LaCl3 treatment improves Agrobacterium-mediated immature embryo genetic transformation frequency of maize

We report an optimized transformation system that uses a LaCl3 pretreatment (a Ca2+ channel blocker) for enhancing Agrobacterium-mediated infection of immature embryos and improving the genetic transformation frequency of maize. Agrobacterium-mediated genetic transformation of immature embryos is important for gene-function studies and molecular breeding of maize. However, the relatively low genetic transformation frequency remains a bottleneck for applicability of this method, especially on commercial scale. We report that pretreatment of immature embryos with LaCl3 (a Ca2+ channel blocker) improves the infection frequency of Agrobacterium tumefaciens, increases the proportion of positive callus, yields more positive regenerated plantlets, and increases the transformation frequency from 8.40 to 17.60% for maize. This optimization is a novel method for improving the frequency of plant genetic transformations mediated by Agrobacterium tumefaciens.


Introduction
Maize is a monocotyledon food crop, as well as a feed and energy crop. It is the most widely grown and productive crop in the world. Approximately, one-third of the world's population depends on corn as a staple food. Maize is a C 4 plant, meaning that it is a model plant for photosynthesis studies (Mookkan, et al. 2017). Owing to the time and labor consumption, and the existence of interspecific reproductive barriers, which prevent the introduction of target traits into recipient plants, the utilization of excellent germplasm resources is limited to a certain extent by conventional breeding (Ahmar, et al. 2020). However, transgenic technology has significantly promoted the process of obtaining various resistance candidate genes and new varieties based on the gene-function studies. Still, the efficient, short-cycle, and stable genetic transformation system of maize remains a hindrance.
Transgenic technology is a powerful method for cultivating high-yield and high-quality crops resistant to biological and abiotic stress. During the development of the maize transgenic technology, scientists have invented many transformation methods, such as electroporation (Fromm, et al. 1986), particle bombardment (Klein, et al. 1988), polyethylene glycol (PEG) treatment of protoplasts (Golovkin, et al. 1993), silicon carbide fibers (Kaeppler et al. 1994), and Agrobacterium-mediated transformation (Ishida et al. 1996). Among these transgenic transformation methods, Agrobacterium-mediated transformation not only has a clear mechanism, simple operation, low cost, but it also exhibits stable inheritance of exogenous genes and low-copy number (Liu et al. 2017). Owing to its many advantages, Agrobacteriummediated transformation is the most widely used genetic transformation method, especially in commercial production (Chen et al. 1998;Hiei et al. 1997).
In 1987, Grimsley et al. first used Agrobacterium for infecting maize, and demonstrated that this method could be used for transforming maize (Grimsley et al. 1987). Subsequently, in 1996, Ishida et al. established a relatively stable agrobacterium-mediated genetic transformation system Communicated by Yuree Lee. with immature maize embryos as explants for the first time (Ishida et al. 1996). There have been many studies on the Agrobacterium-mediated optimization of the immature maize embryo genetic transformation system. Many factors, such as the vector, the explant genotype, the explant pretreatment condition, Agrobacterium strains, the Agrobacterium solution concentration, the infection duration, the co-culture duration, the infection medium, and the coculture medium, affect the genetic transformation frequency (Sheikholeslam and Weeks 1987;Cho et al. 2014;Frame, et al. 2006;Hiei et al. 2006;Sivanandhan et al. 2015;Vega et al. 2008). Recently, a ternary vector system carrying extra copies of Vir genes has been shown to increase the transformation frequency of maize (Anand et al. 2018;Zhang et al. 2019). The application of morphogenic regulator genes such as BABY BOOM (BBM) and WUSCHEL (WUS) was a significant breakthrough in the genetic transformation of maize, significantly improving the transformation frequency and enabling to overcome the dependence on genotypes and explants to a certain extent (Salvo et al. 2014;Lowe et al. 2016Lowe et al. , 2018Mookkan et al. 2017). However, its application in commercial-scale production still has some problems. Although Agrobacterium-mediated genetic transformation frequency of maize immature embryos has significantly improved owing to continuous system optimization, and the method has been widely used in the commercialization of maize breeding, the low transformation frequency remains a bottleneck that precludes practical application of maize gene-function studies and molecular breeding.
It is well known that Agrobacterium tumefaciens, a naturally occurring Gram-negative bacterium, contains a tumorinducing (Ti) plasmid, which contains transfer deoxyribonucleic acid (T-DNA) that can be integrated into recipient plant genomes after being horizontally transferred into plant cells. Hence, Ti plasmid molecules are modified to transform target genes into the plant genome, enabling the transformation of the target genes in the recipient species; Agrobacterium tumefaciens has been termed "the smallest genetic transformation engineer in nature" (Yuan and Williams 2012). In addition, evidence suggests that Agrobacterium tumefaciens triggers the activation of multiple mitogen-activated protein kinases (MAPKs), a defense mechanism that is rapidly triggered by the host perception of pathogen-associated molecular patterns (PAMPs) (Djamei, et al. 2007). However, A. tumefaciens can also induce the formation of plant crown galls (Escobar et al. 2003); thus, it is an exogenous pathogen in sessile plants (Cho and Winans 2005). When exogenous pathogenic microorganisms infect plant receptors, the innate immune response of the receptors is triggered to defend against pathogens and maintain growth (Gómez-Gómez 2004). During the plant-pathogen interaction, a sequence of signal transduction events occurs in plants, including an increase in the Ca 2+ concentration, accumulation of reactive oxygen species (ROS), and activation of signaling cascades mediated by mitogen-activated protein kinases (MAPKs) and Ca 2+ -dependent protein kinases (Lamb and Dixon 1997;Boller and Felix 2009). Toyota et al. investigated the mechanisms of the long-distance transmission of Ca 2+ -dependent defense signals in plants; They applied LaCl 3 , a Ca 2+ channel blocker, to the leaves of Arabidopsis thaliana, blocking the transmission of danger signals from caterpillars to nearby and distal parts of the plants (Toyota, et al. 2018). Inspired by this, here we propose that the Agrobacteriummediated transformation frequency can be improved by dampening the innate immune response of plants to Agrobacterium tumefaciens. In this study, we developed an efficient optimization system that used a Ca 2+ channel blocker for pretreating immature maize embryos before the Agrobacterium infection. Optimization of the receptor pretreatment conditions revealed that immature maize embryos pretreated with 10 mM LaCl 3 yielded twice as many positive regeneration plantlets as those in the control group, and stability was further validated using testing vectors.

Plant materials
Immature embryos of transformed receptors were from maize inbred lines ND101 and ND88 created by the Center for Crop Functional Genomics and Molecular Breeding of China Agricultural University (Liu et al. 2021;Zhang et al. 2019). Maize plants were grown in a greenhouse under a 16/8 h light/dark cycle at 20-25 °C. Immature embryos were collected from fresh ears 9-12 days after pollination for genetic transformation.

Agrobacterium strains and binary vectors
The Agrobacterium tumefaciens strain EHA105 was used for maize transformation. The binary vector contained the Ds-Red gene as the reporter driven by the ubiquitin-1 promoter, and bar as the herbicide resistance selection marker gene. The test vectors contained a single target and double targets, provided by the Center for Crop Functional Genomics and Molecular Breeding of China Agricultural University. The vectors' structure and related construction methods were described by Xing et al. (2014). Gene models were obtained from the Gramene database (https:// ensem bl. grame ne. org/ Zea_ mays/ Info/ Index). Single-guide ribonucleic acids (SgR-NAs) were designed using the CRISPOR database (http:// crisp or. tefor. net/).

Agrobacterium-mediated transformation and immature embryo pretreatment
Maize transformation followed published protocols with minor modifications (Sidorov and Duncan 2009). The 9-12 DAP (day after pollination) ears of maize inbred lines ND101 or ND88 were sterilized in 70% ethanol solution for 1 min after removing the bracts. Intact immature embryos with a length of 1.5-2.0 mm were isolated, and placed in a 2-mL centrifuge tube containing infection solution (2.16 g/L MS basal salt mixture, 10 ml/L 100 × MS vitamins, 68.5 g/L sucrose, 36 g/L glucose, 0.115 g/L L-proline, pH 5.2). Following the collection of immature embryos, the infection medium in the centrifuge tubes was removed and a fresh infection medium containing LaCl 3 was quickly added to them. Then, the centrifugal tubes were placed in a 45 °C water bath for a 5-min-long pretreatment. After the LaCl 3 pretreatment, the supernatant was removed from the centrifuge tubes to the maximal possible extent, and a fresh Agrobacterium solution with OD 600 in the 0.6-0.8 range was added to the tubes. Under dark conditions, the centrifuge tubes containing immature embryos and the Agrobacterium solution were incubated at 22 °C for 30 min. After that, the Agrobacterium fluids were removed to the maximal possible extent. To facilitate subsequent observations and quantitative analysis of the RFP expression fluorescence, after the immature embryos were transferred int the co-culture medium (2.16 g/L MS basal salt mixture, 10 ml/L 100 × MS vitamins, 3 mg/L 2,4-D, 10 g/L glucose, 20 g/L sucrose, 0.115 g/L L-proline, 200 μM acetosyringone, 3.4 mg/L silver nitrate, pH 5.2), sterile filter paper was used for gently absorbing the fluid around the embryos, to reduce background noise during photography. After cultivation for 2 days under dark conditions at 22 °C, the embryos were transferred to screening medium (4.33 g/L MS basal salt mixture, 10 ml/L 100 × MS vitamins, 30 g/L sucrose, 1.38 g/L L-proline, 0.5 g/L casamino acids, 3.0 g/L phytagel, 0.5 mg/L 2,4-D, 2.2 mg/L picloram, and 3.4 mg/L silver nitrate, 250 mg/L carbenicillin and 5 mg/L bialaphos sodium salt, pH 5.8) for 14 days cultivation under dark conditions at 28 °C to induce calluses. Then, the calluses were transferred to pre-regeneration medium (4.33 g/L MS basal salt mixture, 1 ml/L 1,000 × Fromm vitamins stock, 3 mg/L 6-benzyladenine, 3.0 g/L phytagel, 30 g/L sucrose, 1.36 g/L L-proline, 0.05 g/L casamino acids, 250 mg/L carbenicillin and 5 mg/L bialaphos sodium salt, pH 5.8) and cultured for 14 days under light conditions at 28 °C, and then were transferred to regeneration medium (4.33 g/L MS basal salt mixture, 10 ml/L 1,000 × Fromm vitamins stock, 10 g/L glucose, 20 g/L maltose, 0.15 g/L asparagine monohydrate, 0.1 g/L myoinositol, 3.0 g/L phytagel, 250 mg/L carbenicillin and 5 mg/L bialaphos sodium salt, pH 5.8) and cultured for 20 days under light conditions at 28 °C. When the plantlets were 7-8 cm long, they were transplanted to the rooting medium (1/2 MS medium) for cultivation under light conditions at 28 °C.

Analysis of the transient RFP expression and statistical analysis of the RFP fluorescence intensity
The red fluorescence distribution of immature embryos and resistant calluses was observed using a multifunctional zoom microscope (Nikon AZ100), for wavelengths in the 510-560 nm range. The fluorescence signal values were computed using a custom script by ourselves. Statistical significance was assessed using Student's t-test.

Statistical analysis of the infection frequency, rate of callus, rate of positive callus, regeneration rate, and transformation frequency
After 2 days of co-culturing, the infection frequency was calculated as follows: the infection frequency (%) = number of embryos with RFP transient fluorescence expression/number of infected embryos × 100.
After 10 days of culturing using the selection medium, the rate of RFP-positive callus was calculated as follows: the rate of RFP-positive callus (%) = number of calluses with RFP transient fluorescence expression/number of total calluses × 100.
After 14 days of culturing using the selection medium, the rate of callus was calculated as follows: the rate of callus (%) = number of calluses/number of embryos in the co-culture medium × 100.
After 20 days of the first differentiation culture, the regeneration rate was calculated as follows: the regeneration rate (%) = number of calluses with shoots/number of total callus clumps × 100.
After all primary regeneration plantlets (T 0 ) were obtained, the transformation frequency was calculated as follows: the transformation frequency (%) = number of bar-positive T 0 events/number of infected embryos × 100.
After all bar-positive plantlets (T 0 ) were obtained, the single copy frequency was calculated as follows: the single copy frequency (%) = number of single copy T 0 plantlets/ number of infected embryos × 100, and the low-copy frequency was calculated as follows: the low-copy frequency (%) = number of one/two-copy T 0 plantlets/number of infected embryos × 100.
All the above statistical significance analyses were assessed using Student's t-test.

Molecular identification and copy number analysis of transgenic plantlets
Genomic DNA of transgenic plantlets was extracted using magnetic beads. Wild-type lines and putative transformations were confirmed by polymerase chain reaction (PCR) analysis with selection marker gene primers (forward primer: ATG AGC CCA GAA CGA CGC; reverse primer: TCA AAT CTC GGT GAC GGG ). Copy number analysis of transgenic plantlets was performed by the duplexed TaqMan assays (Liu et al. 2021).

Pretreatment of immature embryos with LaCl 3 improves the infection frequency of Agrobacterium tumefaciens
To test the hypothesis above, we simultaneously pretreated immature embryos of ND101 with infection media containing different concentrations of LaCl 3 at 45 °C for 5 min before the Agrobacterium infection; the infection medium without LaCl 3 was used as the control. Twenty-five immature embryos were used for each treatment and kept in the infection medium for less more than 60 min. After the pretreatment with 10 mM LaCl 3 , immature embryos were infected with Agrobacterium tumefaciens EHA105 harboring the binary vector with the RFP reporter gene (Fig. S1). Then, they were transferred to a co-culture medium for co-cultivation.
To detect the infection frequency, fluorescence microscopy was used for investigating the transient expression of RFP after co-cultivation for 2 days, and the infection effect was assessed by the statistical analysis of the images' fluorescence intensity. The results suggested that the fluorescence intensity after the pretreatment with 10 mM LaCl 3 was significantly higher than that for the control group and those for the other tested concentrations (Fig. 1A and B); in addition, the infection frequency for the pretreatment with 10 mM LaCl 3 was the highest (Fig. 1C). After that, we observed the callus induction after 14 days under the selection condition, and calculated the rate of callus; observation of pre-differentiated callus clumps cultured for 12 days were also followed ( Fig. 1A and D). The results of these analyses indicated that the pretreatment with 10 mM LaCl 3 yielded the best performance for the pre-differentiation state, had no effect on the callus formation, and significantly improved the infection frequency mediated by Agrobacterium.

Pretreatment of immature embryos with LaCl 3 improves the rate of positive callus
Our experimental results confirmed that the pretreatment of immature embryos with 10 mM LaCl 3 improves the infection frequency of Agrobacterium, but whether the recipient cells integrating the exogenous genes from the T-DNA of Agrobacterium undergo dedifferentiation and re-differentiation for forming embryogenic callus is a hinge that affects the transformation frequency.
To evaluate whether the transformed cells could form embryogenic callus through dedifferentiation, after 10 days of callus induction in the selection medium under dark conditions, we observed the transit expression of RFP in calluses using fluorescence microscopy, and counted the proportion of RFP-positive callus. We observed that a number of red-fluorescent adventitious buds appeared in embryogenic calluses following the pretreatment with 10 mM LaCl 3 , compared with the control group ( Fig. 2A), and the proportion of RFP-positive callus was consistent with this ( Fig. 2B). The results suggested that the pretreatment of immature embryos with LaCl 3 increased the rate of RFPpositive callus, and did not negatively affect the formation of embryogenic callus.

Pretreatment of immature embryos with LaCl 3 improves the transformation frequency
To investigate the effect of the pretreatment with 10 mM LaCl 3 on the transformation frequency of immature embryos via Agrobacterium tumefaciens, we tracked the re-differentiation process of all calluses. After 14 days of callus induction in the selection medium under dark conditions, all calluses, including non-embryonic calluses, were transferred to the pre-differentiation medium for resistance screening under low light conditions for 12 days, and then were transferred to the regeneration medium for resistance selection under light conditions for 20-30 days. We compared the differentiated shoots developed in the experimental and control groups after culturing for 20 days in the regeneration medium, and it was obvious that the experimental group pretreated with 10 mM LaCl 3 fared better than the control group (Fig. 3A). Furthermore, we estimated the regeneration rate as the proportion of tissues differentiated with elongated shoots relative to the overall number of calluses. The results showed that after the LaCl 3 treatment, the regeneration rate increased from 13.20 to 27.20%, which was Immature embryos were treated with 10 μM, 100 μM, 1 mM, 10 mM, and 100 mM of LaCl 3 , respectively, for 5 min before the Agrobacterium infection. A. Transient expression of RFP in immature embryos after 2 days of co-cultivation; calluses induced in the selection medium after 14 days, and callus clumps cultured after 12 days of pre-differentiation cultivation. The red squares represent calluses in the randomly selected magnified area below. B. RFP fluorescence statistics for the transient expression. C. Statistical analysis of the infection frequency. D. Statistical analysis of the callus' rate. Error bars are mean ± SEM. Statistical differences were analyzed using Student's t-test, n = 3 The white asterisks indicate red-fluorescent adventitious buds that appeared in calluses. B. Statistical analysis of the rate of RFP-positive callus. Error bars are mean ± SEM. Statistical differences were analyzed using the Student's t-test, n = 10 more than twofold than the control group's rate (Fig. 3B). Subsequently, we identified bar-positive T 0 plantlets using PCR, and calculated the transformation frequency. The results revealed that the transformation frequency increased from 8.40 to 17.60% after the LaCl 3 pretreatment (Fig. 3C  and D). The low-copy frequency also increased from 8.00 (20/250) to 15.20% (38/250). Moreover, the number of barpositive T 0 plantlets transplanted into the nutrition bowl of the LaCl 3 pretreatment was twice that of the control group, consistent with the above conclusion (Fig. 3E). Our results indicated that pretreatment of immature embryos with LaCl 3 improved the Agrobacterium-mediated transformation frequency in maize.
We also used EDTA and EGTA (two Ca 2+ chelators, although they also chelate other divalent metal cations, such as Mg 2+ and Mn 2+ ) to pretreat immature embryos of ND101. Preliminary results indicated that the transformation frequency increased from 7.47 (5 bar-positive T 0 plantlets/32 immature embryos of ND101) to 27.78% by EDTA treatment (10 bar-positive T 0 plantlets/36 immature embryos of Fig. 3 Pretreatment of immature embryos with 10 mM LaCl 3 improves the transformation frequency in maize. A The morphology of calluses cultured for 20 days in the regeneration medium. B Statistical analysis of the regeneration rate. C PCR-based detection of T 0 plantlets. The red arrowhead is bar-gene specific amplified PCR product. D Statistical analysis of the transformation frequency. E Barpositive T 0 plants, transplanted into vegetative soil. Error bars are mean ± SEM. Statistical differences were analyzed using the Student's t-test, n = 10 ND101) and 31.43% by EGTA (11 bar-positive T 0 plantlets/35 immature embryos of ND101), respectively (Fig.  S2A). In addition, we pretreated immature embryos of ND88 in the same manner, yielding a recalcitrant maize inbred line. The infection frequency nearly doubled from 46.88% (32 immature embryos of ND88) to 100% (EDTA: 29 immature embryos of ND88, EGTA: 32 immature embryos of ND88), and fluorescence microscopy-based analysis also suggested that the fluorescence quantity of the RFP transient expression in immature embryos after 2 days of co-culturing was significantly higher than that for the control group ( Fig. S2B  and C). These results suggest that the repression of calcium increasing triggered by Agrobacterium tumefaciens infection enhances the maize embryo transformation frequency.
To address whether the LaCl 3 pretreatment of immature embryos affected the morphology and fertility of regenerated plants, we followed the growth, development, and fructification of T 0 generation plants. As expected, no significant difference was observed in terms of the plant growth and development between the LaCl 3 pretreatment group and the control group (Fig. S3).
Based on the above, we established an optimized system for improving the frequency of the Agrobacterium-mediated genetic transformation by pretreating immature maize embryos with LaCl 3 (Fig. 4).

Testing of the effectiveness and stability of the optimized genetic transformation system in maize
To test the validity of the optimized protocol, we selected four adenosine triphosphate (ATP)-binding cassette transporter genes (Zm00001d036986, Zm00001d047534, Zm00001d018522, and Zm00001d046662) and constructed six vectors, namely CAUC1828, CAUC1829, CAUC1831, CAUC1832, CAUC1834, and CAUC1871 (Fig. S4). After that, we introduced five of the vectors into the maize inbred line ND101, using the optimized protocol, while the remaining vector was transformed using the non-optimized protocol as the control. Then, we estimated the regeneration rates and the transformation frequencies for the different transformation protocols. The results suggested that the regeneration rate increased from 11.09 to 20.52-25.21% after the optimization, while the average regeneration rate was 23.03%. Consistently, the transformation frequency increased from 7.17 to 11.98-12.95% after the optimization, while the average transformation frequency was 13.25%. The regeneration rate and the transformation frequency both doubled compared with the corresponding control values (Table 1). Copy number is an important selection index in both gene-function research and production breeding. Therefore, we further analyzed the low-copy frequency. The results of our data analyses showed that the low-copy frequency in the control group was 4.35%, while it ranged from 7.18 to 12.18% for five of the vectors in the LaCl 3 treatment group (Table 1). The average low-copy frequency for the LaCl 3 treatment group was 8.84%, which was more than double that for the control group. This result showed that the protocol optimized with LaCl 3 increased the transformation frequency without affecting the copy number of T 0 plants. Thus, the results showed that the regeneration rate, the transformation frequency, and the low copy frequency were all doubled compared with the control group values.
In conclusion, we used LaCl 3 to inhibit the Ca 2+ -dependent signal transduction triggered by Agrobacterium-infected immature maize embryos, effectively improving the frequency of Agrobacterium-mediated infection and increasing the transformation frequency.

Discussion
Based on the hypothesis that partial inhibition of Ca 2+ transduction triggered by the Agrobacterium infection explants improves the infection frequency and thus possibly improves the transformation frequency, we successfully established a transformation system for pretreatment of immature embryos with LaCl 3 . This method can improve the infection frequency of Agrobacterium, the regeneration rate, and the transformation frequency. We introduced six vectors for system verification, which revealed that the proposed protocol indeed effectively improved the regeneration rate, the transformation frequency. Hence, in the present study, inhibition of the Ca 2+ signal transduction triggered by the Agrobacterium infection in explants improved the transformation frequency.
Ca 2+ is a universal second messenger that plays an important role in signal transduction in many physiological processes, including stress and immune responses in plants and animals (Ma et al. 2019). For an ever-increasing number of environmental stresses, pathogen attacks, drought stress, cold/heat stress, oxidative stress, and slat stress, it has been found that temporally and spatially defined rapid changes in the cytoplasmic Ca 2+ concentration differ in the Ca 2+ elevation duration, intensity, amplitude, frequency, and other aspects. Moreover, Toyota et al. showed that caterpillar feeding or wounding with scissors induced rapid [Ca 2+ ] cyt increased that propagated to distal parts. However, when plants were treated with LaCl 3 , systemic [Ca 2+ ] cyt was blocked. In addition, the relative expression levels of woundinduced defense marker genes, such as JAZ5, JAZ7, ZAT12, OPR3, and RBOHD, significantly decreased (Toyota et al. 2018). The downregulated expression of defense genes also weakened the plants' defense against the invasion of exogenous pathogens, which made it easier for pathogens to infect plants. Based on this theory, LaCl 3 was used to inhibit the increase in [Ca 2+ ] cyt and block this infection signal, allowing Table 1 The data analysis of optimized maize genetic transformation system CAUC1828, CAUC1829, CAUC1831, CAUC1832, and CAUC1834 were treated with 10 mM of LaCl 3 ; CAUC1871 was used as the control group. The low-copy events include one-copy and two-copy conversion events immature embryos to take the edge off their defense against Agrobacterium. Therefore, Agrobacterium-containing target genes or editing systems can more efficiently transfer Ti plasmids into plant receptor cells, and increase the integration opportunity of target genes and editing systems on the receptor genome. Finally, the genetic transformation frequency of immature maize embryos mediated by Agrobacterium is improved. In the LaCl 3 concentration test experiment, we found that a high concentration of LaCl 3 affected the formation of embryonic callus and reduced the rate of callus ( Fig. 1A  and D). This result implied that the optimal concentration of LaCl 3 was key to this optimization, especially in other maize genotypes. We also inquired whether pretreatment of explants with other Ca 2+ inhibitors, such as EDTA and EGTA, could improve the frequency of the Agrobacterium infection, and the similar results as LaCl 3 treatment were obtained. These results further suggested that inhibition of the Ca 2+ signal transduction pathways in explants could improve the infection frequency and transformation frequency.
In summary, the proposed optimized protocol provides a novel approach to improving the genetic transformation frequency of maize. In future studies, by further optimization, we expect to be able to overcome the genotype-dependent obstacles of the Agrobacterium-mediated genetic transformation during operation. This is expected to open new vistas for further basic research on the crop gene function and molecular breeding.