Abstract
Elephant foot yam (Amorphophallus paeoniifolius (Dennst.) Nicolson), is an important edible tropical tuber crop, belonging to the family Araceae. Corms produced by this plant is very big and they are rich in starch, protein, mineral, vitamins, and dietary fiber but has acridity problem. This crop is susceptible to virus and phytoplasma diseases which affects crop growth and corm yield. Even though this crop has high commercial value, the problems like susceptibility to viral diseases, acridity problems, and lack of genetic diversity made hindrance in their exploitation. These issues can be resolved only by improving the characters through genetic transformation. To achieve genetic transformation in this important crop, a study was conducted to optimize various parameters for efficient Agrobacterium-mediated genetic transformation using embryogenic calli with vectors having gus reporter gene. Calli were developed using petiole and leaves of in vitro plantlets of elephant foot yam cultivar Gajendra and experiments were conducted to evaluate the sensitivity of calli to different doses of antibiotics viz. geneticin, hygromycin, ticarcillin. It was observed that complete death and discoloration of the calli were obtained with 25 mgl−1 geneticin and 10 mgl−1 hygromycin. The lowest lethal concentration of ticarcillin against Agrobacterium growth was found to be 500 mgl−1 which did not affect calli growth. Optimized parameters for efficient transformation in elephant foot yam include 100 μM acetosyringone concentration with 2 days of co-cultivation at temperature 22 °C using LBA4404 strain. The putative transformants were characterized for the integration of the gus gene using PCR and nucleic acid spot hybridization. The optimized protocol is simple and reproducible and may be adapted for other cultivars also.
Supplementary Information
The online version contains supplementary material available at 10.1007/s13205-021-02824-6.
Keywords: Elephant foot yam, Genetic transformation, GUS assay, Molecular analysis
Introduction
Elephant foot yam (EFY) (Amorphophallus paeoniifolius (Dennst.) Nicolson), is an important tuberous vegetable belonging to the family Araceae and sub family Aroideae. Its origin is considered to be in South-east Asia and grown widely in the Philippines, Malaysia, Indonesia, and Southeast Asian countries. It is widely cultivated in many states of India, owing to its high production potential, medicinal, nutritional, and commercial value (Ravi et al. 2009). It is cultivated in an area of 30,000 ha with a production of the potential of 774,000 mt in some elephant foot yam growing states like Kerala, Andhra Pradesh, and Odisha in India. The corms of elephant foot yam are popularly used as a vegetable in various delicious cuisines and preparation of indigenous ayurvedic medicines (Misra et al. 2002). The corms are rich in starch, protein, mineral, vitamins, and dietary fiber. Among the diseases affecting this crop, mosaic disease caused by dasheen mosaic virus is an important one. The occurrence of mosaic disease of elephant foot yam in India has been reported as early as 1960s (Capoor and Rao 1969). A major concern of the viral infection is the reduction in the tuber yield, due to the perpetuation of the virus through infected planting materials. In India, 24–88% mosaic incidence with a maximum yield loss of upto 38% has been reported from major elephant foot yam growing areas (Nehalkhan et al. 2006; Babu e al 2011; Kamala et al. 2015). Recently, phytoplasma disease too has been reported in elephant foot yam from the samples collected from Uttar Pradesh (Rao et al. 2020).
Considering the commercial potential of this crop, efforts should be directed to develop disease and pest resistance, enhance the nutritional quality, and improving the tuber palatability by developing non-acrid varieties. Low germplasm variability of elephant foot yam is the major limitation to address these issues. The need to transfer desirable genes to elephant foot yam for addressing the above limitations necessitates the use of transgenic technology.
In vitro regeneration protocol for this crop has been well established and being utilized for producing virus-free plants (Kamala and Makeshkumar 2014, 2015). However, till now only a few attempts have been made for genetic transformation of elephant foot yam, which showed a very low frequency of transformation (Ban et al. 2009; Kamala and Makeshkumar 2013). To overcome these constraints, the present study was undertaken to optimize the parameters to achieve efficient transformation in elephant foot yam.
Materials and methods
The test variety of Amorphophallus paeoniifolius (Dennst.) Nicolson chosen for genetic transformation study Gajendra was procured from the plant field of ICAR-Central Tuber Crops Research Institute (CTCRI), Kerala, India. Callus was developed from the petiole of explants and leaves of in vitro plantlets in modified Murashige and Skoog (MS) medium (Murashige and Skoog 1962) supplemented with 0.5 mg l−1 of 6-benzyl aminopurine (BAP), 2,4-dichlorophenoxyacetic acid (2,4-D) and α-naphthaleneacetic acid (NAA), respectively. The calli were subcultured periodically and maintained under a light intensity of 2500 lx with a 16 h light/8 h dark cycle (at 25 ± 2 °C) (Kamala and Makeshkumar 2013). Embryogenic calli of 6 weeks old were used for the transformation experiments.
Generally, two types of antibiotics were used in genetic transformation studies, which included antibiotics for selecting transformed tissues—geneticin and hygromycin and antibiotics for killing the Agrobacterium cells after the co-cultivation—ticarcillin. The sensitivity of the elephant foot yam calli was monitored and recorded by culturing them in the callus induction medium (CIM) under different concentration of geneticin (0, 5, 10, 15, 20, and 25 mg l−1) or hygromycin (0, 10, 20, 30 40, and 50 mg l−1) as antibiotics for selection. Similarly, the effect of ticarcillin for suppressing the growth of Agrobacterium and sensitivity on callus was also observed and recorded in CIM containing different concentrations (0, 250, 500, and 750 mg l−1). In all these cases, the response of the calli was scored at weekly intervals as + Turning black and dead; + + Partially discolored; + + + Healthy tissue.
Three strains of Agrobacterium tumefaciens viz, AGL0, LBA4404, GV3103 with 2 different binary vectors (viz, pOYE153, pCAMBIA 1305.2) were used for the study. The T-DNA of the pOYE153 vector contains the gusA reporter gene under the control of CaMV35S promoter and the selectable marker gene nptII (selection using 80 mg ml−1 kanamycin) and pCAMBIA 1305.2 vector contain the gusPlus reporter gene under the control of CaMV35S promoter and the selectable marker gene hpt (selection using 80 mg ml−1 hygromycin). Agrobacterium strains were cultured in YEB medium with 2 mM MgSO4 (AGL0) and 20 mg l−1 rifampicin (LBA4404 and GV3103). Strains AGL0 and LBA4404 had pOYE153 vector and GV3103 had pCAMBIA 1305.2 vector.
The suspension of Agrobacterium for co-cultivation was prepared in a 5 ml YEB medium containing MgSO4 (2 mM) with appropriate antibiotics. The cultures were incubated at 28 °C at 200 rpm for 72 h. The culture of 3 days old was inoculated to 25 ml YEB with MgSO4 (2 mM) and appropriate antibiotics and incubated overnight at 200 rpm at 28 °C. The Agrobacterium culture with OD600 = 1.0 was centrifuged at 5000 rpm at 4 °C for 10 min. The pellet was washed twice in liquid MS further the pellet was suspended in the same volume liquid MS (Kamala and Makeshkumar 2013). Embryogenic callus for transformation was pre-cultured in CIM one day before co-cultivation with agrobacterium. Agrobacterium suspension (500 μl) was slowly added (drop by drop for 3 min) to embryogenic pre-cultured in CIM one day before co-cultivation. Excess Agrobacterium suspension was pipetted out from the calli and incubated at 22 °C in dark for 2 days.
Different parameters affecting transformation efficiency i.e., acetosyringone concentration, incubation temperature, number of days of co-cultivation, and efficient Agrobacterium strain for transformation were observed. For the optimization of acetosyringone concentration, a co-cultivation medium with five different concentrations of acetosyringone i.e., 0, 100, 200, 300, and 400 μM was used for transformation. The co-cultivated calli were incubated at five different incubation temperatures viz., 20, 22, 24, 26, and 28 °C for the optimization of suitable incubation temperature of transformation. For the optimization of the number of days of co-cultivation, the calli were co-cultivated for 0 days (The calli washed just after transformation), 1, 2, 3, and 4 days. Three replications were maintained for each treatment. The percentage of transient gusA gene expression in each experiment was evaluated by GUS assay to show the efficiency of transformation.
After transformation, the calli were transferred into a selection medium containing antibiotics to select the putative transformants and suppress the Agrobacterium growth. After two weeks, the calli were transferred to a selection medium containing geneticin/ hygromycin and the data were recorded. The putative transformants were monitored weekly and the surviving calli were used for further molecular analysis. These putative transformants were maintained and subcultured on a fresh medium for continuous selection and molecular analysis to confirm the presence of the transgene.
Putative transformants were evaluated by GUS histochemical assay as described by Jefferson et al. (1987). Putative transformed tissues were incubated in GUS buffer containing 50 mM Na2HPO4, 1 mM Na2EDTA, 1 mM Fe + + + /Fe + + CN and 0.1% Triton X-100. The pH was adjusted to 7.0 with NaOH. The stock solution of 25 mg ml−1 XGluc (5-Bromo-4-chloro-3-indolyl-β-glucuronide) was prepared in dimethyl sulfoxide and was stored at − 20 °C. The GUS assay buffer was prepared freshly by mixing one part of X-Gluc with 49 parts of GUS buffer. The explants were incubated overnight in GUS assay buffer at 37 °C and were washed with 70% ethanol. GUS expressing cells were observed as blue spots on the explants under a microscope. Each spot was scored as one transformation event (Fig. 1).
Fig. 1.
Gus assay of transformed calli
Total genomic DNA was isolated from putative transformants for further molecular analysis. CTAB method of DNA extraction (Doyle and Doyle 1990) with slight modifications was used for genomic DNA isolation (Kamala and Makeshkumar 2013). PCR and nucleic acid spot hybridization analyses were carried out to confirm the transformation events. For PCR analysis of AGL0/pOYE153 and LBA4404/pOYE153, transformants were analyzed for the nptII and gus marker genes with the primers, NPTII-loc (Forward) 5ʹ-GCA CGT ACT CGG ATG GAA GCC-3ʹ and NPTII-ups (Reverse) 5ʹ-TCG CCG CCA AGC TCT TCA GC-3ʹ. GUS gene was amplified using GUSI (Forward) 5ʹ-GGG CAT TCA GTC TGG ATC-3ʹ and GUSII (Reverse) 5ʹ-GTG CGG ATT CAC CAC TTG-3ʹ primers. The analysis of transformants of GV3103/pCAMBIA 1305.2, performed using hpt specific primer HygF (Forward) 5ʹ-CGT CTG CTG CTC CAT ACA AG -3ʹ and HygR (Reverse) 5ʹ-ATT TCA TAT GCG CGA TTG CT-3ʹ. GUS gene amplified using GusplusF (Forward) 5ʹ-TTA ACG AAG CGAGCA ATG TG-3ʹ primer and GusplusR (Reverse) 5ʹ- GAC GCT TGG ATG GTT CTT GT-3ʹ. The PCR amplified products were analyzed in 0.8% agarose gel electrophoresis. Statistical analysis of the data was performed by ANOVA and means were compared by the Duncan's multiple range test (DMRT) using SAS version 9.3 (SAS 2010).
Results
The response of calli to different concentrations of antibiotics (geneticin, hygromycin, and ticarcillin) was evaluated weekly. The growth and proliferation of elephant foot yam calli were inhibited (62.7%) by geneticin at lower concentrations (10 mg l−1), and complete death of the cells occurred at a concentration of 25 mg l−1 (Table 1). Medium supplemented with geneticin 20 mg l−1 showed a partial discoloration from the first week (5.16%) itself leading to complete dead and black tissues during the second week. (Supplementary Fig. 1). Hence, 20 mg l−1 was found to be the minimal inhibitory concentration of geneticin for elephant foot yam calli (Table 1). Elephant foot yam calli were highly sensitive to hygromycin, the calli showed total inhibition of growth at 10 mg l−1 from sixth-week treatment (Supplementary Fig. 2), (Table 1).
Table 1.
Effect of different concentration of antibiotics on elephant foot yam calli
| Antibiotics | Concentration of antibiotic used (mgl-1) | % Survival of explants | Vigor and survival of the explants* |
|---|---|---|---|
| Geneticin | 0 | 100 ± 0.00 | + + + |
| 5 | 99.36 ± 0.85 | + + | |
| 10 | 62.76 ± 0.25 | + + | |
| 15 | 35.16 ± 0.37 | + | |
| 20 | 5.16 + 0.37 | + | |
| 25 | 0 | + | |
| Hygromycin | 0 | 98.6 | + |
| 10 | 0 | + | |
| 20 | 0 | + | |
| 30 | 0 | + | |
| 40 | 0 | + | |
| 50 | 0 | + | |
| Ticarcillin | 0 | 99.46 ± 0.75 | + + + |
| 250 | 99.90 ± 0.17 | + + + | |
| 500 | 99.6 ± 0.52 | + + + | |
| 750 | 77.99 ± 0.21 | + + |
* + Turning black and dead; + + Partially discolored; + + + Healthy tissue
A successful Agrobacterium-mediated plant transformation requires an efficient procedure for suppressing bacteria following co-cultivation. Generally, ticarcillin is used in the medium to kill the bacterium. The present study on the impact of ticarcillin antibiotic on elephant foot yam calli showed that 500 mg l−1 was the lowest lethal concentration for the calli (Table 1). Concentration between 250 and 500 mg l−1 could be used for the successful elimination of Agrobacterium without affecting the regeneration potential of explants (Supplementary Fig. 3). Agrobacterium strains were cultured in a YEB medium containing different concentrations of acetosyringone and they were used for the transformation of elephant foot yam calli (Table 2). Among the different treatments, transformation with AGL0/pOYE153 and GV3103/pCAMBIA 1305.2 resulted in the maximum number of GUS expressed calli (28.24 and 21.59 respectively) at 400 µM acetosyringone, whereas transformation with LBA4404/pOYE153 strain showed the highest average calli (34.29) at 100 μM acetosyringone (Table 2). Among the strains, LBA 4404 showed significantly highest average of GUS expressed calli with a mean vlaue of 19.8318.
Table 2.
Effect of acetosyringone concentration on transformation efficiency of elephant foot yam calli
| Agrobacterium strains | Average number of GUS expressed calli as affected by acetosyringone concentration | Mean of Agrobacterium strains | ||||
|---|---|---|---|---|---|---|
| 0 μm | 100 μm | 200 μm | 300 μm | 400 μm | ||
| AGL0/pOYE153 | 0.92 (1.39)g | 4.98 (2.45)f | 16.07 (4.13)cde | 19.17 (4.49)bcd | 28.24 (5.40)ab | 11.7622 (3.5724)b |
| LBA4404/pOYE153 | 17.09 (4.25)cde | 34.29 (5.94)a | 26.17 (5.21)abc | 17.09 (4.25)cde | 8.99 (3.16)ef | 19.8318 (4.5641)a |
| GV3103/pCAMBIA1305.2 | 0 (1.0)g | 0 (1.0)g | 6.98 (2.82)f | 10.92 (3.45)def | 21.59 (4.75)bcd | 5.7916 (2.6060)c |
| Mean of acetosyringone concentration | 3.9004 (2.2137)d | 8.7875 (3.1285)c | 15.456 (4.0566)b | 15.5266 (4.0653)b | 18.7180 (4.4405)a | |
Figure in the parenthesis shows the square root of transformed values
The data is average of three replicates
The values sharing same alphabet in the superscript in the respective mean values of Agrobacterium strain (A) and concentration of acetosyringone (C) and A × C interaction doesn't differ significantly
The time of co-cultivation is one of the main factors affecting Agrobacterium-mediated gene transformation. The effect of the number of days of co-cultivation on transformation was evaluated based on the GUS expression of 14-day old transformed calli. All the transformed calli were pooled from three separate replications after the GUS assay and counted for the GUS stained callus. The highest number of GUS expressed calli (23.9 and 15.5) was obtained for the strains LBA4404 and AGL0 respectively from calli co-cultivated for 2 days. In strain GV3103, the highest GUS expression (5.97) was observed in calli co-cultivated for 3 days. Statistical analysis showed that the maximum number of transformants was obtained from the explants co-cultivated with LBA4404/pOYE153 and AGL0/pOYE153 strains for 2 days (Table 3). Temperature is an important factor affecting the efficiency of Agrobacterium to transfer the T-DNA to plant cells. The present study showed that the highest GUS expression in elephant foot yam calli was in AGL0 (31.8) at 28 °C, followed by GV3103 (27.05) at 24 °C and LBA 4404 (24.61) at 22 °C. The strain LBA4404 showed a reduction in GUS expression with increase in temperature, whereas this was not the same for the other two strains (Table 4).
Table 3.
Effect of co-cultivation duration on transformation efficiency of elephant foot yam calli
| Agrobacterium strains | Average number of GUS positive calli co cultivated for number of days | Mean of Agrobacterium strains | ||||
|---|---|---|---|---|---|---|
| Day 0 | Day 1 | Day2 | Day3 | Day 4 | ||
| AGL0/pOYE153 | 2.91 (1.98)ef | 7.50 (2.92)cd | 15.49 (4.06)ab | 10.99 (3.46)bc | 4.43 (2.33)def | 7.70132 (2.9498)a |
| LBA4404/pOYE153 | 1.33 (1.53)fg | 5.77 (2.60)cde | 23.96 (4.99)a | 7.94 (2.99)cd | 2.17 (1.78)efg | 6.7256 (2.7795)a |
| GV3103/pCAMBIA1305.2 | 0 (1.0)g | 0 (1.00)g | 1.29 (1.52)fg | 5.97 (2.64)cde | 1.69 (1.64)fg | 1.4317 (1.5594)d |
| Mean of cocultivation duration | 1.25510 (1.5017)d | 3.7214 (2.1729)c | 11.4213 (3.5244)a | 8.1857 (3.0308)a | 2.6791 (1.9181)c | |
Figure in the parenthesis shows the square root of transformed values
The data is average of three replicates
The values sharing same alphabet in the superscript in the respective mean values of Agrobacterium strain (A) and cocultivation duration (C) and A × C interaction doesn't differ significantly
Table 4.
Effect of Co-cultivation temperature on transformation efficiency of elephant foot yam calli
| Agrobacterium strains | Average number of GUS positive calli co cultivated at | Mean of Agrobacterium strains | ||||
|---|---|---|---|---|---|---|
| 20 °C | 22 °C | 24 °C | 26 °C | 28 °C | ||
| AGL0/pOYE153 | 1.33 (1.52)c | 14.06 (3.88)b | 8.99 (3.16)b | 5.97 (2.64)b | 31.28 (5.68)a | 11.5117 (3.5372)a |
| LBA4404/pOYE153 | 0 (1.0) | 24.61 (5.06)a | 8.99 (3.16)b | 1.33 (1.52)c | 0 (1.0)c | 10.4122 (3.3782)a |
| GV3103/pCAMBIA1305.2 | 0 (1.0)c | 5.95 (2.64)b | 27.05 (5.29)a | 12.99 (3.74)b | 24.14 (5.01)a | 4.5210 (2.3497)c |
| Mean of cocultivation temperature | 0.3822 (1.1757)c | 13.8926 (3.8591)a | 13.9993 (3.8729)a | 5.94586 (2.6355)a | 14.1998 (3.8987)a | |
Figure in the parenthesis shows the square root of transformed values
The data is average of three replicates
The values sharing same alphabet in the superscript in the respective mean values of Agrobacterium strain (A) and cocultivation temperature (C) and A × C interaction doesn't differ significantly
It can be inferred that each strain has different conditions for its best performance. AGL0 strain showed the highest transformation efficiency with 400 μM acetosyringone, co-cultivation for 2 days at 28 °C, whereas LBA4404 strain showed the highest transformation efficiency with 100 μM acetosyringone, co-cultivation for 2 days at 22 °C and GV3103 strain showed highest transformation efficiency with 400 μM acetosyringone, co-cultivation or 3 days at 24 °C.
The genetic transformation was carried out with the parameters optimized for each strain. Transformants were selected on regeneration medium containing ticarcillin 500 mg l−1, geneticin 20 mg l−1 (for calli co-cultivated with AGL0/pOYE153 and LBA4404/pOYE153 strains), and hygromycin 10 mg l−1 (for calli co-cultivated with GV3103/pCAMBIA1305.2). The tissues were maintained by sub-culturing once a month. In eight weeks, the transformed tissue developed green sprouts. The transformed tissues were grown in a medium containing antibiotics for 8 months (Fig. 2).
Fig. 2.
LBA4404/pOYE153 transformed calli in selection medium CIM with geneticin 20 mg l−1. a After 2 months. b After 6 months. c Regenerated plantlet
The PCR results showed the presence of the nptII gene, hpt gene, and gus gene in the putative transformants. The GUS assay of transformed calli showed blue color and further confirmation was done by PCR analysis with specific primers. nptII primer yielded a 280 bp single band (Fig. 3) and the GUS primer yielded two bands of size 880 and 700 bp (Fig. 4). hpt primer gave a band of size 300 bp (Fig. 5).
Fig. 3.
PCR analysis of transformed elephant foot yam calli DNA using nptII (loc/ups) primer. L-100 bp ladder, 1–13 LBA4404/pOYE153 transformed callus, 14- Untransformed callus, 15- Non Template Control
Fig. 4.
PCR analysis of LBA4404/ pOYE153 transformed elephant foot yam Calli DNA using GUS specific primer. L-100 bp ladder, 1-Untransformed callus, 2–15 LBA4404/pOYE153 transformed callus
Fig. 5.
PCR analysis of GV3103/pCAMBIA1305.2 transformed elephant foot yam calli using hpt specific primer. L-100 bp ladder, 1–5 GV3103/pCAMBIA1305.2 transformed callus, 6—Untransformed callus
Discussion
Advances in transgenic technologies provide new opportunities for manipulation of the elephant foot yam genome for the introduction of specific genes, especially for disease resistance, overcoming acridity problems and enhancing the starch quality, and shortening the time required for the production of new varieties. Currently, this crop lacks genetic diversity. Among all the available gene transfer methods, the Agrobacterium-mediated transformation has several advantages compared to direct gene transfer. A successful transformation depends on many factors such as temperature, the strain of Agrobacterium used, and co-cultivation time. The efficiency of transformation was optimized by investigating different parameters affecting gene delivery using calli as explants based on the earlier studies (Ban et al. 2009; Kamala and Makeshkumar 2013). Till date, no studies have been reported on the optimization of Agrobacterium-mediated transformation in elephant foot yam. In the present study, different parameters were optimized for efficient transformation of EFY using three different Agrobacterium strains.
The plant cells are sensitive to antibiotics. The selectable marker genes introduced into the plant genome makes the callus resistant to antibiotics and allows to distinguish transformed cells from untransformed ones. Several factors influence the efficiency of an antibiotic as a selection agent. The antibiotic used must be toxic to untransformed plant cells but should not affect the growth of the transformed cells. Thus, the most effective antibiotic is the one which inhibits the growth of the untransformed tissue and allows the transformed tissues. It is observed that the elephant foot yam calli were sensitive to geneticin and hygromycin at lower concentrations. Complete death of the calli was observed with geneticin 20 mgl−1, at the sixth week after the treatment. The result is in agreement with several previous reports. Kamala and Makeshkumar (2013) reported successful transformation of elephant foot yam calli with geneticin at a concentration of 15 mg l−1. Geneticin was the most suitable for transformation in orchids and has effectively killed protocorm-like bodies at 30 mg l−1 (Gnasekaran et al. 2014). Efficient cassava transformation could be achieved at 20 and 25 mg l−1geneticin using nptII as a resistance gene Niklaus et al. (2011). However, the results that hygromycin (22.5 mg l−1) inhibited the shooting ability of the elephant foot yam calli is contrary to the observations by Ban et al., (2009). Raja et al. (2010) used 50 mg l−1 hygromycin for the successful selection of transformants of wheat (Triticum aestivum L.). This indicates that there is variation in the sensitivity of calli to antibiotic depending on the genotype, physiological condition, and size of the explants used. For use of ticarcillin, similar to the present findings, Kamala and Makeshkumar (2013) reported successful elimination of Agrobacterium in elephant foot yam transformation using 500 mg l−1 ticarcillin. Ticarcillin/potassium clavulanate was an effective antibiotic at concentration of 150 mg l−1 in the media to eliminate Agrobacterium strain LBA4404 in Agrobacterium-mediated transformation of tomato (Lycopersicon esculentum Mill.) (Ling et al. 1998).
The addition of acetosyringone in co-cultivation media can improve the transformation efficiency. Acetosyringone is a type of phenolic compound that is well known to increase Agrobacterium vir gene activity. In monocots, acetosyringone played a key factor for transformation which was not present naturally. Elephant foot yam, being a monocot it was assumed that acetosyringone is not secreted by the plant. Acetosyringone has been reported to improve the efficiency of transformation in cassava (Bull et al. 2009). In the present study, 100 μM was found to be effective on EFY.
The time of co-cultivation is one of the main factors affecting Agrobacterium-mediated gene transformation. In one day co-cultivation period, percent mortality was less but the percent GUS expression was reduced considerably. Two days co-cultivation duration was better for the strains LBA4404 and AGL0 and three days for strain GV3103. More than 3 days of co-cultivation promoted the overgrowth of bacteria and led to the necrosis of the transformed tissue. Therefore, two days of co-cultivation was determined to be the best for transformation in elephant foot yam. Ban et al. (2009) also reported successful transformation with 2 days of co-cultivation in Amorphophallus konjac for resistance to soft rot disease. The temperature has been reported as a factor affecting the capacity of Agrobacterium to transfer the T-DNA to plant cells (Karami 2008; Sreeramanan and Xavier 2010). The present investigation on the effect of temperature and co-cultivation on genetic transformation in elephant foot yam calli revealed that temperature plays an important role in transformation efficiency with respect to three strains of Agrobacterium. Higher temperature, 28 °C was found to be optimal to support the highest transient transformation frequency in the strain AGL0, followed by 24 °C for the strain GV3103 and 22 °C for the strain LBA4404. Dramatic reduction in transient expression of GUS occurred when temperature decreased from 22 to 20 °C. The results are contradictory to various reports in which optimal temperatures for transformation ranged from 19 to 25 °C. However, due to high inconsistency across plant species and tissue used, it was possible to affirm that the optimal temperatures varied. Movahedi et al. (2014) reported significant differences in efficiency between the temperatures tested, co-cultivation at 28 °C resulted in the highest transformation efficiency in poplar. There were differences in transformation efficiency with respect to the different strains of Agrobacterium. The maximum percent of GUS expression (31.2%) of transformants was obtained with the strain AGL0, followed by strain GV3103 (27.04%) and strain LBA4404 (24.6%). Thus the difference in transformation efficiency according to the bacterial strain was evident. The Agrobacterium infectivity is because of the interaction between the plant cell and the bacterial cell. The host range is a much more complex process, which is under the genetic control of multiple factors within both the bacterium and the plant host. The infectivity is improved by the use of the right strain of bacteria, varying host genotype, manipulating explants physiology, etc. Bakhsh et al. (2014) compared the transformation efficiency of five Agrobacterium tumefaciens strains in Nicotiana tabacum L. and showed that the highest transformation rate (20%) was obtained with the Agrobacterium strain LBA4404, followed by other strains viz., EHA105, GV2260, C58C1, and AGL1. Bull et al. (2009) reported successful transformation in friable embryogenic calli of cassava using LBA4404 strain.
Geneticin and hygromycin select the rare integration events from the stable transformed tissues. Transformed calli survived in the antibiotic treatment, whereas untransformed calli were effectively killed. Among the calli that resisted antibiotics, few negative GUS expression were observed. DNA was isolated from transformed and untransformed calli (Control). The DNA samples were amplified by PCR with the nptII, hpt and GUS and GUS plus primers designed for nptII, hpt, and GUSA genes in the vector. The amplification achieved with these primers further confirms the presence of the transgene in the genomic DNA. We optimised various parameters for improved transformation efficiency in elephant foot yam, which is simple and reproducible, and may be adapted for developing transgenic EFY varieties for resistance to diseases.
Supplementary Information
Below is the link to the electronic supplementary material.
Acknowledgements
Authors thank Director, ICAR-CTCRI for providing the necessary facilities to carry out this work and Dr. Stephan Winter, DSMZ, Germany for providing the Gus vectors for this study.
Author contribution
All authors contributed equally.
Declarations
Conflict of interest
The authors declare that they have no conflict of interest in the publication.
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