Agrobacterium tumefaciens-Mediated Transformation of Wild Tobacco Species Nicotiana debneyi , Nicotiana clevelandii , and Nicotiana glutinosa

Studies on Agrobacterium tumefaciens-mediated transformation of wild tobaccos Nicotiana debneyi, Nicotiana clevelandii, and Nicotiana glutinosa were conducted. Leaf disks were infected and co-cultivated with A. tumefaciens strain EHA105 carrying the binary vector pBISN1 with an intron interrupted β-glucuronidase (GUS) reporter gene (gusA) and the neomycin phosphotransferase gene (nptII). Selection and regeneration of kanamycin resistant shoots were conducted on regeneration medium containing 8.88 μM 6-benzylaminopurine (BAP), 0.57 μM indole-3-acetic acid (IAA), 50 mg∙L−1 kanamycin and 250 mg∙L−1 timentin. Kanamycin resistant shoots were rooted Murashige and Skoog (MS) medium containing 100 mg∙L−1 kanamycin and 250 mg∙L−1 timentin. Using this protocol, kanamycin-resistant plants were obtained from all three wild tobaccos at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa. Transcripts of nptII and gusA were detected in kanamycin-resistant T0 transformants (i.e., 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii) by the reverse transcript polymerase chain reaction (RT-PCR), and histochemical GUS assays confirmed expression of gusA in both T0 plants and T1 seedlings. The results indicate that the protocols are efficient for transformation of wild tobacco N. debneyi and N. clevelandii. Corresponding author.


Introduction
Of over 75 Nicotiana species, Nicotiana tabacum (cultivated tobacco) and N. benthamiana (wild tobacco) are two major model species for genetic transformation due to their high susceptibility to Agrobacterium tumefaciens and high regeneration capacity of leaf explants through shoot organogenesis [1]- [4].N. tabacum (2n = 4x = 48) is an amphidiploid of interspecific hybrids and a desirable model plant for studying fundamental biological processes through functional genomics and biotechnology approaches [3]- [5].Allotetraploid N. benthamiana (2n = 4x = 38) is widely used for studying plant-microbe interactions and functional gene analyses through virus-induced gene silencing [6] [7].Draft genome sequences for both N. tabacum and N. benthamiana have been released [5] [7].In contrast, few studies have been conducted on other wild tobacco species.
N. debneyi (2n = 4x = 48) is a desirable material to study hybridity and N. debneyi-derived resistance to black root rot disease in Nicotiana species [8]- [10].N. clevelandii (2n = 4x = 48) is susceptible to a large number of plant viruses and N. glutinosa (2n = 2x = 24) shows resistance to a broad range of tombusviruses [11], both species are desirable wild germplasm for studying genetic control of tobmusviruses.Reliable transformation protocols for N. clevelandii, N. debneyi, and N. glutinosa, which have not been reported, will facilitate studies on disease resistance in these wild species.We developed A. tumefaciens-mediated protocols for highly efficient transformation of both N. tabacum genotypes (e.g., "Xanthi" and "Samsun") and N. benthamiana with transformation frequencies ranging from 50% to 90% when nptII was used as a selectable marker.In this study, the protocols were applied for transformation of N. clevelandii, N. debneyi, and N. glutinosa.

Plant Materials and Culture Media
Seeds of N. clevelandii, N. debneyi, and N. glutinosa were surface sterilized in 50% (V/V) Clorox (about 3% sodium hypochlorite) for 10 min in 1.5 ml-eppendorf tubes and were then rinsed five times with sterile distilled water.The seeds (50 -100/dish) were cultured for 2 wk in a Petri dish (60 × 15 mm) on 10 ml Murashige and Skoog medium (MS) [12]containing 30% sucrose, and solidified with 0.6% (w/v) Bacto agar.Sterile seedlings (5/box) or the internode with a single auxiliary bud (1 per box for subculture) were cultured in a Magenta TM GA-7 box containing 50 ml MS medium for 6 to 8 wk to obtain leaf explants.The subculture was conducted every 8 wk.All cultures were grown at 25˚C under a 16-h photoperiod of 45 µmol•m −2 •s −1 from cool white fluorescent tubes.

Regeneration Experiments
Leaves were cut into about 0.6 cm 2 using scissors.Five explants were placed onto 30 ml RM in each Petri dish (100 × 15 mm) with either adaxial side up or abaxial side up.The Petri dishes, three for each of the N. clevelandii, N. debneyi, and N. glutinosa, were cultured in the dark for one wk prior to the culture under a 16-h photoperiod for 3 wk.The explants were then transferred to Magenta TM GA-7 boxes, 5 explants/box and each containing 50 ml RM, and cultured for 4 wk.The number of explants that produced at least one shoot (≥0.5 cm in length) and the total number of shoots were recorded.The experiment was repeated three times.The percentage of explants that produced at least one shoot and the average number of shoots of regenerated explants were calculated.

Transformation Protocols
A single colony of EHA105:pBISN1 was grown in a 50-ml Corning tube containing 15 ml liquid YEP medium, 100 mg•L −1 kanamycin and 30 mg•L −1 rifampicin.The culture was grown at 28˚C for 48 hours with constant shaking at 300 rpm.The cells were collected by a 5 min centrifugation at 2500 × g and suspended to an OD 600 of 0.5 in liquid co-cultivation medium (liquid RM plus 100 μM acetosyringone).
Leaf disks were prepared by punching stacked leaves in a Petri dish (100 × 15 mm) using a sterile cork borer (10 mm in diameter).Newly prepared disks (60/species) were inoculated in EHA105:pBISN1 suspension cells for 1 -2 min, blotted dry on sterile filter paper, and then placed onto a piece of sterile filter overlaid on 30 ml solidified co-cultivation medium (RM plus 100 μM acetosyringone) in a Petri dish (100 × 15 mm).Co-cultivation was carried out at 25˚C in the dark for 4 d.
After co-cultivation, the explants were transferred to a 50 ml Corning tube and washed three times (1 min per time), twice in liquid RM and once in liquid RM supplemented with 500 mg•L −1 timentin, to remove excess Agrobacterium.The washed explants were blotted dry on sterile filter paper and placed, 10 disks/dish, on 30 ml selection RM supplemented with 50 mg•L −1 kanamycin and 250 mg•L −1 timentin in a Petri dish (100 × 15 mm).Selection and regeneration were conducted in the dark for one wk followed by a 16-h photoperiod for the rest process.After 4-wk culture in Petri dishes, the explants (5/box) were transferred on 50 ml selection RM in a Magenta TM GA-7 box and cultured for 4 wk.
After 8-wk on selection RM, regenerated shoots (one or two per explants) were excised and transferred to 50 ml MS medium containing 100 mg•L −1 kanamycin and 250 mg•L −1 timentin in a Magenta TM GA-7 box for rooting at 25˚C under a 16-h photoperiod for 3 wk.The number of leaf disks that produced kanamycin resistant plants was recorded.The transformation experiments were repeated three times.
Well rooted transformants were washed to remove agar, and individual plants were planted in 4-inch pots and zipped in a one-gallon size bag for one wk at 25˚C under a 16-h photoperiod.The bags were then opened to acclimate the plants for one wk before they were transferred to the greenhouse.Seeds of each plant were harvested separately.
Surface-sterilized T 1 seeds were placed onto MS medium (without sucrose) containing 200 mg•L −1 kanamycin.Seed germination was investigated after 3 wk culture at 25˚C under a 16-h photoperiod.

Histochemical GUS Assay
The GUS assay in transformed and non-transformed tissues was determined histochemically according to Jefferson et al. [17] and was used as a quick screen of the putative transformants.All tissues (i.e.inoculated leaf explants, T 0 leaves and T 1 seedlings) were stained in 2 mM X-Gluc (PhytoTechnology Laboratories, Shawnee Mission, KS, USA) overnight at 37˚C.The GUS expression was documented by photography.

Reverse Transcript Polymerase Chain Reaction (RT-PCR)
Isolation of total RNA from young leaves of greenhouse-grown T 0 plants was performed using a RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA).After DNase treatment, purified RNA (1 μg for each sample) was transcribed into cDNA using SuperScript II reverse transcriptase (Invitogen, Carlsbad, CA, USA).The resulting cDNA was used for PCR amplification of a 377 bp fragment of the coding region of gusA (primers: 5'gatcctcgcattacccttacgc-3' and 5'-gtgagcgtcgcagaacattac-3') and a 600-bp fragment of nptII with the primers 5'-GAGGCTATTCGGCTATGACTG-3' and 5'-ATCGGGAGCGGCGATACCGTA-3'.The PCR reaction conditions were 94˚C for 2 min, 35 cycles of 94˚C for 10 s, 58˚C for 1.5 min and 72˚C for 2 min, with a final 10 min extension at 72˚C.PCR products were separated on 1.0% agarose gel containing ethidium bromide, visualized and photographed under UV light.

Shoot Regeneration Capacity
The RM (8.88 µM BAP plus 0.57 µM IAA) used in this study is different from the commonly used one [4.44 µM BAP plus 0.54 µM α-naphthalene acetic acid (NAA)] [1] [2], it was highly efficient for shoot production from leaf explants (100% leaf explants with over 10 shoots/explants) of N. tabacum genotypes (e.g., "Xanthi" and "Samsun") and N. benthamiana.When it was used for Nicotiana debneyi, N. clevelandii, and N. glutinosa, multiple shoots mainly from the cut edges were produced after 8 wk at frequencies of 66.7% for N. glutinosa, 95.6% for N. clevelandii, and 100% for N. debneyi.The average number of shoots of regenerated explants was 3.1 for N. glutinosa, 5.2 for N. clevelandii, and 6.5 for N. debneyi.Apparently, N. debneyi and N. clevelandii are more efficient than N. glutinosa in shoot regeneration from leaf explants (Figure 1).These results suggest that optimal regeneration medium is species/genotype-dependent.Genetically, N. debneyi is believed to be a parent of N. benthamiana (N.suaveolens × N. debneyi).Since the RM is the optimal regerneation medium for N. benthamiana, high genetic similarities to N. benthamiana could contribute to efficient shoot regeneration of N. debneyi on the RM.
The explant orientations on RM (i.e., adaxial side up or abaxial side up) did not result in significant difference in shoot production, and therefore, the orientations were not a consideration in our transformation experiments.

Transformation
The pBISN1 containing a potato ST-LS1 intron interrupted gusA to inhibit GUS expression in Agrobacterium cells was routinely used to optimize our transformation protocols for different plant species [18]- [22].In this study, transient GUS expression assay was conducted after 4-d co-cultivation.Under our co-cultivation conditions, all leaf disks tested for each of N. glutinosa, N. clevelandii, and N. debneyi showed strong blue staining in cutting edges and weak blue foci in non-wounded surfaces.The result indicates EHA105 is efficient in delivering the transfer DNA (T-DNA) of the pBISN1to these species.In addition, by using RM as co-cultivation medium and a piece of filter paper to prevent overgrowth of EHA105, the 4-d co-cultivation did not cause any necrosis in leaf explants (Figure 2(a)).With the washes after co-cultivation, 250 mg•L −1 timentin was able to eliminate EHA105, and no EHA105 overgrowth was observed during the entire selection process.
After 4-wk selection, kanamycin resistant clusters of callus/shoot appeared mainly along the cut edges of the explants (Figure 2(b), Figure 2(c)).In the Magenta TM GA-7 boxes containing selection RM, multiple shoots were observed from some explants and rooted plants were also observed on the explants of N. debneyi (Figure 2(d), Figure 2(e)).All rooted plants survived and were morphologically normal after they were transplanted to soil (Figure 2(f)).Transgenic plants from separate explants were considered to be independent transgenic events.With this transformation system, transformation frequencies based on the percentage of explants that produced at least one kanamycin resistant plants were 75.6% (136/180) for N. debneyi, 25.0% (45/180) for N. clevelandii,  and only 2.8% (5/180) for N. glutinosa.The results indicate that the protocols described enable efficient transformation of both N. debneyi and N. clevelandii and have potential application for transformation of other wild Nicotiana species.The lower transformation frequency (2.8%) of N. glutinosa is due to its lower regeneration ability.

Expression of Transgenes
T 0 transformants, 2 for N. glutinosa and 5 for each of the N. debneyi and N. clevelandii, were analyzed using RT-PCR.The fragments of gusA (377 bp) and nptII (600 bp) were present in all 12 transformants and were absent in nontransgenic plants (Figure 3(a)).The results confirmed transcription of both the gusA and nptII.
Inheritance of the gusA and nptII was investigated in T 1 seedlings of one transgenic event for each of N. debneyi, N. clevelandii and N. glutinosa.On MS (without sucrose) supplemented with 200 mg•L −1 kanamycin, both transgenic and nontransgenic seeds germinated after one wk; however, two wk later all seeds from nontransgenic plants turned white, in contrast, the majority seedlings from transgenic plants remained green and kept growing (Figure 3(b)).In histochemical GUS assays, blue staining was present in all kanamycin resistant seedlings but was absent in nontransgenic seedlings (Figure 3(c)).The results indicate inheritance of both the nptII and gusA in transgenic seedlings.

Conclusion
We developed protocols for transformation of N. debneyi, N. clevelandii and N. glutinosa.Briefly, leaf explants were soaked in A. tumefaciens cells and co-cultivated in the dark for 4 d.Selection and regeneration of trans- formants were conducted on regeneration medium containing 50 mg•L −1 kanamycin and 250 mg•L −1 timentin for 6 -8 wk.Rooting of the transformants was carried out on MS medium containing 100 mg•L −1 kanamycin and 250 mg•L −1 timentin for 2 -3 wk.Production of kanamycin resistant plants from inoculated explants was obtained at frequencies of 75.6% for N. debneyi, 25.0% for N. clevelandii, and 2.8% for N. glutinosa.The protocols described have potential application for transformation of other Nicotiana species.