Optimization of OEC and FEC induction, and shoot regeneration in KU50
We compared the induction efficiency for OEC and FEC from KU50 and 60444 determined per the number of starting axillary bud explants as described by Utsumi et al. (2017) (Fig. 1). OEC were induced from axillary buds (AB) of KU50 within 2 weeks culture on MS medium supplemented with 12 mg/L picloram (CIM). The morphology of OEC produced from KU50 (Fig. 2c) was similar to that seen from 60444 (Fig. 2a). The OEC induction efficiency from AB after 2 weeks on CIM ranged from 47-67% with an average of 60% in KU50, which was slightly lower than that of 60444 which averaged 71% (Table 1). The efficiency of FEC induction from OEC was assessed after 6 months culture on FIM. FEC induction was less efficient at 12% in KU50, compared to 50% for 60444 (Table 1). The morphology of FEC generated from KU50 was similar to that produced by 60444 (Fig. 2b, d), but took at least 3 months to produce, compared to only one month from 60444.
Although the basic procedures of cassava transformation employed in the present study were similar to that previously reported, optimization of regeneration conditions was found to be necessary for KU50. We investigated the optimal concentrations of NAA and BAP necessary for stimulating regeneration of cotyledon-stage embryos from FEC and for germination of mature embryos, respectively. Six colonies of FEC from KU50 were cultivated on MS media supplemented with various concentrations of NAA and subcultured every two weeks as shown in Fig. 1a. Cotyledon-stage embryos were formed (Fig. 1b), with the optimal concentration of NAA found to be 1.0 mg/L (Fig. 1c). The cotyledon-stage embryos produced were incubated on MS media supplemented with various concentrations of BAP and evaluated for the shoot regeneration efficiency over a 2-month period (Fig. 1d, e). The optimal concentration of BAP was found to be 0.4 mg/L (Fig. 1f), being similar to that reported for 60444 (Bull et al. 2009; Taylor et al. 2012).
Agrobacterium-mediated transformation for KU50
Agrobacterium-mediated transformation of KU50 followed the process reported for 60444 (Utsumi et al. 2017). To perform the transformation experiments, approximately 100 axillary buds were prepared from approximately 30 two month old in vitro plants. Stems were placed on MS medium containing 10 mg/L BAP to induce swelling of the axillary buds, followed by excision and culture on MS basal medium containing picloram. (Utsumi et al. 2017). OEC production occurred at about 74% and 64% per total number of starting axillary buds for 60444 and KU50, respectively (Table 1). OEC produced from axillary buds was incubated on DKW media with 12 mg/L of picloram for the growth during 2 weeks. In this step, no significant differences in the growth and OEC formation were observed between 60444 and KU50. OEC (2–3 mm in diameter) was manipulated under a microscope using fine tweezers with OEC from one bud arranged as one group on the FIM containing 12 mg/L picloram to assess efficiency of FEC induction.
FEC can be generated effectively by culturing primary OEC on FIM containing 12 mg/L picloram. First observation of FEC after culturing on FIM media with picloram was at 4 weeks and 12 weeks in 60444 and KU50, respectively. In KU50, about 1.2 g of FEC was generated from 100 axillary buds by sequential culturing on FIM for 9 weeks, whereas about 5.4 g of FEC was generated from 100 axillary buds in 60444 ovr the same period. The type and appearance of FEC generated from KU50 was similar to that from 60444. However, the lower frequency of production, longer culture period required and lesser amount of FEC produced indicates the lower potential for FEC production in KU590 in comparison to the highly amenable variety 60444 (Table 1).
Cassava transformation was carried out using the pMDC111 vector which carried the visual marker including mgfp6 gene. FEC was collected and co-cultured with Agrobacterium suspension (OD600 nm, 0.1). The OD of Agrobacterium suspension did not affect the copy number of T-DNA in transgenic plants or the transformation process (Taylor et al. 2012). To prevent re-growth of Agrobacterium, careful manipulation of washing process was required. The transformed FEC was evenly spread over FIM with hygromycin and carbenicillin. In KU50, selection on FIM with hygromycin for 3 weeks was not sufficient for full selection of the transgenic calli. Therefore, FEC was spread evenly over MSN medium containing hygromycin and NAA, and subcultured every 21 days to induce formation of cotyledon-stage embryos. Table 2 shows the efficiency of cotyledon-stage embryo production and plantlet formation per 1 g fresh weight of starting FEC (normalized for the amount of starting tissues used for Agrobacterium co-cultivation). Cotyledon-stage embryos appeared about one month after culturing on MSN with hygromycin and continued over a period of 3 months. Fifty-eight and 151 cotyledon-stage embryos were generated for 60444 and KU50 respectively. Cotyledon-stage embryos were transferred to CEM containing hygromycin and BAP for the induction of shoots. Shoot formation was observed within 1 month at the earlies, with plantlets formed from 22% of the cotyledon-stage embryos for KU50 (Table 2).
In this study, mGFP6 was used as the selection marker. mGFP6 generates a stronger fluorescence signal due carrying substitutions of ten amino acids from the soluble-modified GFP (smGFP) (Born and Pfeifer. 2019). GFP fluorescence was observed in the process of selection on FIM and plantlet-inducing stage on CEM (Figs 2e and 2f). However, it was found to be difficult to select GFP expressing tissues from transgenic FEC due to autofluorescence from FEC at the early stage after the co-culture with Agrobacterium. Therefore, we evaluated transformation efficiency by confirming GFP florescence in leaf veins and stems of regenerated plants (Table 2). The transformation efficiency (calculated as the number of GFP-positive plantlets per number of cotyledon-stage embryos regenerated from 1 g of FEC) was 45% and 22% for 60444 and KU50, respectively (Table 2). In addition to GFP expression, the transgenic nature of regenerated plants was confirmed by PCR and Southern blotting analyses (Figs. 2n, o, p). Southern blotting confirmed integration of single copies of the T-DNA in 60444 and KU50 transgenic plant lines.
Acclimatizing in vitro cassava plantlets can result in high mortality, due often to damage of the fragile roots. We therefore reduced the agar concentration in MS micropropagation media from 2.0% to 0.5% (w/v) prior to transfer to soil. Using this method, plants with well-developed root systems were transferred from the culture vessel to soil pot without root damage and kept under high humidity. GFP signal was confirmed in plants growing in soil. GFP fluorescence was observed in leaf veins and roots of the transgenic lines (Figs 2j and 2l), with no such signals seen for non-transgenic control plants (Figs 2g and 2h).
Lower FEC induction efficiency in KU50 might be due to excess cell-wall loosing and increased stress response compared with 60444
To compare the physiological status between FEC produced by KU50 and 60444, gene expression analysis was performed by oligo-microarray, using total RNA extracted from these tissues. Using the criteria of FDR ≦ 0.00005 by BH method and a two-fold change in expression, 2,213 differentially expressed genes (DEGs) were identified by comparing 60444 and KU50. Among these, 1,287 genes were up-regulated and 926 genes down-regulated in KU50 compared with 60444 (Supplementary Table 2). The DEGs annotated based on the Arabidopsis thaliana genome sequence were functionally classified with agriGO. The GO terms of “cellular component (Supplementary Fig. 1 legend), “molecular function” and “biological process” were significantly enriched (Supplementary Figure 1-5 and Supplementary Table 3-6).
In the “cellular component”, the genes with GO terms including “GO:0005618 Cell wall”, “GO:0044459 Plasma membrane part” and “GO:0005576 Extracellular region” were enriched among the upregulated genes in FEC from KU50 (Supplementary Fig. 1 and Supplementary Table 3). The following cell wall modification-related genes existed: pectinesterase (PE) which is involved in dimethyl esterification of pectins (Willats et al. 2001), polygalacturonase (Park et al. 2015) that modifies cell wall structure, xyloglucan endotransglucosylase (XTH) which encodes a cell wall-modifying enzyme and expansin which plays an important role in plant cell growth where cell wall loosening occurs (Li et al. 2002). Within “molecular function”, the genes with GO terms including “GO:0035251 UDP-glucosyltransferase activity” were upregulated in FEC from KU50 (Supplementary Fig. 2), suggesting that increased stress response through upregulation of several UDP-glucosyltransferases (Rehman et al. 2018) might occur in FEC from KU50 (Supplementary Table 4). The GO terms enriched in the up-regulated genes in KU50 had “response to abscisic acid” (GO:0009737), “response to osmotic stress” (GO:0006970), “response to oxidative stress” (GO:0006979) and “response to wounding” (GO:0009611) (Supplementary Fig. 3) in the “biological process”. The expression of the following genes was increased in KU50 compared with 60444: P5CS2 (AT2G17840) which is involved in proline biosynthesis (Funck et al. 2020), ADC2 (AT4G34710), involved in the first step of polyamine biosynthesis (Urano et al. 2004), ZAT10 (AT1G27730) involved in photooxidative stress response, ERD7 (AT2G17840) involved in remodelling cell membrane lipid composition during cold stress (Barajas-Lopez et al. 2020), chitinase A (AT5G24090) whose expression is induced by various abiotic and biotic stress conditions (Takenaka et al. 2009), SAG14 (AT5G20230) which encodes a glycosylphosphatidylinositol-anchored protein that regulates lignin biosynthesis (Ji et al. 2015), and CYP94B1 (AT5G63450) which is involved in apoplastic barrier formation through suberin biosynthesis (Krishnamurthy et al. 2020) (Supplementary Table 5).
We also compared the genes upregulated in KU50 FEC with genes up- or down-regulated in 60444 FEC in comparison with somatic embryos (SE) (Ma et al. 2015) and the genes upregulated in 60444 grown under FIM (Utsumi et al. 2017) (Table 3). The expression of several upregulated genes in KU50 FEC included PPCK1 and PPCK2 which plays a key role in control of plant metabolism by phosphorylation of phosphoenolpyruvate carboxylase, XTH16 (AT3G23730) which is involved in loosening of cell wall structure, and non-specific phospholipase C4 (NPC4)(AT3G03530 which is important for supply of inorganic phosphate. Finally, diacylglycerol from membrane-localized phospholipids (Peters et al. 2010) was also up-regulated in 60444 grown on CIM with reduced nutrients and excess vitamin B1 (Utsumi et al. 2017) (Table 3). Several upregulated genes in KU50 FEC, such as acetyl-CoA synthetase were also upregulated in fresh 60444 FEC compared with SE (Ma et al. 2015). There were no differences in KU50 FEC with the genes involved in callus induction or repression (Ikeuchi et al. 2013) (Supplementary Table 7) and the DEGs involved in the cell cycle related process (Supplementary Table 8) was not observed.
Although growth of KU50 FEC is likely to be stimulated by the use of media containing auxin, the expression of genes related to abiotic stress responses and loosening the cell wall was upregulated in KU50 in comparison with 60444. It is possible therefore that FEC growth in KU50 might be inhibited by excess cell wall-loosening process in comparison with that of 60444, and result in the observed increased expression of stress response-related genes.