Research Article

Overexpression of Arabidopsis CBF1 Gene in Transgenic Furenzhi banana (Musa spp. AA group) Improves Resistance to Low Temperature  

Chunhua Hu* , Kai Liu* , Yuerong Wei , Guiming Deng , Chunyun Li , Ruibing Kuang , Qiaosong Yang , Ganjun Yi
Fruit Tree Research Institute, Guangdong Academy of Agricultural Science/Key Laboratory of Biology and Genetic Resource Utilization of Fruit Trees in South Subtropics, the Ministry of Agriculture, Guangzhou, 510640, China
Author    Correspondence author
Molecular Plant Breeding, 2016, Vol. 7, No. 34   doi: 10.5376/mpb.2016.07.0034
Received: 05 Sep., 2016    Accepted: 26 Oct., 2016    Published: 16 Dec., 2016
© 2016 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Preferred citation for this article:

Hu C.H., Liu K., Wei Y.R., Deng G.M., Li C.Y., Kuang R.B., Yang Q.S., and Yi G.J., 2016, Overexpression of Arabidopsis CBF1 gene in transgenic Furenzhi banana (Musa spp. AA group)improves resistance to low temperature, Molecular Plant Breeding, 7(34): 1-10 (doi: 10.5376/mpb.2016.07.0034)

Abstract

Cold is an environmental factor that limits the growing season of banana and adversely affects fruit quality and productivity. C-repeat/dehydration responsive element binding factor 1 (CBF1) play important roles in the responses of plants to low-temperature and cold. To test the function and potential use of cold-induced factor CBF1 in improving the chill tolerance of banana, AtCBF1 driven by a cauliflower mosaic virus 35S promoter was introduced into banana plants by Agrobacterium-mediated transformation of embryogenic cell suspensions (ECSs) of banana cultivar Furenzhi (AA). The presence of AtCBF1 transgenes in regenerated plants was confirmed by polymerase chain reaction (PCR) and Southern blots. ß-glucoronidase (GUS) histochemical assays reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR analysis demonstrated that foreign genes were stably expressed in regenerated plants. Transgenic banana showed growth retardation, thicker leaves and higher chlorophyll content than non-transformed plants. While relative electrolyte leakage (REL) and malondialdehyde (MDA) content were significantly lower in transgenic banana plants than in non-transformed plants under low temperature stress. An obviously higher cold tolerance was observed among the transgenic plants in the cold detection. These results suggest that over-expression of AtCBF1 in transgenic banana plants plays an important role in improving tolerance to low-temperature.

Keywords
Banana; AtCBF1 gene; Transgenic; Cold resistance

Introduction

The transcription network regulated by C-repeat-binding factors (CBFs) plays important role for increased freezing and drought tolerance in plants (Jaglo et al., 2001; Hsieh et al., 2002), the CBF pathway is transiently induced by cold and transitive downstream genes by binding to the CRT/DRE motif (CCGAC) in target gene promoters with the conserved AP2/EREBP DNA binding domain (Liu et al., 1998). Previous reports have indicated that overexpression of the Arabidopsis CBF1 (AtCBF1) gene in transgenic Arabidopsis induced the expression of multiple CRT/DRE-containing genes and resulted in an increase in freezing and drought tolerance in both non-acclimated and cold-acclimated plants (Liu et al., 1998; Kasuga et al., 1999), similar results were observed with the constitutive overexpression of the AtCBF1 gene in transgenic brassica napus, sweet cherry, potato, tobacco, poplar, strawberry and eucalyptus plants (Jaglo et al., 2001; Kitashiba et al., 2004; Pino et al., 2008; Benedict et al., 2006; Navarro et al., 2011), the results indicate that transformation of AtCBF1 gene may represent a strategy in freezing tolerance breeding for freezing-sensitive crops.

 

Banana (Musa sp.) is the fourth important global food crop after rice, wheat and maize in terms of gross value of production (Tripathi et al., 2004). It is widely cultivated in the tropical regions, and some south subtropical regions such as Yunnan, Guangxi and Guangdong province in China, are also engaged heavily in the production of banana. But banana is extraordinary sensitive to low-temperature, when the temperature decreases to 8°C, banana growth is arrested in the interior of the pseudostem and injury occurs (Zhang et al., 2011). Periodic cold stresses during winter or early spring are the main environmental constraints to the banana industry in south subtropical regions; enormous economic losses were incurred each year due to cold injuries (Kang et al., 2003). Therefore, genetic improvement on freezing tolerance is one of the major breeding goals of banana, especially for the banana cultivation in China. However, the most important commercial banana cultivars are triploid and sterile, and no true cold–resistant germplasm is available although plantain is relatively colder tolerant (Zhang et al., 2011), it is very difficult to obtain cold-resistant cultivars through conventional breeding methods. Genetic engineering is obviously the best choice for banana breeding, transgenic technology allows the target plants to obtain wanted gene (s) immediately, avoiding a long period of the selection process involved in conventional breeding practice.

 

The aim of this study was to introduce the AtCBF1 gene into banana, and determine whether introgression AtCBF1 enhanced chilling stress tolerance in transgenic banana. Our study led to further development of molecular responses in Musa to colds tress and establishes a foundation for cloning cold resistance relative genes in Musa.

 

1 Results

1.1 Regeneration of transgenic plants

AtCBF1 overexpression construct driven by CaMV 35S promoter was introduced into ECS of Furenzhi by Agrobacterium-mediated transformation. Transformed cells were GUS stained 30 days post selection, GUS expression could be detected in majority of the selected cells pointed out to good transformation efficiency. After liquid selection for 30 days, the liquid selected ECS (Figure 1A) were subcultured to M3 medium and embryos developed after 2 months (Figure 1B). The mature embryos were germinated on M4 medium (Figure 1C) and rooted on rooting medium (Figure 1D). After which, the shoots were transferred to RM medium and adventitious roots developed in 2 weeks. Histochemical assay for GUS activity revealed that all of the 55 regenerated plants were positive (Figure 1F).

 

 

Figure 1 Regeneration of transgenic banana plants

Note: (A) Agrobacterium-mediated transformation of the ECS of banana cultivar Furenzhi (AA) was selected in liquid M2 medium. (B) The resistant embryos were induced on M3 medium. (C) The germination of resistant embryos was on M4 medium. (D) The resistant plants were rooted on RM medium. (E) The transgenic and non-transgenic plants were transplanted in a greenhouse. (F) GUS staining of tissues of transgenic plants

 

The regenerated plants were tested for the presence of transfer DNA (T-DNA) using PCR with primers specific for AtCBF1 gene, About a fragment of 640 bp was amplified from leave DNA of transgenic plants and the constructed plasmid p1301-AtCBF1 (positive control) with AtCBF1 specific primers, but not be found in no-transformed plants (negative control) (Figure 2A). All of the resulted in PCR-positive clones were selected for propagation. During the propagation procedure, most of the transgenic buds grew much slowly and not have good multiplication or root formation ability as compared to the control. 12 of the 55 lines succeeded in regenerating at least 50 whole plants. These putative transgenic plants were transplanted in pots and grown in greenhouse. Unfortunately, Two months after the transfer from in vitro to soil, it was observed that all the transgenic lines showed severely retarded growth, obviously altered morphology, and lower survival rates compared with wild-type phenotype (Figure 1E), based on their comparative ease of manipulation to produce plantlets, only transgenic line T1 and T4 can get enough plantlets for further molecular analyses and physiological and biochemical assays. The result of Southern blot analysis indicated that a single AtCBF1 gene copy was present in the transgenic line T1 and T4, whereas the untransformed plants had no hybridization signal (Figure 3).

 

 

Figure 2 AtCBF1 were confirmed from transgenic and non-transgenic plants by PCR, RT-PCR and qRT-PCR, respectively

Note: (A) PCR with specific primers; (B) RT-PCR; (C) qRT-PCR. M: DNA marker; P: AtCBF1 detected from plasmid p1301-AtCBF1; C: Non-transgenic banana plants; T1, T2, T4, T5, T6, T7 and T9: Seven transgenic lines. Arrow indicated GUS gene, AtCBF1 gene and banana actin gene, respectively. Data in C are means ± SD from two independent biological replicates with three repeated trials

 

 

Figure 3 Southern blot analyses for AtCBF1 gene transgenic clones of Furenzhi banana. CK wild-type plant; T1 and T4 transgenic lines

 

1.2 Transgene expression analysis

The expression of both AtCBF1 and GUS in transgenic lines was detected by using RT-PCR (Figure 2B). Real time quantitative PCR was used for further analysis of the expression levels of foreign AtCBF1 gene. Both T1 and T4 showed the expression of the integrated gene, the constitutive expression of AtCBF1 was indistinguishable between transgenic line T1 and T4 (Figure 2C). The results further indicated that the AtCBF1 gene has been already transformed into the banana genome and were significantly more highly expressed in transgenic seedlings.

 

1.3 Cold resistance analysis of transgenic banana

To confirm whether the cold tolerance of transgenic banana plants improved, the same old and robust transgenic T1 and T4 line and non-transformed seedlings were chosen for cold treatment (Figure 4A). When plants transferred to a cold chamber at 4 °C for three days, leaves of no-transformed plants displayed severe dehydration and wilting (Figure 4B). However, the transgenic plants only exhibited dehydration of several leaves grown near the top of the plants. After returned to room temperature for 3 days, the transgenic banana overcame dehydration and achieved complete recovery (Figure 4C). However, the wild-type banana displayed severe chlorosis and wilting and suffered some level of irreversible damage, when recovered for 7 days, all the no-transformed plants died. These results intuitively indicated that transgenic banana over-expression of AtCBF1 showed improved cold tolerance and enhanced recovery from cold treatment.

 

 

Figure 4 Detection of the tolerance of transgenic and non-transgenic banana plants to low temperature

Note: (A) Under normal condition (non-stress); (B) Treated at 4 °C for 3 days; (C) Recovered at 25°C for 3 days

 

1.4 Chilling tolerance in AtCBF1 transgenic banana plants

The REL of transgenic T1 and T4 line and non-transformed plants were measured at different low-temperature treatments. The REL of transgenic lines was lower than that of non-transgenic plants (Figure 5) at same condition; the results indicated that the plasma membranes were damaged more serious in banana wide type than transgenic lines under different freezing conditions.

 

 

Figure 5 Changes of relative electrolyte leakage of banana leaves under different low temperature

Note: CK: Non-transgenic plants; T1, T4: Transgenic lines. Data are means ± SD from three independent replicates

 

MDA concentration in non-transformed plants was higher than in the transgenic banana plants before the cold treatment (Figure 6), and there was a significant increase in the MDA concentration in no-transgenic plants after 3 days of chilling stress treatment, but changed little in transgenic lines. The increase of MDA paralleled the increase in electrolyte leakage during cold stress in no-transformed plants; this finding suggested that higher oxidative lipid injury as compared to transgenic plant which had lesser MDA concentration.

 

 

Figure 6 Changes of malondialdehyde (MDA) in transgenic lines and non-transgenic banana plants under 7°C condition for five days

Note: CK (●): Non-transgenic plants; T1(○), T4(▼): Transgenic lines. Data are means ± SD from three independent replicates

 

2 Discussion

Banana is very sensitive to low temperature, which is easily damaged by cold wave. Therefore, in the present work, using Agrobacterium-mediated transformation, the full- length AtCBF1 gene was introduced into banana cultivar Furenzhi in order to obtain cold resistance banana. The PCR and Southern blot analysis showed that the transgene had integrated into the banana genome. GUS histochemical assays, RT-PCR and real-time PCR analysis also indicated that foreign genes were constitutively expressed in transgenic lines. The transgenic banana over-expression of AtCBF1 gene showed better chilling tolerance in comparison to no-transformed plants. But the CBF1 overexpression negatively affected the growth of transgenic plants, similar results were observed in former studies (Kasuga et al., 1999; Hsieh et al., 2002). In this study, we obtained a batch of transgenic buds regenerated from mature embryos. The buds grew much slowly and not have good multiplication or root formation ability. Moreover, when the in vitro plantlets transferred to soil, most of the transgenic plants died compared to the control, it is difficult to obtain enough transgenic plants. The survived plants showing dwarf phenotype in greenhouse, compared with no-transformed banana, the length between leaves of the transgenic plants is shortened, causing dwarf phenotype (Table 1). It was previous reported that over-expression of AtCBF1 or hyper accumulation of CBF1 protein affected GA biosynthesis in the transgenic tomato plants (Hsieh et al., 2002). More direct reason was that overexpression of AtCBF1 gave rise to accumulation of DELLAs protein which hindered GA biosynthesis in transgenic Arabidopsis thaliana plants (Achard et al., 2008). Therefore, the reason of dwarf phenotype might be also that GA biosynthesis was hindered in transgenic banana plants, but needed to verify further. However, the dwarf phenotype of transgenic plants overexpressing AtCBF1 could be reversed by GA3 treatment (Hsieh et al., 2002) or using stress inducible promoter instead of constitutive promoter CaMV35S (Lee et al., 2003; Singh et al., 2011) to either eliminate the negative agronomic characters or improve capacity to cold tolerance. Besides, the leaves of transgenic potato overexpressing AtCBF1 became thicker and dark green compared with non-transgenic plants, which showed that heterologous expression of AtCBF1 could change cell structure and chlorophyll synthesis of leaves (Pino et al., 2008). The number of layers of palisade tissue increased in transgenic Arabidopsis thaliana plants over-expressing AtCBF1 relative to non-transgenic plants (Gilmour et al., 2004). Similar results acquired in this research showed that thicker leaves and higher chlorophyll contents were in transgenic banana plants than in non-transgenic plants (Table 1).

 

 

Table 1 The effects of AtCBF1 on the phenotype of transgenic banana plants

 

When plants are encountered with abiotic stress such as low temperature, the structure of the cell membrane may be damaged, semipermeabilities of plasma membrane disappear and ions or organics leak out from cells resulting in increasing conductivity of surrounding medium. At the same time, chilling stress affects the integrity of plant membranes and lipid composition through accumulation of ROS such as O2-and H2O2, which causes peroxidation of membrane lipids to produce MDA (Moran et al., 1994). Therefore, ion leakage rate and MDA content are sensitive diagnostic indexes of stability of biological membrane systems. In this study, the REL and MDA contents of leaves in transgenic banana lines overexpressing AtCBF1 were lower than in non-transgenic plants under low-temperature stress, which indicated the integrity of transgenic plants membranes were better than that of non-transgenic plants under chilling stress. Our results here were parallel to the previous work (Hsieh et al., 2002; Benedict et al., 2006; Lee et al., 2003; Yang et al., 2010; Zhang et al., 2011), which demonstrated that over-expression of AtCBF1 in transgenic plants could protect cell membrane effectively from damaging and maintain stability under low-temperature.

 

Plants adapting chilling stress occur various changes including characteristics of physiology, morphology and gene expression (Pino et al., 2008). In this study, transgenic banana plants over-expressing AtCBF1 started to recover growth after treated under 4 °C for three days and at 25 °C for three days, but non-transformed plants had been died (Figure 5). These results showed that cold resistance of transgenic banana plants had been improved and also could infer that heterologous expression of AtCBF1 induced some genes expression of transgenic banana plants and leaded to physiological and biochemical reactions in connection with winter hardiness. In our preliminary work, we acquired a clp gene by Suppression Subtractive Hybridization (SSH) from transgenic banana plants, which was relative to cold stress (data not shown). It also confirmed further that over-expression of AtCBF1 regulated certain genes expression of banana. And meanwhile, there was possible a similar cold inducible CBF signal pathway in banana to improve cold tolerance, but needed to be further confirmed.

 

There were few reports on genetic engineering technology to improve abiotic stress resistance of banana. To our knowledge, the research reported here is the first to describe over-expression of AtCBF1 in an economically important fruit crop banana cultivar Furenzhi to improve tolerance to low temperature. The upcoming work, we will excavate stress inducible promoter instead of CaMV35S used in present study in order to either improve tolerance to low temperature or avoid negative agronomic characteristics of transgenic banana plants caused by over-expression of AtCBF1.

 

3 Materials and Methods

3.1 Plant materials

Embryogenic cell suspensions (ECS) of banana cultivar Furenzhi (Musa spp. AA group) were obtained from immature male flower (Hu et al., 2013) and maintained in MS M2 liquid medium containing MS basal medium supplemented with 100 mg/l glutamine, 100 mg/l malt extract, 1 mg/l biotin, 1 mg/l 2,4-D and 45 g.l-1 sucrose. The pH of the medium was adjusted to 5.3 prior to autoclaving. ECS were sub-cultured in M2 medium every 2 weeks by transferring about 0.5 ml packed cell volume (PCV) into 30 ml fresh M2 medium. ECS sub cultured in M2 medium for 7 days were harvested by centrifuging at 1 000 rpm for 10 min, and 1 ml packed cells were prepared for transformation experiments.

 

3.2 Transformation, selection and regeneration of ECS

EHA105 strain of Agrobacterium tumefaciens harboring the plasmid pCAMBIA1301-AtCBF1 carrying the desired CBF1 gene driven by cauliflower mosaic virus (CaMV) promoter were used in this study (Figure 7), The Agrobacterium was initially activated and incubated on solid LB medium(Yang et al., 2011), supplemented with 50 mg/l kanamycin (Sangon, Shanghai, China) at 27 ± 1°C for 48 h to acquire single colonies. A positive single colony of this Agrobacterium was incubated in 20 mL of liquid LB medium containing 50 mg/l kanamycin and 25 mg/l rifampicin (Sangon, Shanghai, China) at 27 ± 1°C with shaking at 200 rpm overnight. The Agrobacterium was collected by centrifugating under 6 000 rpm for 5 min and resuspended to OD600 of 0.2 with fresh LB medium containing the same antibiotics at the same concentrations, and then placed back on the shaker at 200 rpm and 27 ± 1 °C to the OD600 of 0.6-0.8. Finally, the cultured Agrobacterium were pelleted immediately by centrifugation at 6 000 rpm for 10 min and resuspended in M2 medium. The bacterial density was adjusted to an OD600 of 0.2 and added 100 μM Acetosyringone (ACS) (Sigma-Aldrich, USA) as engineering bacteria for transformation.

 

 

Figure 7 Plant expression vector of AtCBF1 gene

 

About 1 ml PCV of ECS was immersed in 10 ml bacterial suspension with 100 μM acetosyringone (Aldrich Chemical Co) for 30 min on the shaker at 50 rpm and 27 ± 1°C in darkness. After infection, the explants ECS were rinsed two times with 40 ml fresh M2 medium, and then transferred to 30 mL M2 medium for co-culture, which was carried on a rotary shaker at 27 ± 1°C in the dark for three days. After that, the co-cultured ECS was transferred to liquid selected medium M2 containing 300 mg/l cefotaxime and 20 mg/l hygromycin for a month and sub-cultured every two weeks. Then the selected ECS was transferred to embryo induction medium M3 mediums (pH 5.3) contained SH macronutrients, SH micronutrients(Liu et al., 2012), MS vitamins, 1 mg/l biotin, 100 mg/l glutamine, 100 mg/l malt extracts, 230 mg/l proline, 0.2 mg/l NAA, 0.1 mg/l kinetin, 45 g.l-1 sucrose, 10 g.l-1 lactose and 2.1 g.l-1 phytagar, and supplemented with 300 mg/l cephalosporin and 20 mg/l hygromycin for inducing resistant embryos. The cultures were sub-cultured every three weeks in the dark at 27 ±1 °C for three months. Then, putatively transformed somatic embryos were transferred to germination medium (M4 medium) with 200 mg/l cephalosporin containing MS macronutrients, MS Micronutrients, 1 mg/l 6-Benzylaminopurine (BAP), 0.2 mg/l NAA, 30 g.l-1 sucrose and 2.1 g.l-1 phytagar. The pH of M4 media was adjusted to 5.8 prior to autoclaving. The cultures was carried out at 27 ± 1°C under a photoperiod of 16 h (light) / 8 h (dark) at 100 μmol photons.m-2.s-1 for about 1 month. The developed shoots were then transferred to rooting medium (RM medium) (pH 5.8) containing MS macronutrients, MS Micronutrients, 0.1 mg/l NAA, 30 g.l-1 sucrose and 2.1 g.l-1 hytagar for a month with the same growth conditions to embryo germination. The regenerated plantlets were hardened by transferring to a sterile culture medium mixed with humus and soil at 1: 1 ratio. The hardened plants placed in greenhouse for further growth which would be used for molecular analysis and for the evaluation of resistance to chilling.

 

During each transformation experiment, about 1 ml PCV of ECS were not treated with Agrobacterium, but went through the whole regeneration process along side the infected ones. The regenerants obtained were used as the control in further analyses.

 

3.3 Molecular confirmation of transgenic plants

ECS, somatic embryos, germinated somatic embryos, leaf and root sections from both non-transformed and transformed cultures were tested for GUS expression. The GUS-positive plants were analyzed by polymerase chain reaction (PCR) with primers specific for the AtCBF1 gene. Genomic DNA was isolated from leaves of the putative transgenic plants and the non-transformed plants using DNA easy plant mini kit (Qiagen, GmbH, Germany). The primers for the AtCBF1 gene were: 5’-ctctagaatgaactcattttcagc-3’ and reverse 5’-cggagctcttagtaactccaaagcgac-3’. The following thermal cycling conditions were set for amplification: 94 °C for 4 min followed by 35 cycles each with 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min with a final extension 72°C for 10 min.

 

The integration of AtCBF1 gene of the PCR-positive plants was performed by southern blot analysis. Genomic DNA was isolated from in vitro-grown plants and digested with restriction enzyme EcoRI, separated by electrophoresis on a 1% agarose gel and transferred to a Hybond-N nylon membrane (Amersham Pharmacia Biotech, USA). The probe of the AtCBF1 gene was obtained by PCR amplification and labeled by PCR DIG Labeling Mixplus Kit (Roche Diagnostics, Switzerland). Hybridization and detection were carried out using DIG High Prime DNA Labeling and Detection Starter Kit II (Roche Diagnostics, Switzerland), according to the manufacturer’s instructions.

 

3.4 Expression analysis of AtCBF1 gene in transgenic banana plants

Expression of AtCBF1 and GUS gene in transgenic plants was confirmed using RT-PCR. Total RNA was isolated using Column Plant RNAOUT (TiandzInc, Beijing, China) from non-transformed and putative transgenic plants. One microgram RNA was used for the retro-transcription in a 20μL reaction solution containing 1×M-MLV buffer, 10 mM dNTPs, 10 μM oligo-dT primer, 15 U RNasin, 100 U M-MLV(Takara, Japan) incubated for 1 h at 42°C and 5 min at 95°C. The first cDNA was used was amplified with the specific AtCBF1 primers under the same conditions described for PCR amplification.

 

Real-time PCR was further performed to analyze the expression levels of the transgenes using the SYBR green PCR master mix (Takara, Osaka, Japan) in optical 96-well reaction plates (Roche, Germany) on LightCycler®480II (Roche, Germany). Primers of inserted AtCBF1 used for real-time PCR amplification were 5’-ggcgttggcttttcaagatg-3’ and 5’-aagtcggcatcccaaacatt-3’, RPS2 gene was used as internal marker (5’-tagggattccgacgatttgttt-3’ and reverse 5’-tagcgtcatcattggctggga-3’). The reaction system included 20 μl contained 10 μl SYBR Premix Ex TaqTM II, 2 μl of diluted cDNA and 0.8 μl of each specific primer (10 μM) and 6.4 μl sterile dH2O. Cycling was performed using the conditions: 94°C for 2 min; 40 cycles of 94°C for 20 s, 60°C for 20 s, 72°C for 20 s. Data were normalized using the △△Ct method. Three repeated trials were done using two independent biological replicates.

 

3.5 Bioassay for cold stress tolerance

Cold stress tolerance test, Both transgenic and wild type plantlets were transplanted in pots and placed in a growth cabinet under normal growth conditions (28°C/22°C day/night, 75% relative humidity, 12-h photoperiod with 50 μmol m-2 s-1 illumination), Two months after the transfer from in vitro to soil, six well-grown uniform and healthy saplings from each selected transgenic lines were chosen at random for the present experiment, These saplings were treated with 4°C/4°C day/night for 3 days, and recovered at 28°C/22°C day/night with the same light/dark regime and relative humidity mentioned above. The experiment was replicated three times. Physiological and biochemical parameters such as relative electrolyte leakage (REL) and malondialdehyde (MDA) content were also assayed in both transgenic and non-transformed plants under normal (non-stress) and stress conditions.

 

Relative electrolyte leakage (REL) assay, Three plants from the two months transgenic and non-transgenic plants were chosen at random to measured for REL content under various low-temperature treatment. The same position leaves from each group were washed by deionized water and dried using absorbent paper. These leaves were punched into 0.6 cm diameter segments, 0.3 g of which were treated under 0 °C, -4 °C, -8 °C and -12 °C for 30 min respectively. Afterwards, 10 mL deionized water was added into each processed tube for incubating at 4 °C for 30 min and at room temperature for 1 h and then the freezed conductivity (S1) of which was determined with a conductivity meter (WTW, Germany). Hereafter, they were transferred to a boiling water bath for 10 min and complemented with deionized water up to 10 ml. After they cooled in running water, the boiled conductivity (S2) of which was measured. The relative electrolyte leakage was calculated by the following formula: REL= S1 / S2 × 100. For statistical analysis, the mean value of three tested plants of each group and for each treatment was calculated and used for comparing with the non-transformed plants.

 

Malondialdehyde (MDA) content assay, the comparative rate of lipid peroxidation was assayed from the transgenic and non-transformed leaves by determining the level of MDA at 7 °C for five days, which was determined by the thiobarbituric acid (TBA) reaction according to the method of Pan et al. (2006). 0.5 g leaves were thoroughly ground with a precooled mortar and pestle in 5 mL 10 % (m/v) trichloroacetic acid (TCA) reagent. The homogenate was centrifuged at 6 000 rpm for 10 min at 4 °C and the supernatant was collected as crude MDA extract used for measurement. The reaction mixture contained 2.0 mL MDA extract and 2.0 mL 0.6% (m/v) TBA reagent (TBA dissolved in 10% (m/v) TCA), and 2.0 mL deionized water instead of MDA extract was added in as a blank control. The reaction mixture was boiled for 15 min, cooled in ice-water bath and again centrifuged at 6 000 rpm for 10 min. The supernatant was subjected to assay with spectrophotometer (PerkinElmer, USA) and absorbance was read at 450, 532 and 600 nm, which were used to calculate the concentration of MDA. Statistical analysis was same as above.

 

Morphological analysis, Morphological recorded on plant growth, plant height, number of leaf, thickness and color of leaf were studied in transgenic banana as compared to non-transgenic plants under normal condition. The chlorophyll contents of fully expanded leaves were determined referring to the method described by Liu et al. (2012). Leaves from the same position of transgenic and non-transgenic plants were made into cryo-sectioning, observed and measured using inverted fluorescence microscope (ZEISS, Germany). 15 plants of transgenic lines and non-transformed plants were observed and recorded.

 

Authors' contributions

Hu Chunhua carried out the studies and drafted the manuscript. Liu Kai participated in its design, performed the transformation experiment and helped to draft the manuscript. Wei Yuerong and Deng Guiming participated in the design of the study and performed the morphological analysis. Li Chunyun, Kuang Ruibing and Yang Qiaosong participated in the evaluation of the transgemic banana. Yi Ganjun conceived of the study, participated in its design and helped to draft the manuscript. All authors read and approved the final manuscript.

 

Acknowledgements

This research was supported by the Science and Technology Plan Project of Guangdong Province (2015A050502037, 2015A030302045), the Natural Science Foundation of China (C150102), the Natural Science Foundation of Guangdong (2016A030310326), the Project of Livelihood Science and technology from Guangzhou Science and information Bureau (2014Y2-00519), the Project of Tropical and Subtropical Fruits Germplasm Innovation and Breeding Technology Platform Building (2014B070706018), and the Rural Areas of Science and Technology Project from Science and Technology Department of Guangdong Province (2016A020208007).

 

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