPA vs wild type: 55 DPA; Figures 1e, S1). This suggests that vacuole-located betalain (Figure S2) was degraded during the final maturation stage. Cotton bolls were collected at around 10, 15, 20, 25, 30, 40, 50 and 60 DPA, and boll coats were either cut open or removed entirely followed by 48 h of freeze-drying, and the pink colour was retained (Figures 1f, S1). Mature fibre or freeze-dried immature fibre (>46 DPA) from controls and five independent T0 plants with the fibre-specific betalain expression were measured by Cottonscope for fibre quality. The results suggested the transgenic lines have the potential to present similar maturity ratio and fineness as the wild type (Line 44, Figure 1g). The seed numbers and fibre yield were less in the T0 compared with wild type, which is common to see in T0 generation (Figure S3). Subsequent generations of transgenic plants could potentially retain wild-type-like yield and fibre quality alongside strong pigment accumulation, noting that Coker 315 can be introgressed into modern elite varieties for yield and quality. New colours may be generated by crossing the betalain lines with existing naturally coloured genotypes (Ke et al., 2022). In conclusion, we present a novel example of introducing the exogenous multi-gene betalain pathway to generate a plant-made pink cotton fibre which remains pink until the very late stages of fibre development. Using the betalain pathway is advantageous because the pigment is stable over a wide pH range (Jackman and Smith, 1996), potentially improving colour stability and consistency. Future research could investigate reducing pigment degradation (potentially via cross-linking) and introducing new colours. We thank Lissette Perez and Dina Yulia for technical support, Drs Danny Llewellyn and Phil Hands for helpful advice and support, and the CSIRO Synthetic Traits Group for Golden Gate vectors and support. The authors declare no conflict of interest. This research was funded by the CSIRO SynBio Future Science Platform. MM, VR, CM and FP contributed to the conceptualization. MM, VR, CM, FP and XL contributed to the methodology. XL contributed to the formal analysis. SA, MM, XL, VR, CM and FP contributed to the investigation. XL contributed to the writing—original draft. XL, MM, CM, FP and VR contributed to the writing—review and editing. CM, FP and VR contributed to the supervision. FP, MM, CM and XL contributed to the project administration. FP and CM contributed to funding acquisition. The data are available in the article and accompanying supplementary material. Table S1 Betalain-pathway gene coding sequences (CDS) and LTP promoter. Figure S1 Images: cotton plants, seeds, mature bolls, freeze-dried immature bolls, stem sections and T1 plants. Figure S2 Betalain fluorescence. Figure S3 Yield of T0. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

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