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A lack of stability in the expression of Bacillus thuringiensis genes (CRY) and the dialaninophosphat e resistance gene (BAR) in transgenic rice plants can lead to the loss of important characters. The genetic stability of transgenic expres- sion in high-generation lines is thus critically important for ensuring the success of molecular breeding efforts. Here, we studied the genetic stability of resistance to insect pests and herbicides in transgenic rice lines at the molecular and phenotypic levels in a pesticide-free environment. Southern blot analysis, real-time polymerase chain reaction, and enzyme-linked immunosorbent assays revealed high stability in the copy numbers and expression levels of CRY1C, CRY2A, and BAR in transgenic lines across different generations, and gene expression levels were highly corre - lated with protein expression levels. The insecticide resistance of the transgenic rice lines was high. The larval mortal- ity of Chilo suppressalis was 50.25% to 68.36% higher in transgenic lines than in non-transgenic control lines. Percent dead hearts and percent white spikelets were 16.66% to 22.15% and 27.07% to 33.47% lower in transgenic lines than in non-transgenic control lines, respectively. The herbicide resistance of the transgenic rice lines was also high. The bud length and root length ranged were 2.53 cm to 4.20 cm and 0.28 cm to 0.73 cm higher in transgenic lines than in non-transgenic control lines in the budding stage, respectively. Following application of the herbicide Basta, the chlo- rophyll content of the transgenic lines began to recover 2 d later in the seedling and tillering stages and 3 d later in the booting and heading stages, by contrast, the chlorophyll content of the non-transgenic lines did not recover and continued to decrease. These findings revealed high genetic stability of the resistance to insect pests and herbicides across several generations of transgenic rice regardless of the genetic background. Keywords Bacillus thuringiensis, Genetic stability, Transgenic rice, Insect resistance, Herbicide resistance Introduction Yue Sun and Zhongkai Chen contributed equally to this work Rice is the world’s most important staple food crop. *Correspondence: Rapid and continuous changes in the environment driven Haohua He email@example.com by anthropogenic activities have increased the inten- Xiaosong Peng sity and frequency at which rice plants are exposed to firstname.lastname@example.org abiotic and biotic stresses, such as heat and cold stress, Key Laboratory of Crop Physiology, Ecology, and Genetic Breeding, Ministry of Education /College of Agronomy, Jiangxi Agricultural drought, heavy metals, herbicides, pests, and diseases, University, Nanchang, Jiangxi, China and this has accelerated rice yield losses (Li et al. 2016). Hainan Yazhou Bay Seed Laboratory, Sanya, Hainan, China Chilo suppressalis is an economically significant pest College of Agronomy, Hunan Agricultural University, Changsha, Hunan, China that can induce substantial damage to rice plants, and Pingxiang Center for Agricultural Sciences and Technology Research, it has been estimated to be responsible for losses that Pingxiang, Jiangxi, China amount to 10–30% of total rice production (Jian et al. © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Sun et al. Rice (2023) 16:8 Page 2 of 18 2014). Bacillus thuringiensis (Bt) proteins are highly determining whether foreign genes have been integrated effective biocides against insect pests, and they function into plant chromosomes. The copy number and inser - by destroying the midgut cells (Vaeck et al. 1987). The tion mode of exogenous genes can be determined using Bt toxin is highly specific and thus non-toxic to various southern hybridization analysis (Maghuly et al. 2007), animals, such as reptiles, birds, and mammals (including and the expression level of foreign genes can be deter- humans) (Stewart et al. 1996). The most commonly used mined using real-time PCR (Zhang et al. 2017). The Bt genes in transgenic crops include CRY1Ab/Ac, CRY1C, proteins expressed in transgenic plants can be identified and CRY2A (Wünn et al. 1996; Chen et al. 2005; Tang using enzyme-linked immunosorbent assays (ELISAs) in et al. 2006). A biosafety certificate was issued for MH63 the event that the exogenous genes are protein-coding (CRY1Ab/Ac) in 2009, and the commercial production genes. Robust assessments of genetic stability require the of MH63 (CRY1Ab/Ac) began in China that same year; accurate identification of foreign genes (Xu et al. 2018). several studies have examined transgenic rice with the The development of genetically stable high-generation CRY1Ab/Ac gene and its derivatives (Chen et al. 2011). lines is essential for generating transgenic rice hybrids Efforts to test the potential utility of the transgenic rice with high yield and quality and resistance to insects, dis- lines MH63 (CRY1C) and MH63 (CRY2A) in China were eases, and herbicides. Here, we characterized the insect accelerated in 2021, and the genetic stability of key traits and herbicide resistance of newly bred Bt-transgenic rice in transgenic rice is critically important for evaluating parental lines with the CRY1C, CRY2A, and BAR genes in the ability of transgenic traits to make significant contri - different genetic backgrounds. Specifically, our aim was butions to ongoing breeding programs (Gahakwa et al. to assess the genetic stability of Bt-transgenic rice lines 2000). The genetic stability of transgenic traits is typically from different generations at the DNA, RNA, and pro - evaluated over at least two generations to ensure the suc- tein levels as well as their insect resistance and herbicide cess of the breeding of commercially important varieties resistance phenotypes. using genetic engineering approaches. Following the introduction of foreign genes into recipi- Results ent cells, callus induction, adventitious bud differen - Genetic Background Analysis tiation, adventitious root formation, and other culture We analyzed the response rate of the genetic background processes are required for asexual propagation. In the using six BC F and B C F populations. A total of 512 4 8 4 9 sexual stage, plants germinate, grow, and flower, and this molecular markers were used for genotyping, and the is followed by meiosis, pollination, fertilization, zygote linkage map of SSR fingerprint markers was generated formation, and embryo development; various vegeta- (Fig. 1). A total of 31, 10, 23, 11, 21, and 25 polymorphic tive growth and reproductive growth processes also take markers between CH121(1C), CH871(1C), CH891(1C), place in this stage (Azhakanandam et al. 2000). Next, CH891(2A), CHT025(1C) and CHT025(2A) and their foreign genes are transferred to cultivars with desirable parents were identified, and the percentage of polymor - traits through sexual hybridization (i.e., transfer process), phic markers was 6.05%, 1.95%, 4.49%, 2.15%, 4.10%, and and these cultivars are then used to cultivate new varie- 4.88%, respectively. The low number of polymorphic ties (Bavage et al. 2002). These processes, especially the markers identified suggests that the genetic background transfer process, challenge the genetic stability of for- of the six transgenic recovered lines was similar to that eign genes. At the molecular level, the integration site, of their recurrent parents. The response rate of the gene fragment size, copy number, methylation, repeat actual genetic background of CH121(1C), CH871(1C), sequences, trans-inactivation, and co-inhibition of for- CH891(1C), CH891(2A), CHT025(1C), and CHT025(2A) eign genes can all have an effect on their stable inherit - was 96.97%, 99.02%, 97.75%, 98.93%, 97.95%, and 97.56%, ance during asexual and sexual reproduction (Fernandez respectively (Additional file 1: Table S1). The actual et al. 2009; Marjanac et al. 2009; Mette et al. 2000; Que response rate of the genetic background was higher than et al. 1997). Polymerase chain reaction (PCR) screen- the theoretical response rate of the genetic background. ing of marker genes or reporter genes is one of the main methods used to detect foreign genes. Although rapid PCR and Southern Blot Analysis and convenient, this method is prone to producing false The six high-generation transgenic rice lines from positives. PCR can be used to determine whether the the BC F and B C F populations were shown to be 4 8 4 9 target gene has been integrated into the chromosomes transgene-positive according to PCR analysis. The 799- of recipient cells; however, this method is also prone to bp, 600-bp, and 479-bp molecular weight bands were the effects of DNA contamination, and this increases the amplified from the CRY1C, CRY2A, and BAR genes in difficulty of detecting multilocus insertions. Molecular the six transgenic lines, respectively, via PCR (Fig. 2). hybridization is currently the most effective method for We characterized the integration sites of the CRY1C Sun et al. Rice (2023) 16:8 Page 3 of 18 Fig. 1 The linkage map of CH121(1C), CH871(1C), CH891(1C), CH891(2A), CHT025(1C), and CHT025(2A). The red, purple, sky blue, green, yellow, and blue bars indicate the Minghui 63 chromosomal segments in CH121(1C), CH871(1C), CH891(1C), CH891(2A), CHT025(1C), and CHT025(2A), respectively. The scale of the ruler indicates the genetic distance in “cM.” and CRY2A genes with variable copy numbers (Fig. 3). Analysis of Gene and Protein Expression Patterns Southern blot analysis with the CRY1C gene revealed The relative expression levels of CRY1C, CRY2A, and transgenes with two copies of the transforming DNA, BAR in fresh leaves, stems (at the seedling, tillering, and these transgenes were also consistently detected booting, heading, and filling stages), and endosperm in the progeny analysis. NcoI and HindIII enzymes and (at the booting, heading, and filling stages) of the high- uncut genomic DNA from transgenic plants showed generation transgenic rice lines (BC F and BC F ) 4 8 4 9 different banding patterns at high molecular weights. were measured. The expression of CRY1C and CRY2A Southern blot analysis of the CRY2A gene revealed a was significantly higher in different tissues and at single copy of the transforming DNA in different gener - developmental stages in transgenic rice lines (Addi- ations and lines. BamHI and EcoRI enzymes and uncut tional file 1: Table S2) compared with their non-trans- genomic DNA from transgenic plants showed different genic parents (Additional file 1: Table S3). In various banding patterns at high molecular weights. The copy generations of all strains, the expression of CRY1C first numbers of target genes were analyzed using Southern increased and then decreased from the tillering stage to blot (Fig. 4). The Southern blot analysis with the BAR maturity, and its expression was highest at the heading gene revealed two bands for SmaI enzyme-digested stage. This gene was also expressed stably in the leaf, stem, and panicle (Fig. 5). The expression of BAR was samples, indicating the presence of two copies of target significantly higher in different tissues and at devel genes in the rice genome. Southern blot-positive plants - containing fragments consistent with the expected size opmental stages in transgenic rice lines (Additional of the CRY1C, CRY2A, and BAR genes were used in file 1: Table S4) compared with their non-transgenic analyses of gene and protein expression and bioassay parents (Additional file 1: Table S5). In the leaves of all activity. transgenic lines, the expression of BAR first increased Sun et al. Rice (2023) 16:8 Page 4 of 18 Fig. 2 Marker-assisted selection of target genes in the transgenic restorer lines. M indicates marker; P indicates the donor parent Minghui 63; N indicates the receptor parent (recurrent parent); a 1–8 correspond to the transgenic CRY1C gene-resistant restorer lines. b 1–4 correspond to the transgenic CRY2A gene-resistant restorer lines. c 1–12 correspond to the transgenic BAR gene-resistant restorer lines Sun et al. Rice (2023) 16:8 Page 5 of 18 Fig. 3 Southern blot analysis of the total genomic DNA of BC F and BC F transgenic plants. Southern blot of a CHT025(1C), b CHT025(2A), 4 8 4 9 c CH891(1C), d CH891(2A), e CH871(1C), and f CH121(1C) in BC F and BC F transgenic plants. The DNA samples were digested with NcoI and 4 8 4 9 HindIII and hybridized with the prepared radioactive probe (NcoI and HindIII restriction sites are absent within the T-DNA region). The size of the probe was 4.3 and 5.5 kb for NcoI and 4.1 kb for HindIII and was a fragment of the CRY1C gene. The DNA samples were digested with BamHI and EcoRI and hybridized with the prepared radioactive probe (BamHI and EcoRI restriction sites are absent within the T-DNA region). The size of the probe was 2.5 kb for BamHI and 3.5 kb for EcoRI and was a fragment of the CRY2A gene. M: DNA marker; 1 and 4: negative control; 2 and 5: positive control; 3 and 6: BC F and BC F transgenic plants, respectively; C: PCR product 4 8 4 9 and then decreased, and its expression was highest at generations, indicating that the genetic environment the heading stage. However, the expression of BAR in has an effect on BAR expression (Fig. 6). CH871(1C) was highest in the stem and panicle at the The content of Cry1C and Cry2A in the six transgenic tillering stage, and the expression of BAR was high- rice lines at various developmental stages and in sev- est in the stem and panicle at the booting stage in eral tissues was measured via ELISAs (Additional file 1: CHT025(2A) and CH121(1C). The genetic background Table S6, Fig. 7). The concentration of Cry1C in the had an effect on the relative expression levels of BAR . four transgenic lines ranged from 3.21 µg/g leaf fresh The relative expression levels of BAR also varied among weight in the BC F line of CH871(1C) at the boot- 4 9 ing stage to 16.16 µg/g leaf fresh weight in the B C F 4 9 Sun et al. Rice (2023) 16:8 Page 6 of 18 Fig.4 Southern blot analysis of the total genomic DNA of BC F and BC F transgenic plants. Southern blot of a CHT025(1C), b CHT025(2A), 4 8 4 9 c CH891(1C), d CH891(2A), e CH871(1C), and f CH121(1C) in BC F and BC F transgenic plants. The DNA samples were digested with SmaI and 4 8 4 9 hybridized with the prepared radioactive probe (SmaI restriction sites are absent within the T-DNA region). The size of the probe was 6.8 and 3.0 kb for SmaI and was a fragment of the BAR gene. M: DNA marker; 1 and 6: negative control; 2 and 7: positive control; 3 and 8: BC F and BC F 4 8 4 9 transgenic plants, respectively; 4 and 9: PCR product. 1–4: southern blot analysis BC F in 2020; 1–4: southern blot analysis BC F in 2021 4 8 4 9 Fig. 5 The relative expression levels of CRY1C and CRY2A were determined using real-time PCR. All tests were conducted in three replicates. The relative expression of CRY1C and CRY2A in the leaves (a–f), stem (g–l), and panicles (m–r) of six transgenic rice lines was measured at different developmental stages. Osactin1 was used as an internal control. Values are mean ± standard error. The red line indicates the donor parent (positive control), the blue line indicates the high-generation transgenic rice line (BC F ), the yellow line indicates the high-generation transgenic rice line 4 8 (BC F ), and the green line indicates the receptor parent (negative control) 4 9 Sun et al. Rice (2023) 16:8 Page 7 of 18 Fig.6 The relative expression levels of BAR were determined using real-time PCR. All tests were conducted in three replicates. The relative expression of BAR in the leaves (a–f), stem (g–l), and panicles (m–r) of six transgenic rice lines was measured at different developmental stages. Osactin1 was used as an internal control. Values are mean ± standard error. The red line indicates the donor parent (positive control), the blue line indicates the high-generation transgenic rice line (BC F ), the yellow line indicates the high-generation transgenic rice line (BC F ), and the green 4 8 4 9 line indicates the receptor parent (negative control) Fig. 7 The Cry1C and Cry2A content was determined using ELISAs. The correlations between the content of Cry1C and Cry2A in transgenic rice for the two years of the field trials are shown. All tests were conducted with three replicates. The Cry1C and Cry2A content in (a–f) leaves, (g–l) stems, and (m–r) panicles of rice plants at different developmental stages. Values are mean ± standard error. Scatter plot of s CHT025(1C), t CHT025(2A), u CH891(1C), v CH891(2A), w CH871(1C), and x CH121(1C) in leaves, stems, and panicles at different developmental stages line of CH871(1C) at the heading stage; the concentra- of Cry2A was higher than that of Cry1C in leaf tissue tion of Cry2A ranged from 34.39 µg/g leaf fresh weight (Fig. 7a–f ); the same pattern was also observed in the in the BC F line of CHT025(2A) at the maturation stem (Fig. 7g–l). The concentration of Cry1C decreased 4 9 stage to 73.33 µg/g leaf fresh weight in the BC F line as plants transitioned from vegetative growth and 4 9 of CH891(2A) at the heading stage. The expression reproductive growth. In the panicle, expression levels Sun et al. Rice (2023) 16:8 Page 8 of 18 of Cry1C were low (Fig. 7m–r). Expression patterns of genes and proteins were high, with the exception of proteins and genes inferred from ELISAs and real-time CH891(1C) (Fig. 8s–x). PCR, respectively, were similar, and the expression of Cry1C and Cry2A was highest at the heading stage. Resistance of Transgenic Rice Lines to C. suppressalis Correlations between the expression levels of genes and in the Laboratory and the Field proteins were high, with the exception of CH871(1C) The resistance of the six transgenic plant lines and (Rs = 0.719) (Fig . 7s–x). The content of the Bar protein recurrent parents to C. suppressalis was tested in the in the six transgenic rice lines in several tissues and at laboratory (P < 0.05, Additional file 1: Table S8) and various stages was determined using ELISA (Additional field (P < 0.05, Additional file 1: Table S9), and larval file 1: Table S7, Fig. 8). The expression of Bar was con - mortality, percent dead hearts, and percent white spike- sistent across various generations (BC F and BC F ); lets were determined. Stems at the heading stage (when 4 8 4 9 the content of Bar first increased and then decreased, borer damage is most common) were used for indoor and its expression was highest at the heading stage. In insect-feeding tests. The mortality rate of C. suppres - the six transgenic lines, the concentration of Bar ranged salis ranged from 71.11% to 88.89% and from 82.22% from 0.49 µg/g to 6.58 µg/g leaf fresh weight and from to 97.06% on the stems of the transgenic lines at the 0.03 µg/g to 3.42 µg/g stem fresh weight (Fig. 8a–f ). The heading stage in 2020 and 2021; the transgenic plants expression pattern of the Bar protein first increased thus showed high insect resistance (Fig. 9). In 2020, the and then decreased as development progressed in both percent dead hearts of the recurrent parents ranged the leaves and stems; its expression was highest at the from 20.86% to 22.86%, indicating that they were dam- heading stage (Fig. 8g–l). The expression of the Bar pro - aged to various degrees by C. suppressalis; however, tein was relatively stable during vegetative growth and the six transgenic lines only experienced slight damage reproductive growth (Fig. 8m–r). The expression pat - (0.31–0.71%) from C. suppressalis (Fig. 10a). Recur- terns of the Bar protein and the BAR gene inferred from rent parents were also extensively damaged by C. sup- ELISA and real-time PCR, respectively, were basically pressalis at the heading stage, and the percent white consistent, and the expression of Bar was highest at the spikelets ranged from 27.53% to 34.10%; however, the heading stage. Correlations between the expression of six transgenic lines only experienced slight damage (0.03–0.71%) from C. suppressalis (Fig. 10b). In 2020, Fig.8 The Bar content was determined using ELISAs. The correlations between the content of Bar in transgenic rice for the two years of the field trials are shown. All tests were conducted with three replicates. The Bar content in (a–f) leaves, (g–l) stems, and (m–r) panicles of rice plants at different developmental stages. Values are mean ± standard error. Scatter plot of s CHT025(1C), t CHT025(2A), u CH891(1C), v CH891(2A), w CH871(1C), and x CH121(1C) in leaves, stems, and panicles at different developmental stages Sun et al. Rice (2023) 16:8 Page 9 of 18 Fig. 9 Insect resistance of BC F and BC F transgenic plants in the laboratory. Larval mortality of C. suppressalis in laboratory bioassays in 2020 (a) 4 8 4 9 and 2021 (c). All tests were conducted with ten replicates, and one replicate comprised 20 s instar larvae. Values are mean ± standard error. Data followed by different lowercase letters denote significant differences between the mortality (%) rates of C. suppressalis at the 5% level according to least significant difference tests. Laboratory insect feeding tests of the stems of CHT025T(1C), CHT025T(2A), CH891T(1C), CH891T(1C), CH871T(1C), and CH121T(1C) at the heading stage in 2020 (b) and 2021 (d) the insect resistance of the six transgenic lines was sim- differences in the abundances of insect pests across ilar and significantly (P < 0.01) higher than that of the the two years of the experiments. Overall, resistance to recurrent parents (Fig. 10c). The percent dead hearts of insect pests was stable (Fig. 10f ). the recurrent parents ranged from 17.00% to 19.50%, indicating that they were damaged to various degrees Herbicide Resistance of Transgenic Rice Lines by C. suppressalis, and transgenic lines only experi- in the Laboratory and Field enced slight damage (0.31–2.14%) (Fig. 10d). Recurrent Weed management is essential for maximizing the pro- parents were also severely damaged by C. suppressa- ductivity of rice cultivation. We tested the herbicide lis at the heading stage, and the percent white spike- resistance of transgenic rice plants from five growth lets ranged from 27.53% to 34.40%; the six transgenic stages (P < 0.05, Additional file 1: Tables S10, S11). The lines only experienced slight damage (0.31–0.66%) by herbicide Basta was used to evaluate the herbicide resist- C. suppressalis (Fig. 10e). The damage induced to the ance of BAR-transgenic rice at the bud stage. Basta appli- recurrent parents by C. suppressalis might stem from cation strongly inhibited the growth of non-transgenic Sun et al. Rice (2023) 16:8 Page 10 of 18 Fig. 10 Insect resistance of BC F and BC F transgenic plants in the field. Percent dead hearts (%) in field trials in 2020 (a) and 2021 (d). Percent 4 8 4 9 white spikelets (%) in field trials in 2020 (b) and 2021 (e). There were three replicates for all tests. Values are mean ± standard error. Data followed by different lowercase letters denote significant differences between mortality (%) rates of C. suppressalis at the 5% level according to least significant difference tests. Insecticide resistance at the heading stage of CHT025T(1C), CHT025T(2A), CH891T(1C), CH891T(1C), CH871T(1C), and CH121T(1C) in 2020 (c) and 2021 (f) lines, and no increases in root length and shoot length values 2 d after Basta application, and herbicide resist- were observed at a Basta concentration of 10 mg/L. By ance at the booting stage and grain filling stage slightly contrast, the resistance of transgenic rice with the BAR differed. Thereafter, the chlorophyll content in the leaves gene to Basta was high, and no inhibition of germination began to return to normal (Fig. 11g–j). After 7 d of Basta was evident under 10 mg/L Basta application at the bud- application, BAR-transgenic rice could still grow nor- ding stage in 2020 and 2021 (Fig. 11a, d). In field experi - mally in each growth period, and leaves showed no signs ments in 2020 and 2021, we first examined herbicide of dryness and yellowing, indicating that they had high resistance at the seedling and tiller stages. After Basta herbicide resistance; no weeds were observed growing in application for 7 d, non-transgenic lines had withered the field (Fig. 11). The results suggested that the cultiva - and yellow leaves, and their growth stagnated; they even- tion of BAR-transgenic rice along with Basta application tually died within 12 d of Basta application (Fig. 11b, c, e, might be an effective strategy for overcoming the delete - f ). However, the chlorophyll content in the leaves of the rious effects of weeds on rice cultivation. six transgenic rice lines began to return to their normal (See figure on next page.) Fig. 11 Herbicide resistance of BC F and BC F transgenic plants in the field. The bud length (cm) and root length (cm) were measured in a 4 8 4 9 hydroponic system at the budding stage in 2020 (a) and 2021 (d). The chlorophyll content (SPAD) of rice plants at the seedling stage in field trials in 2020 (b) and 2021 (e). The chlorophyll content (SPAD) of rice plants at the tillering stage in field trials in 2020 (c) and 2021 (f). The chlorophyll content (SPAD) of rice plants at the booting stage in field trials in 2020 (g) and 2021 (i). The chlorophyll content (SPAD) of rice plants at the booting stage in field trials in 2020 (h) and 2021 (j). There were five replicates for all tests. Values are mean ± standard error. Data followed by different lowercase letters denote significant differences in bud length, root length, and chlorophyll content at the 5% level according to least significant difference tests Sun et al. Rice (2023) 16:8 Page 11 of 18 Fig. 11 (See legend on previous page.) Sun et al. Rice (2023) 16:8 Page 12 of 18 Grain Yield Performance the yield traits showed good genetic stability. On the Different years and different genotype of Bt-trans - other hand, the six Bt-transgenic rice lines had higher genic rice lines showed different yield performances yields than their respective non-transgenic counterparts (P < 0.05, Additional file 1: Table S12). In 2020, yields per in 2020 and 2021, and the yield advantages were mainly plant of the six Bt-transgenic rice lines (CHT025(1C), due to the more grain per panicle and higher weight per CHT025(2A), CH891(1C), CH891(2A), CH871(1C), 1000-grain of Bt-transgenic rice lines comparing with CH121(1C)) were around 34.41 g, 38.96 g, 26.87 g, their respective non-transgenic counterparts. Genotype 25.70 g, 35.26 g, and 31.66 g, respectively. In 2021, had significant effects on yield and relative traits of Bt- the corresponding lines were around 34.08 g, 39.18 g, transgenic rice line. However, year had significant effects, 27.50 g, 25.76 g, 39.07 g, and 32.61 g, respectively. There in addition to some lines of grains per panicle (Fig. 12). was no significant difference in the yield traits of the same Bt-transgenic rice lines in different years. Therefore, Fig. 12 Yields and relative traits of BC F and BC F transgenic plants in the field. Panicles per plant, Grains per plant, Weight per 1000-grain and 4 8 4 9 yield per plant in field trials in 2020 and 2021 (a-d). Percent white spikelets (%) in field trials in 2020 (b) and 2021 (e). There were three replicates for all tests. Values are mean ± standard error. Data followed by different lowercase letters denote significant differences between yields and relative traits at the 5% level according to least significant difference tests Sun et al. Rice (2023) 16:8 Page 13 of 18 Discussion little effect on the insect resistance of transgenic rice In our study, all transgenic rice lines showed higher lines; these findings are consistent with the results of pre - resistance to insect pests and herbicides compared with vious studies (Chen et al. 2005; Tang et al. 2006). In field non-transgenic control lines under pesticide-free con- trials under a pesticide-free environment, the percent ditions. Our findings provided new insights into the dead hearts of the transgenic lines was below 2.5%, and molecular basis of the stable inheritance of these traits. the percent white spikelets was even lower than 1%; this First, the copy number of the foreign genes in all trans- indicates that the damage induced by insect pests was genic rice lines remained unchanged during the two substantially inhibited. The slight variation in the percent years of the field experiments compared with non-trans - dead hearts and white spikelets of non-transgenic control genic control lines. Previous studies have indicated that lines in the two years of the experiment indicated that multiple heterologous transgenes are stably inherited in non-transgenic control lines were affected by variation diverse genetic backgrounds (Beaujean et al. 1998; Scott in the abundance of pests in these two years (Jiang et al. et al. 1998). Second, the expression patterns of exog- 2016). enous genes and proteins were similar among different Herbicide resistance was the second trait of interest transgenic lines. Previous studies have shown that the in our study. The EPSP gene and BAR gene are the most expression patterns of the CRY and BAR genes are simi- widely used genes in herbicide-resistant transgenic crops lar under different genetic backgrounds (Cao et al. 1992; (Green et al. 2014). The BAR gene from Streptomyces Chen et al. 1998; Yan et al. 2007; Zhou et al. 2016), but hygroscopicus used in this study confers high resistance the correlations between gene and protein expression to glyphosate herbicide. During the germination stage, varied among genes (Peach and Velten 1991; Datta et al. Basta application had a stronger effect on shoot growth 1998). Third, the higher expression of Bt genes and pro - compared with root growth, and no effects of Basta teins in the leaves compared with the stem and panicle application on transgenic lines were observed. Before of the transgenic rice lines might stem from differences the tillering stage, the chlorophyll content of transgenic in the location of the tissues on the plant (Fearing et al. lines began to return to normal after 2 d of herbicide 1997; Kranthi et al. 2005). There was a strong correla - application; at the tillering stage, the chlorophyll con- tion between the expression of exogenous genes and pro- tent of transgenic lines began to return to normal after teins, but the external environment had a greater effect 3 d of herbicide application. This difference is likely on the expression of proteins, which explains why protein explained by differences in the energy demands associ - expression differed between the two years of the experi - ated with vegetative growth and reproductive growth. ment (Jiang et al. 2014a, b). Ubiquitin promoter expres- More substances and energy are used for the develop- sion varied in different tissues and was generally higher ment of spikelets and floral organs during reproductive in leaves (Tang et al. 2006). Meanwhile, in maize and rice, growth, and this reduces the expression of the BAR gene where starch is the main storage substance, the expres- in the phloem of leaves and thus leads to a decrease in sion of exogenous genes regulated by constitutive pro- the chlorophyll content and photosynthetic efficiency. moter in the leaves of the source organ is higher than that Basta began to be applied in the 1990s when trans- in the ears of the pool organ and in rice (Jin et al. 2015). genic crops were introduced, and previous studies have However, this did not affect the resistance of the trans - mainly focused on the effects of Basta on photosynthe - genic lines to insect pests because the protein expression sis (Brookes et al. 2011). Previous studies have shown amplitude exceeded the lethal threshold of C. suppres- that Basta decreases the chlorophyll content and photo- salis (Ferré and Rie 2002; Chen et al. 2011). Finally, we synthetic efficiency by impeding ammonia digestion in found that the expression levels of CRY and BAR were plants, which decreases ammonia accumulation. Gener- high at heading stage, which might be related to the ally, it takes 2 to 3 d for Basta to be transmitted to various plant’s own energy metabolism, and the vigorous capac- parts of plants through the cuticle and cytoplasm (Vencill ity metabolism led to the high expression of exogenous et al. 2012). genes (Ye et al. 2010). In our study, the yield of transgenic lines was signifi - Insect resistance was the first trait of interest in our cantly higher compared with non-transgenic lines in a study. The stem and young panicle of rice plants are the pesticide-free environment. Analysis of yield compo- parts most susceptible to damage by C. suppressalis. We nents revealed that the percent dead hearts and percent found that larval mortality was similar among varieties white spikelets were low; thus, the number of undamaged and years when they were exposed to transgenic stem tis- panicles was high in transgenic lines because of their sue in the laboratory, and the resistance of all transgenic high resistance to insect pests and herbicides, and this lines with the CRY1C or CRY2A gene to target pests was high level of resistance was conferred by the CRY1C and over 80%, which indicated that genetic background had CRY2A genes. In addition, few leaves in transgenic lines Sun et al. Rice (2023) 16:8 Page 14 of 18 showed signs of damage, indicating that the introduc- double digested with BamHI + SacI, inserted into the tion of the BAR gene and CRY gene promoted increases CRY1C and CRY2A became the final transformation vec - in the chlorophyll content and photosynthetic efficiency tor. The CRY1C and CRY2A gene was driven by the rice under exposure to insect pests and herbicides (Xiao et al. ubiqultin promoter (Additional file 1: Fig S1). A strain 2007; Wang et al. 2012). Non-targeted effects are also of Agrobacterium tumefaciens EHA105, was used for important to consider. In general, the introduction of the transformation experiments. The callus culture and foreign genes can have deleterious effects on yield traits transformation procedures were carried out as described (Jiang et al. 2014b; Liu et al. 2022). In field trials under by Hiei et al. (1994). The donor parent introduced the normal pesticide management, no negative effects on foreign gene into the recurrent parent, then backcross important traits, such as the number of tillers, grains per and marker-assisted selection were performed to obtain panicle, and weight per 1,000 grain, were observed in all six Bt-transgenic rice lines. transgenic lines with the CRY1C, CRY2A, and BAR genes. This indicates that the yield traits of the transgenic lines Experimental Design were relatively stable compared with non-transgenic lines Field experiments were carried out from May to Octo- and that the transgenes had no substantial effect on yield ber in 2021 in the economic and technological develop- traits. ment zone (28˚48′10″ N, 115˚49′55″ E) of Nanchang City, Jiangxi Province, China. Table 1 shows the mean Methods monthly day and night temperature during the rice grow- Plant Materials ing season. The six Bt-transgenic rice lines CHT025(1C), In this study, we used six Bt-transgenic rice lines, CHT025(2A), CH891(1C), CH891(2A), CH871(1C), and CHT025(1C), CHT025(2A), CH891(1C), CH891(2A), CH121(1C) and the control lines Changhui T025, Chang- CH871(1C), and CH121(1C), and their non-transgenic hui 891, Changhui 871, and Changhui 121 were used in counterparts: Changhui T025, Changhui 891, Changhui field experiments. The field experiment was conducted in 871, and Changhui 121. Changhui T025, Changhui 891, a randomized block design with three replications. Each Changhui 871, and Changhui 121 were backcross lines plot was 5 m × 5 m. Twenty-day-old seedlings were trans- (Table 1). The seeds for these lines were cultivated by planted at a density of 15 cm × 20 cm with one seedling the Education Ministry Key Laboratory of Crop Physi- per hill. The soil type of the experimental site was red - ology, Ecology, and Genetic Breeding at Jiangxi Agri- dish-yellow clay-like paddy soil. Soil in the upper 15 cm cultural University, Nanchang, China. CRY1C, CRY2A at the experimental site had the following properties at and BAR were transferred into the donor parent MH63 –1 the start of the experiment: pH, 5.01; 1.26 g kg , total by Agrobacterium transformation. The pCAMBIA1300 –1 –1 N; 105.6 mg kg , available phosphorus; 125.2 mg kg , was digested with XhoI, the hygromycin phosphotrans- −1 potassium; and 20.56 g kg , organic matter. The exper - ferase (hph) gene was replaced with the phosphinotricin imental field was maintained in a flooded state from acetyltransferase (BAR) gene, flattened and then inserted transplanting until 7 d before maturity. Standard man- into the bar gene to form an intermediate vector. The agement practices were applied to control the spread of BAR gene was driven by CaMV 35S promoter (Cheng pests and diseases and the growth of weeds to minimize et al. 1998; Alam et al. 1999). Then the intermediate vec - yield losses. tor was double digested with HindIII + BamHI and con- nected to the ubiqultin promoter (Tang et al. 2006), After Genetic Background Detection A total of 512 simple sequence repeat (SSR) primers spanning the whole rice genome were used to screen the Table 1 Bt-transgenic rice lines (BC F and BC F ) and their 4 8 4 9 genomes of three transgenic lines and their correspond- respective non-transgenic counterparts used for the experiments ing recurrent parents, and the genetic background recov- in 2020 and 2021 ery rate was determined. DNA was extracted using the Type Bt line Control line cetyltrimethylammonium bromide (CTAB) method. The SSR primers were designed by the China Research Insti- Inbred line CHT025(1C) Changhui T025 tute and synthesized by Shanghai Shenggong Biotechnol- Inbred line CHT025(2A) Changhui T025 ogy Company. The 15-μL PCR reactions included 1.5 µL Inbred line CH891(1C) Changhui 891 −1 of 10 × PCR buffer, 2 µL of 100 ng•μL DNA template, Inbred line CH891(2A) Changhui 891 −1 0.3 µL of 2.5 mmol• L dNTPs, 10 µL of ddH O, 0.5 µL Inbred line CH871(1C) Changhui 871 −1 of 10 μmol• L positive and negative primers, and 0.2 µL Inbred line CH121(1C) Changhui 121 Sun et al. Rice (2023) 16:8 Page 15 of 18 Table 2 PCR primers used in this study Table 3 qRT-PCR primers used in this study. Primer Primer sequence PCR product Primer Primer sequence Description CRY1C-FTTC TAC TGG GGA GGA CAT CG 799 bp CRY1C-FAAA GAA TCG CTG AGT TCG CTAG qRT-PCR CRY1C-RCGG TAT CTT TGG GTG ATT GG CRY1C-RAAG AAG TCC ATC AAG GAT ACGG CRY2A-FCGT GTC AAT GCT GAC CTG AT 600 bp CRY2A-FTTC CTG CTG AAA TAA GGT GGGT qRT-PCR CRY2A-RGAT GCC GGA CAG GAT GTA GT CRY2A-RACG AGC GAG GGT GTC AGT GTT BAR-FGTC AAC CAC TAC ATC GAG ACA AGC BAR-FACA AGC ACG GTC AAC TTC C qRT-PCR 460 bp BAR-RAGC AGG TGG GTG TAG AGC GT BAR-RGAG GTC GTC CGT CCA CTC Actin-FTTC GGA CCC AAG AAT GCT AAG qRT-PCR Actin-RAAC AGA TAG GCC GGT TGA AAAC −1 of 5 U•μL Taq DNA polymerase. The thermal cycling conditions were as follows: 94 °C for 5 min; 30 cycles of 94 °C for 1 min, 57 °C for 1 min, and 72 °C for 1 min; and 6210A, Japan) according to the instructions of the Pri- 72 °C for 5 min. The PCR products were visualized using meScript RT Master Mix Kit. After ten-fold dilution of 8% polyacrylamide gel electrophoresis and rapid silver the cDNA, the target genes CRY1C, CRY2A and BAR and staining. The homozygous bands were labeled as 1, het - the reference gene Actin1 were detected by quantitative erozygous bands were labeled as 2, and missing bands real-time PCR (qRT-PCR) according to the instructions were labeled as 0. of the SYBR Premix Taq II Kit (TaKaRa) (Table 3). The program of the 7500 Real-Time PCR system (Thermo PCR and Southern Blot Analysis Fisher Scientific) was incubation at 95 °C for 2 min, fol - Specific primers for CRY1C, CRY2A and BAR were lowed by 40 cycles of 95 °C for 15 s and 68 °C for 30 s, designed for PCR reactions (Table 2). PCR was con- and a final extension step at 68 °C for 10 min. The purity ducted using the procedure described in the previous of the amplicons was confirmed in the presence of a sin - section. The CTAB method was used to extract genomic gle peak in the melting curve (Agostinetto et al., 2019). DNA. For southern blot analysis, 5 µg of genomic DNA Osactin1 was used as the internal reference gene for qRT- from each sample was digested with NcoI and HindIII, PCR normalization, and the qRT-PCR results were ana- −ΔΔCT separated on a 0.8% agarose gel, and then transferred to lyzed by the 2 method. a nylon membrane. A PCR-amplified fragment of CRY1C was used to prepare the probe. Next, 5 µg of genomic Quantitative ELISA Assay DNA from each sample was digested with BamHI and The amount of Cry1C, Cry2A and Bar proteins in leaves, EcoRI, separated on a 0.8% agarose gel, and then trans- stems, and panicles was measured at the tillering, boot- ferred to a nylon membrane. A PCR-amplified fragment ing, heading, filling, and maturity stages using an ELISA of CRY2A was used to prepare the probe. Lastly, 5 µg of kit (AP003 and AP005 CRBS; EnviroLogix Inc., Port- genomic DNA from each sample was digested with SamI, land, ME, USA). The absorbance value was measured at followed by separation on a 0.8% agarose gel and then 450 nm using a VICTOR Nivo multimode plate reader transfer to a nylon membrane. A PCR-amplified frag - (PerkinElmer, Waltham, MA, USA). Based on the range ment of BAR was used to prepare the probe. of the standard curve, the Cry1C, Cry2A and Bar pro- tein extract was diluted appropriately so that its absorb- Quantitative qRT‑PCR Assays ance value was within the range of the standard curve Total RNA was extracted from tissues at various devel- (Xu et al. 2018). For ELISA, a standard curve was drawn opmental stages by grinding in TRIzol (Merck, KGaA, based on the absorbance of known concentrations of Germany). DNase digestion was performed to avoid Cry1C, Cry2A and Bar standard (AP003 and AP005; contamination from genomic DNA, and the phenol– EnviroLogix). The concentration of each test sample was chloroform method was used to isolate total RNA. The determined from the standard curve, and the Cry1C, integrity of the extracted RNA was determined by 1.5% Cry2A and Bar protein content of the sample was cal- agarose gel electrophoresis, and RNA quantity and qual- culated based on its dilution ratio and the conversion ity were measured using a NanoDrop 2000 spectropho- −1 formula: Cry1C, Cry2A and Bar protein content (μg g tometer (Thermo Fisher Scientific Inc., Waltham, MA, −1 fresh weight) = test sample concentration (ng g ) × dilu- USA) based on the 260/280-nm and 260/230-nm absorb- tion × extract volume/tissue fresh weight (mg). ance ratios. Complementary DNA was synthesized using a PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Sun et al. Rice (2023) 16:8 Page 16 of 18 Insect Resistance in the Laboratory and the Field plants via foliar spraying. The chlorophyll content of rice Indoor insecticidal assays were conducted using 10 rep- plants was measured using a portable chlorophyll meas- licates of each transgenic line and non-transgenic con- urement instrument (SPAD, Soil and Plant Analyzer trol. An artificial diet was administered to C. suppressalis Development) at the seeding stage; chlorophyll content larvae for 9–10 d, and most of the borers developed to measurements were taken twice two days before Basta the second instar stage by 10 d. Second instar larvae were was applied, and once a day at 5:00 p.m. for 7 d follow- fed fresh transgenic rice leaves and stems that were col- ing Basta application. The chlorophyll content in rice lected from rice plants at the tillering and heading stages; leaves was measured at the tillering stage, booting stage, non-transgenic rice tissues at the same growth stages and filling stage after non-transgenic rice plants were were used as negative controls. Second instar larvae were transplanted. placed on individual Petri dishes that contained a piece of leaf (4 g) or stem (5 g), and ddH O was added to the filter Measurement of Grain Yield Traits paper to keep the environment of the Petri dish humid. Transgenic rice plants were planted in a paddy field in Petri dishes were sealed with parafilm membranes to pre - the Transgenic Experimental Plots of Jiangxi Agricul- vent larvae from escaping. All Petri dishes were stored in tural University (Nanchang, Jiangxi, China) for evalu- a hermetic box in the dark at approximately 27 ± 1˚C and ation of agronomic performance. The non-transgenic 70 ± 10% relative humidity. The leaves were weighed after control lines Changhui T025, Changhui 891, Changhui the in-situ assays were initiated; after 48 h, larval mortal- 871, and Changhui 121 were planted in paddy fields adja - ity was determined, and the mortality rate of C. suppres- cent to the transgenic lines. Six randomly chosen blocks salis was calculated. The insect resistance of transgenic of 6 m (2 m × 3 m) were used in field trials. There were plants in the field was assessed by artificially infesting approximately 100 plants in each block, and each plant rice plants with C. suppressalis. Chemical insecticides had approximately 10–15 tillers. The following five agro - that target lepidopteran pests were not applied through- nomic traits were measured for each plant: panicles per out the experimental period. Field assays were conducted plant, grains per panicle, weight per 1,000 grains, and using three replicates of each transgenic line and non- yield per plant. transgenic control. Fifty individual plants were planted in each replicate. At the tillering stage, 15–20 first-instar Data Analysis C. suppressalis larvae were applied to each rice plant. After PCR bands were scored, the data were digitized The number of dead hearts induced by stem borers was in MS Excel, and linkage maps were constructed using counted at the end of the maximum tillering stage, and CASS2.1 software. The recovery rate of the recur - the number of white spikelets was counted at the flower - rent parent background was calculated using the for- ing stage. g+1 mula E[G(g)] = 1-(1/2) . The response rate of the genetic background was calculated using the formula G(g) = [L + X(g)]/2L, where G(g) indicates the response Herbicide Resistance rate of the genetic background in the g generation, g indi- The herbicide resistance of transgenic rice plants was cates the number of generations used for backcrossing, evaluated at the bud stage through the application of the L indicates the number of molecular markers involved herbicide Basta. The seeds of six transgenic varieties and in the analysis, and X(g) indicates the number of band control varieties were germinated for 24 h, and ungermi- markers in the backcross g generation that are the same nated seeds were removed to ensure that the seed vigor as those of the recurrent parents. The gene and protein would be 100% in the experiment. Treatments were con- relative expression levels were processed and analyzed ducted in a hydroponic system. The germinated seeds using Excel 2007 and SPSS 16.00 (IBM Corp., Armonk, were placed into different hydroponic tanks and hydro - NY, USA). The borer mortality rate data were processed ponic solution per national standards. 10 mg/L Basta and analyzed using Excel 2007 and SPSS 16.00. Agro- was added to the hydroponic solution, and plants were nomic traits of transgenic plants were compared with cultured at room temperature for 7 d, with the culture the recurrent parents using one-way analysis of vari- medium replaced every 3 d. Bud length and root length ance (ANOVA). Values were presented as means (± SD). measurements were taken after the 7-d culture period. Data were analyzed by one-way ANOVA, and treatment Several varieties of genetically modified rice and non- means were compared using the least significant differ - genetically modified control seeds were germinated for ence test at P = 0.05. Figures were constructed using Ori- one day, planted in pots, watered every three days, and gin 2017 (OriginLab Corp., Northampton, MA, USA). fertilized on the tenth day after germination until the 27th d when 300 mg/L Basta was applied to the rice Sun et al. Rice (2023) 16:8 Page 17 of 18 and its additional files. All experiment of plant and all field experiments are Conclusion performed in our affiliated university. The newly bred high-generation transgenic rice lines (BC F and B C F ) with CRY1C, CRY2A, and BAR genes Code availability 4 8 4 9 Not applicable. all showed high genetic stability at the DNA, RNA, and protein levels. The high-generation transgenic lines also Declarations showed high resistance to insects and herbicides, and the yield of these transgenic lines was much higher than that Ethics approval and consent to participate of non-transgenic control lines in a pesticide-free envi- Not applicable. ronment. Under normal field management, the heterolo - Consent for publication. gous genes were stably inherited in these transgenic lines, Not applicable. and they had no effects on agronomic traits. Therefore, Competing interests the resistance of the transgenic rice lines to insect pests The authors declare no competing interests. and herbicides shows high genetic stability under various genetic backgrounds. Our findings indicate that CRY1C, Received: 15 September 2022 Accepted: 6 February 2023 CRY2A, and BAR could be used to breed new transgenic varieties. Supplementary Information References The online version contains supplementary material available at https:// doi. Alam MF, Datta K, Abrigo E, Oliva N, Tu J, Virmani SS et al (1999) Transgenic org/ 10. 1186/ s12284- 023- 00624-5. insect resistant maintainer line (IR68899B) for improvement of hybrid rice. Plant Cell Rep 18:572–575 Additional file 1： Table S1. Genetic background response rate Azhakanandam K, McCabe MS, Power JB, Lowe KC, Cocking EC, Davey MR statistics. Table S2. Relative expression level of CRY1C and CRY2A in (2000) T-DNA transfer, integration, expression and inheritance in rice: Bt-transgenic rice lines (BC F and BC F ). Table S3. Relative expression 4 8 4 9 effects of plant genotype and Agrobacterium super-virulence. J Plant level of CRY1C and CRY2A in positive and negative control. Table S4. 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Rice – Springer Journals
Published: Dec 1, 2023
Keywords: Bacillus thuringiensis; Genetic stability; Transgenic rice; Insect resistance; Herbicide resistance
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