1 Introduction

Wild crop relatives are important source of genetic diversity. They can be helpful for developing new varieties which are able to withstand challenging environment. Plant breeders have found different ways to use the genetic diversity of wild relatives to produce new plant varieties (Warschefsky et al., 2014; Brozynska et al., 2015). Genes from wild relatives have increased the productivity of major crops such as wheat, rice, maize and barley. These plants have different traits and showed tolerance to salinity, extreme temperature, drought conditions and resistance to disease (Ford-Lloyd et al., 2011; Porch et al., 2013). Nicotiana plumbaginifolia is specie of tobacco commonly known as Wild Tobacco and Tex-mex Tobacco. It belongs to family (Solanaceae). It is an annual weed herb with hairy stem. It has height of 60 cm with extending radical and thin leaf branches (Davey et al., 2000). Tex-Mex Tobacco is native to the American continent. It has great medicinal properties because of antimicrobial agents. Its dried leaves are used in treatment of several human diseases like rheumatic, swelling in order to relieve pain (Singh et al., 2010). Its methanoic extract has been used as folk medicine for treatment of oxidative damage in liver of chicks (Shah et al., 2016).

By the year 2020, it is estimated that world population will reach to eight billion and food production will have to be doubled. To meet the challenges of increasing population, it is obligatory to make well use of plant genetic diversity. Plant genes from wild crops are the primary source of genetic variability for modern crops that are involved in different traits like higher productivity, exceptional nutritious values and resistance against disease (Rao, 2004). Genetic diversity refers to the total number of different genetic characteristic in a species. Genetic diversity is very crucial for species to survive, to adapt towards new environmental conditions and for evolution of new species. In plants, genetic diversity is important to develop varieties tolerant to biotic as well as abiotic stresses (Kettenring et al., 2014).

A proficient and consistent plant regeneration protocol is essential for the application of routine plant transformation techniques. Exploitation of totipotent genotypes, suitable explants, appropriate media contents and culture conditions are of core importance in optimizing plant tissue culture procedures (Joyia and Khan, 2013). Additionally, the process of dedifferentiation and regeneration under in vitro culture conditions has long been recognized to prompt somaclonal variations (Filipecki and Malepszy, 2006; Sun et al., 2013) which may also be an efficient source of crop improvement. Development of whole plants from single cells under in vitro conditions leads to various perspectives in order to manipulate somatic cells (Conner and Meredith, 1989). Consequently, it provides a powerful tool for molecular, biochemical and physiological studies of different plant characters. Nicotiana plumbaginifolia is the most fascinating model systems which allowed a variety of genetic manipulations at the somatic cell level (Maliga, 1982; Nikova et al., 1991). The establishment of protoplast and cell cultures of this specie is simple and can promptly regenerate into whole plant. Monoploid (n =10) cells can be obtained and easily diploidized to restore fertility. The large progenies of this small plant can be grown in a limited space and can allow rapid genetic analysis.

Genetic transformation is the insertion of foreign gene in genome of an organism. It is the genetic alteration of plant which provides a powerful way to manipulate different characteristic and conservation of desired traits for crop improvement (Paszkowski et al., 1984). A varied range of techniques has been established for DNA transfer into plant cells. Genetic transformation can be accomplished with Agrobacterium transformation method or by electroporation in protoplast or direct DNA uptake using gene gun. Agrobacterium has limited host range and regeneration from protoplast is difficult in some species. Direct DNA uptake through gene gun is valuable for both transient as well as stable gene expression without any limitation of host specificity. Biolistic method is the most powerful method of plant transformation. In spite of different variety of specialized techniques for plant genetic transformation, particle bombardment is considered to be the most rapid, simple and versatile. It can deliver DNA directly to intact cells and tissues, therefore avoids the limitation of host range and sometimes tissue culture (Sanford et al., 1987; Klein et al., 1988). Adequate number of reports has been published on in vitro propagation of N. plumbaginifolia using leaf explant; however there is no report on utilization of these explants for biolistic genetic transformation studies of N. plumbaginifolia. (Li et al., 2003) evaluated potential of shoot organogenesis in 53 Nicotiana species including Nicotiana plumbaginifolia. Similarly, leaf explant-based direct organogenesis in five wild Nicotiana species has been reported by (Sarala et al., 2008). To the best of our knowledge, this is the first report of biolistic transformation of N. plumbaginifolia using gene gun. Hence, we report here, the expression of the Gus reporter gene in N. plumbaginifolia using in vitro grown mature leaves as explant.

2 Materials and Methods

2.1 Plant material

Stem cuttings were obtained from Nicotiana plumbaginifolia plants grown as a weed in the fields of University of Agriculture Faisalabad - Pakistan. Stem cuttings were surface sterilized by successive immersions in tap water, 70% (v/v) ethanol for few seconds followed by washing with 5% commercial bleach with few drops of tween 20 for 15 minutes. Finally, the explant was rinsed 3-4 times with sterile distilled water. The cuttings were cultured in glass jars having solid MS medium without plant growth regulators (PGRs) and incubated at 25˚C ± 2˚C under 16/8 hrs light and dark condition.

The basal medium used for in vitro regeneration, root formation and further in vitro multiplication was MS medium (Murashige and Skoog, 1962). All the stock solutions of vitamins and plant growth regulators (PGRs) were prepared in double distilled water. Sucrose was added as a carbon source at the rate of 30 g/L. pH of culture medium was adjusted to 5.8 and solidified with 2.6 g/L gellan gum powder. The screw capped glass jars of 175 ml capacity were used accommodating 50 ml of culture medium in each jars. The culture medium for all investigations was sterilized by autoclaving at 121˚C at 15psi for 20 minutes. Leaves of the in vitro grown plants were employed as explant in further experimemtation. Explants were cut into small pieces and placed onto regeneration media containing various plant hormones.

2.2 Direct in vitro regeneration

In order to attain direct in vitro regeneration, six regeneration media containing 30 g/L sucrose, 4.33 g/L MS basal medium supplemented with following six different combinations of IAA and kinetin were used. RM1: 0.125 mg/L IAA and 1 mg/L Kinetin, RM2: 0.25 mg/L IAA and 1 mg/L kinetin, RM3: 0.75 mg/L IAA and 1 mg/L Kinetin, RM4: 0.125 mg/L IAA and 2 mg/L Kinetin, RM5: 0.25 mg/L IAA and 2 mg/L Kinetin, RM6: 0.75 mg/L IAA and 2 mg/L Kinetin. All media were solidified with 2.6 g/L phytagel while pH was maintined at 5.8 before autoclaving as described earlier. Regeneration efficiency was calculated as under.

2.3 Root induction

After 5 weeks of culturing the regenerated plantlets were transferred into jars having root induction media medium {MS medium supplemented with different doses of IBA (0.00, 0.25 mg/L, 0.5 mg/L, 1 mg/L)}. Plantlets with developed roots were successfully acclimatized to peat moss mixture in pots covered with polythene bags for maintaining proper humidity and later placed in greenhouse.

2.4 Biolistic transformation of Nicotiana plumbaginifolia

Explants cultured on regeneration media in the center of petri plates under aseptic conditions were used for bombardment. The gene gun (Bio-Rad PDS-1000/He) was properly sterilized with ethanol to avoid contamination. Bombardment was carried out under a partial vacuum (28 mm of Hg) using 1100-psi rupture disc. Gold particles (0.4-0.8 µm) were coated with 5 µg of the plasmid DNA of the p7i-UG vector, following protocol described by (Becker et al., 1994). The stopping screen and macro-carriers were placed into the vacuum chamber at 1^st level and the petri dish containing the target leaf sections were placed at 3^rd level from the top. After bombardment, plates containing bombarded leaves were sealed using cling film and placed at 25°C under dark condition in growth room for 48 hrs.

2.5 Histochemical Gus assay

In order to check the efficiency of biolistic transformation histochemical assay was performed. These explant were analyzed for Glucuronidase (Gus) activity (Jefferson et al., 1987) 8 days after bombardment. Explants were incubated for 48 hrs at 37°C in buffer containing X-Gluc (5-Bromo-4-chloro-3-indolyl-D-glucuronic acid), Potassium buffer (pH 7.0), 0.5% Triton X-100 as a substrate. Gus signals were visually observed and photographed.

2.6 Statistical Analysis

A completely randomized design (CRD) with three replications per treatment was used in this study. Each replication contained three plates of respective media. Mean value of three plates was considered as one replication. Means and standard errors of means (SEM) were calculated and level of significance of the mean values was checked by using LSD (Latin Square Design) test at 5% level of significance (Steel et al., 1997).

3 Results and Discussion

3.1 Optimization of regeneration protocol

Efficient in vitro regeneration methodologies authorizing the development of complete plant from a single cell are of vital significance to both clonal propagation and successful genetic engineering. Therefore, first and the foremost endeavor should be the production of an immense number of regenerable cells as easily and quickly as possible. Present research was designed to establish an efficient in vitro regeneration system which will help in genetic transformation of N. plumbaginifolia L. The in vitro culture system was optimized exploiting leaf explant. This study was focused on the development of direct regeneration for N. plumbaginifolia L. on MS basal culture media using different combinations of kinetin and IAA. Success in direct in vitro regeneration circumvented the callogenesis step, accelerating the process of in vitro regeneration and increasing the chances of obtaining true to type plants.

Fully expanded healthy 4 week old leaves from in vitro grown plants of N. plumbaginifolia were excised under sterilized conditions and cultured on different regeneration media. For enhancing regeneration response different combination of plant growth regulators (PGRs) were used and leaves from in vitro grown plants were cultured on these media. RM1 medium showed 32% regeneration efficiency and most of the explants just increased in size and showed callogenesis instead of direct in vitro regeneration. RM₂ medium having kinetin and IAA 1 mg/L and 0.25 mg/L respectively showed highest regeneration efficiency (94%), where bunch of shoots (4.733 shoots/explant) emerged from every explant within 8 weeks of culturing (Table 1; Table 2; Figure 1). RM₃ medium having kinetin and IAA in 1 mg/L and 0.75 mg/L respectively showed 16% regeneration efficiency and only a few explants regenerated into shoots with an average of 0.8 shoots / explant. In present study, RM₄ medium having kinetin and IAA in 2 mg/L and 0.125 mg/L respectively showed 30% regeneration efficiency with 1.53 shoots / explant. RM₅ medium showed only 6% regeneration efficiency with very poor response to regeneration. RM₆ medium showed 44% regeneration efficiency with 2.2 shoots / explant (Table 1; Table 2; Figure 1).

In present study, RM₂ showed highest regeneration efficiency where the concentration of kinetin and IAA was 1 mg/L and 0.25 mg/L respectively. On the other hand RM₅ medium having 2 mg/L, 0.25 mg/L kinetin and IAA respectively exhibited minimum regeneration efficiency. The study revealed optimal level of IAA and Kinetin required for efficient direct regeneration of N. plumbaginifolia. This indicated the effect of PGRs on regeneration efficiency of leaf explant of N. plumbaginifolia (Figure 1). Analysis of variance revealed significant difference among regeneration efficiency on different regeneration media. LSD demonstrated the relationship between number of shoots produced from each explant and the regeneration media used with highest mean value for RM₂ (Table 1; Table 2). (Rahman et al., 2010) showed similar results for different Bangladeshi tobacco cultivars with maximum efficiency at IAA and Kinetin @ 2 mg/L each. However, (Li et al., 2003) evaluated shoot organogenic potential of 115 Nicotiana accessions bu using Indole-3-acetic acid (IAA) 5.1 mM in combination with 6-Benzylaminopurine (BA) 11.1 mM. Our results showed a change in behavior of leaf explant on different media, as RM₂ indicated higher regeneration as compared to other media as depicted in Table 2. RM₂ medium exhibited regeneration on minimum concentration of IAA (0.25 mg/L), indicated that IAA in the culture medium greatly influence organogenic response and development (Pierik, 1988).

3.2 Plant Transformation

Expression of β-glucuronidase (GUS) reporter gene is an efficient and reliable techniques studying gene regulation (Fior and Gerola, 2009). For genetic transformation of N. plumbaginifolia, transformation vector (Figure 2) coated gold particles (microcarriers) was bombardment under vacuum with a BioRad PDS 1000 (He) gene gun, using 1100-psi rupture discs, stopping screens and macrocarriers having microcarriers. Fully expanded lush green leaves were placed in the centre of a petri plate containing RM₂ medium with the help of sterile foreceps in laminar air flow cabinet. Plates with bombarded leaf sections were wrapped and placed under dark condition in growth room for 48 hrs, which were then transferred to normal light on RM₂. The gus assay was performed after 8 days of bombardment. All the leaves sections were dipped in 20 ml gus buffer (1 mM X-Gluc, 1M Na2HPO4, 0.1% Triton X-100, 0.5M EDTA) in a small beaker and leave the plants in assay solution at 37˚C for 48 hrs. After 48 hrs, gus expressing cells showed blue colour as a result of chromogenic cleavage by X-Gluc, representing successful integration of gus gene and its expression in leaf sections as depicted in Figure 3.

The gus assay was performed 8 days after the bombardment, this was done to minimize the transient expression and increase the chances to see the stable transformation, as transient expression is maximum till 96 hrs of bombardment and after that the chances to appear for the stable expression are more (Janssen and Gardner, 1990). Gus gene showed sufficient expression 8 days after bombardment which means that there are enough chances for the stable transformation. While our non-bombarded control did not show any colour. Similar study was conducted by (Lipsky et al., 2014) where genetic transformation of Ornithogalum via particle bombardment was carried out. In our study all leaf sections were dipped in gus buffer and left the leaves in assay solution at 37˚C for 48 hrs. Temperature is also an important factor for highest activity of GUS assay (Su et al., 2012). After 48 hrs, Gus expressing cells showed blue colour as a result of chromogenic cleavage by X-Gluc, representing successful expression of gus gene (Figure 3). The Gus assay is safe, highly reliable, versatile, and easy to perform and needs no specialized equipment (Jefferson et al., 1987; Jefferson and Wilson, 1991).

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