The framework of plant regeneration in duckweed (Lemna turonifera) comprises genetic transcript regulation and cyclohexane release

Regeneration is important for vegetative propagation of excellent variety, detoxification and the obtain of transgenic plant, but plant regeneration is time-consuming. Here, we found that duckweed regeneration could be enhanced by regenerating callus. Firstly, Genetic transcript regulation has been applied to study the molecular mechanism controlling regeneration. Auxin related genes have been significantly down-regulated in regenerating callus. Cytokinin signal pathway genes have been up-regulated in regenerating callus. Secondly, volatile organic compounds release has been analysised by gas chromatography/mass spectrum during the stage of plant regeneration, and 11 kinds of unique volatile organic compounds in the regenerating callus were increased. Among them, cyclohexane treatment enhanced duckweed regeneration by initiating root. Moreover, Auxin signal pathway genes were down-regulated in callus treated by cyclohexane. All together, these results provide novel mechanistic insights into how regenerating callus promotes duckweed regeneration. Graphical abstract

reproduction, high protein content 5 , and distinguished tolerance for a variety of toxic 48 substances 6-7 , has been applied as a monocotylous modle plant for gene-expression 49 systems. In duckweed, stable transformation mediate by Agrobacterium depends on 50 efficient callus regeneration protocols. 51 Here, we use transcriptome sequencing technology to explore the molecular 52 mechanism of plant hormones regulating callus regeneration 8 . Nevertheless, there is 53 no study focus on the transcriptome analysis during the regeneration in duckweed. In 54 former studies, it has been reported that the growth and development of callus was 55 mediated by many plant hormones 5 . The balance of auxin and cytokinin is the basis 56 for vitro tissue culture 9 . Explants can be incubate to callus on auxin-rich 57 callus-inducing medium (CIM). And on cytokinin-rich shoot inducing medium (SIM), 58 the vigorous callus can be induce to novo shoots. It is emergent to study the 59 mechanism of duckweed regeneration via dynamic hormonal and transcriptional 60 changes. 61 The volatile organic compounds (VOCs) could be produced to defense against 62 herbivores, and it may also play a secondary role in attracting natural enemies, which 63 is allelopathy [10][11] . For example, the VOCs of Artemisia frigida Willd play an 64 allelopathic role on the seed germination of pasture grasses 12 . Does allelopathy play a 65 role during plant regeneration? Interestingly, we found the plant regeneration could be 66 promoted by regeneration callus. Why? The global insight on the signal and VOCs 67 released from regenerating callus needs to be investigated. 68 Here, the main objectives has been studied: (i) the molecular mechanism 69 controlling regeneration by comprehensive transcriptomic comparison between callus 70 and regenerating callus; (ii) which VOCs have been increased during the stage of 71 plant regeneration; (iii) the allelopathic effects of VOCs on the inducement of callus 72 regeneration; (iv) the transcriptome analysis on the regenerating callus which has 73 been promoted by VOCs.

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Promoted effect of regenerating tissue 76 Frond regeneration of duckweed has been promoted when co-cultured with 77 regenerating callus (Co). Frond formed in 14 d with Co treatment, and duckweed 78 regenerated at 21 days with with Co treatment (Fig 1a). In Co group, significant 79 enhancement was found in the percentage of callus regeneration (77.3 %). Compared 80 with that, the callus regeneration percentage without co-culture was 53.6% (Fig 1b). 81 Thus, the callus regeneration has been significantly increased by Co treatment. "cell" "cellpart" and "intracellular" were in biological process with the most 102 up-regulated and down-regulated DEGs. These were followed by "macromolecular 103 complex" and "organelle" in the category of biological process with the most 104 up-regulated and down regulated DEGs. "DNA integration", "pollination", and "cell 105 recognition" were up-regulated DEGs, without down-regulated (Fig.2).  Table 1). The peak area of 1, 3-dimethyl 147 benzene in the regenerating callus was 0.84*10 7 , 3.23 times than that in the callus. 148 And the emission of 1, 3-dimethyl benzene increased the most in the regenerating 149 callus. Besides, the content of 4-methyl-2-pentanol and cyclohexane also have been 150 improved. Compared with the cyclohexane peak area of the callus (0.85*10 7 ), the 151 cyclohexane peak area of the regenerating callus was 1.28*10 7 , 4.3*10 6 higher than 152 that of callus. And the peak area of 4-methyl-2-pentanol was 2.1*10 7 , 2.33 times than 153 that of callus. The numbers in blue represented the mass-to-charge ratio (m/z) of a substance in the 156 histogram.
157 Table 1 The main components of VOCs from regenerating callus and callus 158 Callus regeneration was promoted by cyclohexane 159 In order to explore the effect of VOCs in callus regeneration, 1, 3-dimethyl benzene,

Transcriptome analysis identifies KEGGs and DEGs in callus treated
168 by cyclohexane 169 Transcriptome analysis has been analyed to investigate the potential functions of cyclohexane. As shown in Fig. 7a, "RNA transport" and "glycolysis/gluconeoge, 172 and galaclose metabolism " were in the biological process with the most 173 down-regulated KEGGs. "Ribosome" was the the top-enriched pathway 174 (Richfactor>0.55). It was followed by "photosynthesis", and "oxidative 175 phosphorylation" (Fig. 7b). 176 In order to understand the difference of DEGs in callus treated with cyclohexane, 177 gene ontology enrichment analysis was conducted in callus treated by cyclohexane vs 178 callus. As shown in Fig. 7c, "DNA integration", "ribonucleoprotein complex" and 179 "structrural molecule activity" were in biological process with the most up-regulated 180 DEGs. These were followed by "ribosome biogenesis", "ribonucleoprotein complex" 181 and "ribosome" in the category of biological process with the most up-regulated 182 DEGs. "ribonucleoprotein complex" and "structrural molecule activity" were were in 183 biological process with the most down-regulated DEGs. (Fig. 7c). In order to know molecular factors underlying the participation of hormone in callus 189 regeneration, we first checked gene expression related to auxin signal pathway (Table   190 2). AUX/IAA and GH3 has been down regulated in both callus treated with 191 cyclohexane and in the regenerating callus. A majority of SAUR have been 192 down-regulated during regeneration and treated with cyclohexane (Fig. 8a). ERF3,193 cysteine-rich receptor and Zinc finger has been down-regulated as well. 194 Secondly, we studied the expression of genes related to CTK signal (Fig. 8b). 195 The gene regulation in regeneration and treated with cyclohexane is different. The 196 CRE1 has been up-regulated in the regenerating callus, and that has been   down-regulated in callus regeneration (Fig. 4) (Fig. 6). 257 Here, the regulation of gene expression related to hormone in callus treated with 258 cyclohexane, which promoted regeneration, suggested the role of auxin during 259 regeneration. AUX/IAA and GH3 has been down regulated in both callus treated with 260 cyclohexane, which is similar with that in the regenerating callus (Fig. 8). And 261 adventitious root initiation and enlongation has been promoted by AUX/IAA 8 . 262 Interestingly, the root formation has been enhanced significantly by cyclohexane 263 treatment (Fig. 6).  Fig. 9 System of co-culture and dynamic headspace air-circulation.

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The Co-culture of regenerating callus and callus 299 The callus was cultured on subculture medium for more than two weeks for 300 subsequent experiments. Callus and regenerating callus in the same growth condition 301 were placed in B5 medium (containing 1.5% sucrose) respectively. For fumigate, the 302 regenerating callus and callus were placed together in a closed environment for 303 co-culture described as Fig. 9a.

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VOCs Collection and analysis 305 Shown as Fig. 9b, the VOCs released from callus and regenerating duckweed were 306 collected using the dynamic headspace air-circulation method described by Zuo et al. 307 (2018) 38 . There were 3 conical flasks of callus or regenerating callus for each group. 308 The chemical composition analysis of VOCs was performed by thermal-desorption or low sequencing quality. The filtering contents were followed as our previous study: 324 Removed adapters; Removed reads whose proportion of N is greater than 10%; 325 Remove low-quality reads 6 . The clean reads were assembled by the trinity de novo 326 assembly program with min_kmer_cov set to 2 by default, otherwise it was set to 327 default 39 . Overall, a reference sequence, with an average length of 1928 bp and a total 328 length of 282527137 bp, was obtained for subsequent analysis.