Expression of cytokinin responsive and ethylene biosynthesis genes in rice callus with different regeneration rates

This study aimed to investigate the correlation between callus regeneration rate and the expression of several genes responsible for cytokinin response and ethylene biosynthesis in the Ciherang, Mentik Wangi Susu, Hwayoung and Tarabas rice varieties. The callus regeneration rate of each rice variety was in vitro tested using N6 media, while the gene expression during the callus regeneration stages was examined using quantitative real‐time PCR (qRT‐PCR). Our results showed that the callus of Ciherang and Mentik Wangi Susu showed earlier green spot formation that then turned brown at a later stage, resulting in a low regeneration rate. While Hwayoung and Tarabas showed late green spot formation, high shoot regeneration was observed in both calluses. Gene expression analysis of regeneration media‐grown calluses showed that two cytokinin‐responsive genes, OsRR2 and OsRR6 , were highly expressed in the Ciherang and Hwayoung callus, respectively. We also observed that ethylene biosynthesis genes such as OsACS1 and OsACO1 were highly expressed in the Mentik Wangi Susu and Hwayoung callus, respectively. Moreover, the expression of OsBBM1 was high in Hwayoung and Tarabas. Thus, the positive correlation between the expression of cytokinin‐responsive and ethylene biosynthesis genes with somatic embryogenesis activity likely depends on the induction level of OsBBM1 .


Introduction
Rice is a staple food for more than 3.5 billion people that provide at least 20% of their daily calorie intake (Fraiture et al. 2016). As rice demand increases during the current global climate change, increasing rice productivity has be come a formidable challenge. Hence, applying biotechno logical tools becomes an effective approach to face such challenging problems. Tissue culture is a biotechnolog ical tool that has been recognized as a powerful method for in vitro plant propagation (Pais 2019). Tissue culture can be used in various biotechnological applications such as genetic transformation, synthetic seed production, cry opreservation, and assembling plants with superior geno typic properties to increase yields and be more productive (Martínez et al. 2019).
The initial stage of somatic embryogenesis in rice tis sue culture requires the addition of exogenous auxin to in duce and proliferate callus cells to some extent of growth level (JuárezGonzález et al. 2019). In regeneration media containing high cytokinin: auxin ratio, the initial stage of callus differentiation is visibly marked by the formation of green spots that are gradually transformed into shoots (Vercruyssen et al. 2015). The success of shoot regener ation in rice tissue culture is tightly correlated with the genotype of the explant source (Pais 2019). Rice sub species japonica generally has a high frequency of callus to shoot regeneration, whereas the regeneration frequency of indica subspecies tends to be low due to the occurrence of callus browning during the regeneration stage (Yang et al. 2020; Tran and SananMishra 2015; Han et al. 2021).
It has been identified that several genes belonging to the response regulators (RR) gene family are the key point for cytokinin signaling in plants. The typeB RRs are tran scription factors for regulating cytokinin primaryresponse gene expression, whereas the typeA of RRs are negative regulators that desensitize the plant to cytokinin (Kieber and Schaller 2014). In rice, OsRR21, OsRR22, OsRR23, and OsRR24 have been identified as the most abundant typeB RRs, whereas OsRR1, OsRR2, OsRR3, OsRR5, and OsRR6 are categorized into the typeA of RRs (Duan et al. 2019; Yamburenko et al. 2020. In rice cytokinin insensitive gnt1 mutant that was deficient in tiller bud for mation and failure in calli differentiation, the expression of OsRR2 and OsRR6 were found to be downregulated (Fanata et al. 2013).
In many studies, ethylene is another type of plant hor mone that determines the success of shoot regeneration in rice tissue culture. The positive role of ethylene was illus trated by a significant increase in shoot regeneration rate in indica variety FR13A callus by the addition of ethy lene synthesis inhibitor aminoethoxy vinyl glycine (AVG) into regeneration media, whereas the addition of ethylene precursor 1aminocyclopropane1carboxylic acid (ACC) decreased shoot regeneration (Yasmin et al. 2013). More over, the negative role of ethylene in rice tissue culture has been illustrated in experimental results that the addi tion of putrescine as another type of ethylene biosynthesis inhibitor increases shoot regeneration in rice anther culture (Arisandi et al. 2020; Dewi andPurwoko 2008). The con version of methionine initiates the biosynthesis of ethylene to Sadenosyl methionine (SAM/AdoMet), which is sub sequently converted into ACC by ACC synthases (ACS). At the last step of biosynthesis, ACC is further converted to ethylene by ACC oxidase (ACO), a member of the oxy genase superfamily (Wen 2015).
BABY BOOM (BBM) is another essential transcrip tion factor encoding genes that play a crucial role in so matic embryogenesis by regulating cell totipotency, pro liferation, and regeneration ability (Jha and Kumar 2018). In Arabidopsis thaliana, BBM1 is responsible for the up regulation of hundreds of morphogenesisrelated genes that collaborate to produce somatic embryos, including LEC, SERK, and YUCCA (Khanday et al. 2019). The most recent experiment showed that the somatic embryo genesis was induced in rice callus abundant in OsBBM1 transcripts, and its shoot regeneration was completed if cytokinin was supplemented into the regeneration media (Khanday et al. 2020).
We previously reported that the Tarabas rice vari ety showed high somatic embryogenesis capacity with an equal shoot regeneration rate with the japonica vari ety Hwayoung (Fanata and Qudsiyah 2020). However, the underlying molecular mechanism that supports a high level of somatic embryogenesis in those two rice varieties has not been described. In this study, we characterized the molecular response of Tarabas and Hwayoung callus as well as Ciherang and Mentik Wangi Susu as the low somatic embryogenesis counterpart by analyzing the ex pression of cytokinin responsive, ethylene biosynthesis, and somatic embryogenesis related genes. We found that the expression of cytokinin responsive genes OsRR2 and OsRR6 were high in Ciherang and Hwayoung callus, re spectively, and the ethylene biosynthesis genes OsACS1 and OsACO1 were highly expressed in Mentik Wangi Susu and Hwayoung, respectively. Moreover, the high expres sion of genes related to the somatic embryogenesis Os BBM1 in Hwayoung and Tarabas callus suggests the posi tive role of cytokinin responsive and ethylene biosynthesis genes in the rice somatic embryogenesis might depend on the high expression ofOsBBM1.

Measurement of Chlorophyll Content
Total chlorophyll content was measured based on (Jiang et al. 2017) with a brief modification. Two hundred mil ligrams of CIM grown 2weekold and SIM grown 3 weekold callus were transferred into 2 mL microtube con taining 1.5 mL of absolute ethanol. The mixtures were then shaken using a vortex and incubated in dark condi tions for 24 hours. Absorbance readings were performed using a microplate reader (Corona SH1000) at 649 and 665 nm wavelengths. Total chlorophyll was calculated us ing equation as follow: Chlorophyll a = 13.95 × A 665 6.88 × A 649 Chlorophyll b = 24.94 × A 649 7.32 × A 665 Total Chlorophyll = Chlorophyll a + Chlorophyll b. The results were expressed as mg of chlorophyll g 1 fresh weight.

RNA Total Extraction and Quantitative RT-PCR
Total RNA was extracted from CIM and SIM grown callus using RNAprep pure kit (Tiangen) following the manu facturer's instruction. One microgram of total RNA was used for cDNA synthesis using a Revertra Ace qPCR RT Kit (Toyobo). Quantitative RTPCR was performed using the CFX96TM RealTime PCR Detection System (BioRad) and SsoFastTM Evagreen® Supermix (Bio Rad) qRTPCR mixture under the following conditions: initial denaturation at 94°C for 2 min followed by 40 cy cles of 94°C for 15 s, 60°C for 30 s, and 70°C for 1 min. Relative expression of OsRR2, OsRR6, OsACS1, Os ACO1, and OsBBM1 genes were normalized to OsActin1 as a reference gene.

Callus Morphology
The morphology of callus that was grown in CIM and SIM was analyzed using a stereomicroscope (Leica EZ4HD). The analyzed morphological parameters were callus struc ture, callus color, green spot, and shoot formation.

Statistical analysis
The somatic embryogenesis experiment data (n=4) were statistically analyzed using Analysis of Variance (ANOVA) with Duncan's Multiple Range Test (DMRT) for the posthoc test using Microsoft Excel software version 2019 (Microsoft, USA). The mean values were considered statistically significant if p ≤ 0.05. The gene expression results were statistically analyzed using an unpaired Student ttest.

Callus Induction Rate
Rice mature seed has been known as an excellent explant for callus induction. Using CIM media containing 2 mg/L 2,4D, we observed that callus were efficiently formed from the mature seeds scutellar tissue of Ciherang, Mentik Wangi Susu, Hwayoung, and Tarabas ( Figure 1a). The mi croscopic observation of the twoweekold callus showed that the structure of each variety was compact with yellow ish color and a dry appearance. Among the four rice va rieties, Tarabas and Hwayoung produced the highest rate of callus formation (91% and 90%, respectively), which was significantly higher (p<0.05) compared to Ciherang (and Mentik Wangi Susu (85.6% and 85%, respectively) ( Figure 1b).

Green Spot Formation
Green spot formation in callus has been considered as one of the indicators of the success of shoot regeneration in rice and wheat tissue culture (Amer andBorner 1997; Mostafiz andWagiran 2018). In our result experiment, callus of four rice varieties was grown in SIM supplemented with 2 mg/L Kinetin, 2 mg/L BAP, 0.5 mg/L NAA, and the green spot formation were observed after one and twoweeks incu bation. Mentik Wangi Susu showed the fastest green spot formation among the four varieties, as most of the callus clumps started to show greenish color in the first week of incubation (Figure 2a). At two weeks of incubation, lev els of green spot formation in Mentik Wangi Susu and Ci herang callus were 95% and 90%, respectively, which was significantly higher (p < 0.05) than Hwayoung and Tarabas (70% and 67.5%, respectively).
The results of green spot formation level were further confirmed by the analysis of chlorophyll content of three weeks SIM incubated callus (Figure 2b), which showed that the callus of Mentik Wangi Susu produced signif icantly the highest total chlorophyll (p < 0.05) (11.17 mg.g 1 FW) compared to Ciherang (8.50 mg.g 1 FW), Tarabas (6.24 mg.g 1 FW), and Hwayoung (2.61 mg.g 1 FW). These results suggested that the callus of Mentik Wangi Susu showed the fastest initial response against cy tokinin treatment.

Shoot Regeneration
The shoot regeneration rate was analyzed at five weeks of incubation in SIM. The japonica rice Hwayoung and Tarabas callus showed the highest regeneration rate (100% and 85%, respectively), which was significantly different (p < 0.05) from Mentik Wangi Susu (17.5%), and Ciherang  (Figure 3a). The low regeneration rate of Mentik Wangi Susu and Ciherang callus was correlated to the highlevel callus browning of both varieties (82.5% and 100%, respectively) compared to 0% and 15% browning levels of Hwayoung and Tarabas, respectively (Figure 3b). Moreover, Hwayoung callus produced an average of 9.25 shoots number per clump, which was significantly high est (p < 0.05) compared to Tarabas (3.62 shoots), Mentik Wangi Susu (2 shoots), and Ciherang (no shoot formation) (Figure 3c). Although the shot number of Tarabas cal lus was lower compared to Hwayoung, the average shoot length of Tarabas was 6.75 cm which was significantly dif ferent (p < 0.05) compared to Hwayoung (1.97 cm), Men tik Wangi Susu (1.34 cm), Ciherang (no shoot formation). These results indicated that Hwayoung and Tarabas have a high regeneration rate despite the two varieties showing the late emergence of green spots.

Expression of cytokinin responsive, ethylene biosynthesis, and somatic embryogenesis genes
To deeply understand the molecular response for the dif ferent regeneration rates of four rice varieties, we further investigated the cytokinin signal perception and ethylene production in CIM and SIM grown callus by gene expres sion approach. We analyzed the expression of OsRR2 and OsRR6 to represent cytokinin responsive genes, OsACS1 and OsACO1 for ethylene biosynthesis genes, and Os BBM1 for the somatic embryogenesisrelated genes. Us ing qPCR analysis, the five analyzed genes generally were upregulated in callus subcultured in SIM media ( Figure  4). The expression of OsRR2 was 3folds induced in Ci herang callus after one week of subculture onto SIM, and the expression level was maintained until two weeks of subculture. We also observed that OsRR6 was found to be upregulated (7 folds) in the callus of Hwayoung after one week of subculture onto SIM, although the level was decreased after two weeks of subculture. The pronounced inductions of OsRR6 were also shown by Ciherang, Men tik Wangi Susu, and Tarabas callus, but the level was lesser than Hwayoung. The analysis of ethylene biosynthesis genes showed that OsACS1 expression was 3.8folds induced in the cal lus of Mentik Wangi Susu after one week of subculture in SIM, and the level was maintained at two weeks of sub culture. The OsACS1 basal expression level of Mentik Wangi Susu callus was twofold higher than other vari eties, indicating that the synthesis of ethylene precursor might be increased during callus formation and differenti ation. Moreover, OsACO1 expression was 2.25folds in duced in Hwayoung callus after one week of subculture onto SIM and slightly decreased at two weeks of subcul ture. The OsACO1 expression was apparently induced in Mentik Wangi Susu and Tarabas, but the expression level remained unchanged in Ciherang callus.
The expression of OsBBM1 was five and twofold in duced in Hwayoung and Tarabas, respectively, but the lev els were decreased at two weeks of SIM subculture. In terestingly, OsBBM1 expression level was reduced in Ci herang and unchanged in Mentik Wangi Susu callus after two weeks of subculture in SIM. Thus, the positive corre lation between the expression of cytokinin responsive and ethylene biosynthesis genes likely depends on the induc tion level of OsBBM1.

Discussion
In this study, we used the callus of Ciherang (indica subspecies), Mentik Wangi Susu (javanica subspecies), Hwayoung (japonica subspecies), and Tarabas (japonica subspecies) to investigate the correlation between callus regeneration rate and the expression of several genes re sponsible for cytokinin response and ethylene biosynthe sis. Callus of each rice variety were well developed in CIM containing 2 mg/L 2,4D (Figure 1). The addition of cy tokinin hormones such as kinetin is indispensable for the formation of green spots on the callus (Mostafiz and Wagi ran 2018). Previous research suggests that green spot for mation can be used as an indicator of the existence of em bryogenic callus that can differentiate into coleoptiles in rice and wheat (Umar et al. 2017; Amer and Borner 1997; Mostafiz and Wagiran 2018. The regeneration media containing 2 ppm kinetin, 2 ppm BAP, and 0.5 ppm NAA resulted in the highest re generation rate using callus of indica rice (Ming et al. 2019). Using this hormone, we observed that the callus of Mentik Wangi Susu and Ciherang showed the fastest green spot formation compared to Hwayoung and Tarabas rice ( Figure 2), but their shoot regeneration rate was low ( Figure 3a). The high rate of callus browning in Mentik Wangi Susu and Ciherang (Figure 3b) was suspected as the leading cause of the low shoot regeneration rate. This result follows the previous findings that indica rice has low shoot regeneration due to the high rate of callus browning (Mostafiz and Wagiran 2018). Therefore, the green spot formation is not related to the success of shoot regenera tion in Mentik Wangi Susu and Ciherang callus. Callus browning is tightly linked to the high accumulation and subsequent oxidation of phenolic compounds. The activity of phenylalanine ammonialyase (PAL) is a key factor for the conversion of phenylalanine into phenolic compounds and polyphenol oxidase (PPO) is responsible for the oxi dation of phenol to quinones that generate the brown color pigment (Khosroushahi et al. 2011).
The gene expression analysis of cytokinin responsive and ethylene biosynthesis was conducted since cytokinin signal perception ability and ethylene biosynthesis in cal lus have been known as important factors for shoot regen eration in rice (Fanata et al. 2013; Arisandi et al. 2020; Dewi and Purwoko 2008; Yasmin et al. 2013. In vitro shoot formation is a precise tool for cytokinin response assay (Inoue et al. 2001; Kakimoto 1996. We observed that induction of OsRR2 in Ciherang was maintained at a high level until two weeks in SIM, whereas OsRR6 was highly induced in Hwayoung at one week in SIM, but the level was decreased after two weeks. Since both genes belong to the type A of RRs that negatively regulate the cytokinin response through cytokinin desensitization, the callus of Ciherang might be less sensitive to exogenous cy tokinin treatment due to its high and stable expression level of OsRR2 during the subculture period in SIM. Although Hwayoung callus showed the highest induction of OsRR6, the level decreased after two weeks in SIM, which caused the cytokinin insensitivity not to be present in Hwayoung callus. The constitutive overexpression of OsRR6 in rice has been reported to abolish shoot regeneration, suggest
ing that OsRR6 acts as a negative regulator of cytokinin signaling (Hirose et al. 2007). The overexpression of sev eral typeA RRs in Arabidopsis thaliana genes compro mised the cytokinininduced shoot regeneration capacity (Ren et al. 2009).
The ethylene biosynthesis gene expression revealed the high induction of OsACS1 in Ciherang and the level was stably maintained until two weeks of culture in SIM (Figure 4). The induction of OsACO1 was found to be high in Hwayoung callus and apparent induction was also shown by Mentik Wangi Susu and Tarabas. Moreover, Os BBM1 expression was induced in Hwayoung and Tarabas but decreased in Ciherang callus. Ethylene hormone af fects shoot regeneration differently, depending on species or even cultivar (Yang et al. 2015). The positive role of ethylene for shoot regeneration was shown by reducing of shoot regeneration rate in the ethylene insensitive mu tant of Arabidopsis thaliana, while plants with constitutive ethylene response showed a high level of shoot regenera tion (Chatfield and Raizada 2008). In rice, the addition of ethylene precursor into regeneration media increased shoot regeneration, whereas the opposite effect was shown by ethylene synthesis inhibitor addition (Yasmin et al. 2013). On the other hand, ethylene was identified as a browning inducer in plant cell culture that further represses shoot regeneration (Adkins et al. 1990; Kobayashi et al. 1991. Furthermore, somatic embryogenesisrelated gen such as BBM is vital for shoot regeneration since the ec topic expression of BBM induces somatic embryogene sis in several plant species (Boutilier et al. 2002; Lowe et al. 2016. Therefore, both OsBBM1 and ethylene in duction might synergistically induce shoot formation in Hwayoung callus, whereas a decrease of OsBBM1 level and cytokinin desensitization might be the predominant factor for the absence of shoot regeneration in Ciherang callus.

Conclusions
Analysis of cytokininresponsive gene expression showed that OsRR2 induction was found to be highest in Ciherang callus, whereas OsRR6 expression was found to be highly upregulated in Hwayoung callus. Analysis of the ethy lene biosynthetic gene showed that OsACS1 was highly expressed in Mentik Wangi Susu callus. Thus, the positive correlation between the expression of cytokinin responsive and ethylene biosynthesis genes with the somatic embryo genesis activity likely depends on the induction level of OsBBM1.