Agrobacterium-Mediated Cassava Transformation For The Asian-elite Variety KU50

Agrobacterium-mediated cassava transformation via friable embryogenic calli (FEC) has allowed the robust production of transgenic cassava. So far, transformation has been performed mostly for the model cassava variety 60444 and African varieties due to their good production of, and regeneration from, embryogenic tissues. It is important to develop transformation methods for elite Asian cassava varieties to meet changing needs within one of the world’s major cassava production areas. To date, however, a suitable transformation method for the Asian elite variety Kasetsart 50 (KU50) has not been developed. Here, we report a transformation method for KU50, the cultivar with the highest planting area in Thailand and Vietnam. In cassava transformation, the preparation of FEC as the target tissue for transgene integration is a key step. FEC induction from KU50 was improved by the use of media with reduced nutrients and excess vitamin B1, and somatic embryo and plant regeneration optimized by manipulation of naphthalene acetic acid (NAA) and benzylamino purine (BAP). The transformation eciency for KU50 was 22%, at approximately half of that of 60444 (45%). Transcriptome analysis indicated that expression of genes related to cell wall loosing was upregulated in FEC from KU50 compared with 60444, indicating cell wall production and assembly was out of balance in the Asian variety. The transformation system for KU50 reported here will contribute to molecular breeding of cassava plants for Asian farmers by transgenic and genome-editing technologies. This is the rst report of cassava transformation for Asian elite variety using friable embryogenic calli. hygromycin and 200 mg/L carbenicillin and cultured for one week. A third selection was subsequently performed on FIM supplemented with 20 mg/L hygromycin and 200 mg/L carbenicillin for one week. Regeneration of cotyledon-stage embryos was achieved by transferring FEC colonies to SE emerging medium (MSN) consisting of MS salts supplemented with 1 mg/L NAA, 100 mg/L carbenicillin and 20 mg/L hygromycin and cultured for a maximum of four months. Cotyledon-stage embryos were selected and subcultured onto cassava elongation medium (CEM) composed of MS medium amended with 0.4 mg/L BAP, 100 mg/L carbenicillin and 20 mg/L hygromycin. Embryos were cultured on CEM until shoot and roots were produced. Regenerated plantlets were maintained on MS media supplemented within 50 mg/L of carbenicillin and 5 mg/L of hygromycin. 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.


Introduction
Cassava (Manihot esculenta Crantz) is grown across the tropics and sub-tropical regions for production of its starchy tuberous roots. It provides an important source of daily calories for 100s of millions for people and plays a critical role in global food security and rural economies (Balat and Balat 2009). The acceleration of cassava breeding is desirable to meet demands of the increasing population and evolving market needs (Malik et al. 2020). However, conventional breeding based on sexual hybridization is challenging in cassava due to its high degree of genetic heterozygosity, inbreeding depression, asynchronous owering, self-incompatibility, and low fruit set in some varieties (Ceballos et  In this study, we report the establishment of Agrobacterium-mediated transformation for the Asian elite cassava variety, Kasetsart 50 (KU50). Currently, KU50 is cultivated in Thailand, Vietnam, Indonesia, Cambodia, Myanmar, and the Philippines. This variety has been cultivated in more than 1 million hectares of Thailand and Vietnam alone (Malik et al. 2020).

Plant materials and growth conditions
Cassava cultivars Kasetsart 50 (KU50) and 60444 were obtained from the in vitro germplasm collection of the International Center for Tropical Agriculture (CIAT), Cali, Colombia. In vitro plantlets were maintained by sub-culturing microcuttings every 4-8 weeks on Murashige and Skoog basal salts (Murashige and Skoog 1962) (MS) supplemented with 20 g/L sucrose, 2 µM CuSO 4 , MS vitamins and 3 g/L gelrite. Media was adjusted to pH 5.8 using 1M KOH before autoclaving for 20 min at 121•C (Supplementary table 1). Plantlets were cultured in plastic vessels containing 100 mL media and incubated at 28 • C under a 16h light/8h dark photoperiod at 40 µmol m -2 s -1 intensity.
Axillary buds (AB) produced from nodal stems of in vitro plantlets of KU50 and 60444 were used as explants for the induction of organized embryogenic calli (OEC). In order to induce the formation of the AB, all leaves were removed from the in vitro plantlets (8-10 cm in size) and 20-30 mm stem sections carrying single nodes excised. The single-nodal-stem fragments were placed horizontally on cassava axillary medium (CAM) consisting of MS medium supplemented with 10 mg/L BAP (Bull et al. 2009) and incubated for 4-7 days at 28•C in the dark. Swollen ABs were excised from the stem sections using a sterile syringe, and incubated on callus induction medium (CIM) consisting of MS medium supplemented with 12 mg/L of picloram for 2 weeks at 28•C in the dark. The number of AB that formed the OEC was counted at two weeks after culturing on CIM. When multiple OEC were observed from one axillary bud, number of OEC induced was counted as one. The OEC produced were excised and subcultured onto media containing Driver and Kuniyuki Walnut basal salts (DKW) (Driver and Kuniyuki 1984), supplemented with 20 g/l sucrose, MS vitamins and 12 mg/L picloram (Chauhan et al. 2015 on FIM for 2-3 weeks at 28°C in the dark. Fine FEC as isolated from non-embryogenic tissue using ne tweezers and transferred to fresh FIM. The isolated FEC were stringently selected in this manner every 2-3 weeks for a maximum of six months to produce homogenous colonies of FEC. The number of AB that formed the FEC was estimated at six months after culturing on FIM. When multiple FEC were induced from OEC induced from one bud, number of FEC induced was counted as one.

Agrobacterium -mediated transformation and plant regeneration
The pMDC111 binary plasmid vector (Curtis and Grossnikaus 2003) was used for transformation of KU50 and 60444 FEC tissues. The mGFP6 reporter gene (Born and Pfeifer. 2019) in pMDC111 was under control of the enhanced 35S promoter. A DNA sequence of enhanced 35S promoter was obtained by PCR using the pCAMBIA1300 as the template using the primer pair: (Forward; 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCT ATGGTGGAGCACGACACTCTCG-3') and (Reverse; 5'-GGGGACCACTTTGTACAAGAAAGCTGGGT GGAGAGATAGATTTGTAGAGAG-3'). A DNA fragment of enhanced 35S promoter was cloned into the pDONR/Zeo vector by the BP clonase reaction (ThermoFisher). A DNA fragment in the pDONR/Zeo was ligated into the pMDC111 vector by the LR clonase reaction (ThermoFisher). pMDC111 vector with enhanced 35S promoter::mGFP6 was electroporated into Agrobacterium tumefaciens strain LBA4404 (Hood et al. 1986) and transformation of FEC was performed as described by Utsumi et al.

Microscopic observation
The observation of the GFP uorescence was performed on the rst to third leaves from top of plantlets maintained in MS media and on roots of plants grown in soil. Imaging was done with the M165 FC uorescence microscope (Leica, Wetzlar, Germany) equipped with a cooled CCD camera VB-7010 (Keyence, Osaka, Japan), 10x eyepiece, plan apo 2x Corr. objective or objective planapo 0.63x M series (Leica, Wetzlar, Germany). Images were taken using illumination with excitation laser line 486 nm, with emission lter 518 nm.

Detection of GFP and HPT gene
The rst or second youngest, healthy leaves of in vitro plants were used for genomic DNA preparation. Leaf samples were disrupted using a ShakeMaster and zirconia beads (Hirata Corporation), and genomic DNA extracted according to the protocol of Wizard Magnetic 96 DNA Plant System (Promega) using BIOMEK (Beckman Coulter). Genomic DNA was stored at -80ºC until further use.
Total RNA extraction from FEC samples RNA was extracted from homogenous FEC tissues of KU50 and 60444. Total RNA was extracted from at least 100 mg FW (fresh weight) per biological replicate as described by Utsumi et al. (2017) and then stored at − 80 °С until use.
Oligo-microarray analysis of gene expression Total RNA was used to evaluate gene expression levels with a cassava DNA oligo-microarray comprising more than 30,000 probes as described by Utsumi et al. (2016). Gene expression data represent four independent biological replicates per FEC from KU50 and 60444. A total of eight microarray data sets were analyzed with GeneSpring GX (Agilent Technologies, USA). Speci cally, the data underwent an analysis of Student's t-test with a Benjamini-Hochberg (BH) false discovery rate (FDR) to identify differentially expressed genes (DEGs) among two data sets. The oligo-DNA microarray data were deposited in the NCBI Gene Expression Omnibus (GEO) database and are accessible via a GEO Series accession number GSE169685.
Southern blot analysis to determine T-DNA copy number Genomic DNA was prepared by the CTAB method (Doyle and Doyle 1987). Homogenized leaf samples were prepared by cryogenically grinding tissue in a multibead shaker after chilling in liquid nitrogen. One gram of homogenized tissue was incubated in 6 ml of 2×CTAB {2% CTAB, 100 mM Tris-HCl (pH 8.0), 1.4 M NaCl, 20 mM EDTA} at 55°C for 60 minutes. After centrifuging the homogenate for 20 min at 2,800 x g, the supernatant was transferred to a new tube and gently mixed in 1 volume of chloroform/isoamyl alcohol (24:1) for 20 min at room temperature, followed by centrifugation for 20 min at 2,800 x g to separate the phases. The aqueous upper phase was transferred to a new tube and mixed in 0.1 volume of 10% CTAB solution (10% CTAB, 0.7 M NaCl) and 1 volume of chloroform/isoamyl alcohol (24:1) and incubated for 10 min at room temperature. The aqueous upper phase was collected by centrifugation for 30 min at 2,800 x g. The DNA was precipitated by adding 1 volume of CTAB precipitation buffer {1% CTAB, 50 mM Tris-HCl (pH 8.0), 10 mM EDTA} to the aqueous upper phase collected and incubated at room temperature for overnight. The DNA was collected by centrifugation at 5,000 x g for 10 minutes and dissolved in 100 µl TE buffer {10 mM Tris-HCl (pH 8.0), 1 mM EDTA}. Three microlitres of RNase solution A (10 mg/mL) was added to the DNA solution and incubated at 37°C for 30 min. Southern blot analysis was carried out according to the standard protocol (Southern 2006). Five micrograms of genomic DNA was incubated for 20 hours with 30 units of HindIII, which cuts the T-DNA once, and subjected to electrophoresis on a 0.8 % agarose gel. The agarose gel was soaked in depurination solution (0.2 N HCl) for 10 minutes at room temperature and denatured in denaturation solution (1.5M NaCl, 0.5M NaOH) for 30 min. The agarose gel was then soaked in neutralization solution {1.5M NaCl, 0.5M Tris-HCl (pH 7.5)} for 30 min. The fractionated DNA in agarose gel was transferred to a positively charged nylon membrane using capillary blotting in 20 x SSC {(3M NaCl in 0.3M sodium citrate (pH 7.0)}. The hybridization probe speci c to hpt of T-DNA was DIG-dUTP-labeled using a PCR DIG probe synthesis kit (Roche Diagnostics GmbH, Germany) and used for the hybridization experiment according to the manufacturer's instructions.

Statistical Analyses
All data except for transcriptome data are represented as means ± SD from at least three biological experiments. Statistical analysis of means was assessed by analysis of variance using StatPlus 5 pro (AnalystSoft Inc. USA). Data for the optimization experiments underwent a one-way ANOVA and differences among means were analyzed by Scheffe's method at a 95% con dence level (p ≤ 0.05) with StatPlus 5 pro (AnalystSoft Inc. USA) (Bother 1967).

Optimization of OEC and FEC induction, and shoot regeneration in KU50
We compared the induction e ciency 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 e ciency 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 e ciency of FEC induction from OEC was assessed after 6 months culture on FIM. FEC induction was less e cient 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 e ciency 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 (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 signi cant 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 ne tweezers with OEC from one bud arranged as one group on the FIM containing 12 mg/L picloram to assess e ciency 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 su cient 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 e ciency 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 uorescence signal due carrying substitutions of ten amino acids from the soluble-modi ed GFP (smGFP) (Born and Pfeifer. 2019). GFP uorescence 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 di cult to select GFP expressing tissues from transgenic FEC due to auto uorescence from FEC at the early stage after the co-culture with Agrobacterium. Therefore, we evaluated transformation e ciency by con rming GFP orescence in leaf veins and stems of regenerated plants ( Table 2). The transformation e ciency (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 con rmed by PCR and Southern blotting analyses (Figs. 2n, o, p). Southern blotting con rmed 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 con rmed in plants growing in soil. GFP uorescence 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 e ciency 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 identi ed 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 classi ed with agriGO. The GO terms of "cellular component ( Supplementary Fig. 1 legend), "molecular function" and "biological process" were signi cantly 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 Table 3 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 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-speci c 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.

and Supplementary
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.

Discussion
In this study, we developed a system for Agrobacterium-mediated transformation of the Asian elite variety KU50. The use of picloram and limited nutrient media in addition to excess vitamin B1 resulted in increased induction of FEC, while optimization of BAP and NAA improved somatic embryo regeneration from FEC and germination to produce transgenic plantlets. This is the rst report for establishment of Agrobacterium-mediated cassava transformation system via FEC for KU50.
Many factors affect genetic transformation e ciency in cassava. Effective induction of FEC is crucial as this acts as effective target tissue for transgene integration (Bull et al. 2011;Chavarriaga-Aguirre et al. 2016;Taylor et al. 2012). Successful induction of SE, the rst step in FEC production, in Asian cassava varieties has been reported (Ntui et al. 2015;Saelim et al. 2009), so was not a limiting factor for transformation for KU50. FEC of KU50 was induced during extended culture on FIM. However, the induction e ciency of FEC was 5 times lower in KU50 compared with the model variety 60444 (Table 1). We hypothesis that this may be due to imbalance between cell wall production and assembly in KU50 FEC, because the genes related to cell wall loosening process were found to be up-regulated in KU50 FEC (Table 3 and Supplementary Table 3). Cassava transformation has been reported in most cases using FEC induced by culturing on GD media containing picloram (Bull et al. 2009;Taylor et al. 2012;Utsumi et al. 2017), with FEC formation observed after 2 month-incubation on media with picloram. In KU50 FEC, production takes at least 4 months cultivation on FIM media. Further technical improvement is therefore necessary to shorten this time and reduce the potential frequency of somaclonal variation resulting from longer culture durations. In this report, we determined the optimal NAA concentration for regeneration from FEC to cotyledon-stage embryos and for subsequent shoot formation on medium containing BAP. The optimal concentration of NAA and BAP was 1.0 mg/L (Fig. 1c) and 0.4 mg/L (Fig. 1f), respectively, in a manner similar to results of transformation using 60444 (Bull et al. 2009).
The transformation e ciency of 60444 and KU50 was 45% and 22%, respectively. An average 26 transgenic lines per one gram of FEC from 60444 were obtained, whereas 33 transgenic lines per one gram of FEC from KU50 were obtained within 12 months after Agrobacterium inoculation ( Table 2) The development of a genetic transformation method for KU50 described here will contribute to improvement of key characteristics in this very important Asian cassava cultivar. The use of novel cutting-edge technologies such as CRISPR-Cas9 can now be applied and contribute to molecular breeding (Altpetera et al. 2016). For example the introduction of the Flowering Locus T gene has been demonstrated to be successful for shortening the breeding time, generation of the transgene-free progenies and production of the amylose-free cassava starch, which is a useful trait for the food and industrial applications of cassava (Bull et al. 2018;Malik et al. 2020). This approach now becomes feasible for KU50 also. Such approaches will contribute to expanding cassava diversity towards food security, commodity diversity, and sustainability for global demands Declarations Data availability Novel data generated in this study, including microarray data, have been deposited in the National Center for Biotechnology Information under the accession number GSE169685.