Introduction

Cucumber is an important agricultural as well as horticultural crop which belongs to the family Cucurbitaceae. Cucumis sativus L. is an important vegetable crop worldwide which grows both in tropical and subtropical regions. Cucumber is not only used for food dishes but also different parts such as leaf, fruit, and seeds have been explored for their therapeutic potentials such as cosmetics and wound healing activity. Most recently, leaves and callus tissues from cucumber plant were used for synthesis of biomolecules loaded metallic silver nanoparticles and its wound healing property was studied by preparation of nanodrug-based ointment in rat model (Venkatachalam et al. 2015). Therefore, there is a growing interest for enhancement of cucumber productivity worldwide due to its versatile applications. The ability of cucumber to grow under a wide range of agroclimate and soil conditions, disease and pest problems significantly affected the yield of this important crop (Vengadesan et al. 2005; Grozeva and Velkov 2014). Plant tissue culture technology is an important tool for genetic manipulation which is essential to overcome crop yield losses due to various biotic and abiotic stresses (Kumar et al. 2015). Somaclonal variation is considered as one of the important phenomena which is a potential factor detected in tissue culture of plants with reduced levels of desired secondary metabolites such as cafestol and kahweol in coffee (Sridevi and Giridhar 2014). Therefore, development of an efficient protocol for high frequency plant regeneration system with no risk of somaclonal variations is a major focus for genetic improvement of horticultural crops including cucumber. In vitro regeneration has many advantages, such as higher rates of multiplying clean (pest and disease-free) planting material and a little space is required to multiply large number of plants. Plant regeneration has been achieved via organogenesis in cucumber using different explants such as cotyledons (Gambley and Dodd 1990; Selvaraj et al. 2007), shoot tips (Vasudevan et al. 2004), embryonal axis (Vasudevan et al. 2007), hypocotyls (Selvaraj et al. 2006), nodal segments (Ahmad and Anis 2005; Kontas and Kintzios 2003). Somatic embryogenesis was also reported in cucumber by Mashayekhi et al. (2008). Plant regeneration from one cultivar may be different from that of another cultivar within the same species (Vasudevan et al. 2007). Therefore, efficient plant regeneration protocol should be developed for each cultivar (Walden and Wingender 1995). ‘Green Long’ is one of the most popular cucumber cultivars in India, especially in Tamil Nadu (Vengadesan et al. 2005). According to the earlier report, the regeneration rate was found to be low and it was largely dependent on the type of cultivar, nature of the explants and growth regulators (Selvaraj et al. 2007). There is an increasing interest to use the cotyledonary node which is a juvenile meristematic part from the seedlings, as alternative explant for in vitro regeneration in the recent past. Earlier, Singh et al. (2007) demonstrated that about 3–4 shoots were obtained with BAP at lower concentrations and multiple shoot regeneration was inhibited at higher concentrations of BAP. For breeding purpose, an efficient protocol for high frequency plant regeneration is still essential for development of cucumber transgenic plants with ‘Green Long’ cultivar.

Analysis of in vitro derived plants for genetic variability via clonal fidelity is one of the prerequisites to ensure the desired superior genotypes for agronomically important traits including good growth, tolerance to both biotic and abiotic stresses (Kumar et al. 2015). Though various molecular marker techniques were applied to detect the genetic fidelity of in vitro derived clones, the Random Amplified Polymorphic DNA (RAPD) technique has proven to be effective in detecting genetic variability and it was successfully applied for identification of genetic similarities among micropropagated plants until recently (Williams et al. 1990; Hussain et al. 2008; Martins et al. 2004; Thiyagarajan and Venkatachalam 2012; Kumar et al. 2015).

In view of the above, the development of an efficient regeneration protocol is one of the prerequisites for production of uniform cucumber plants that could be used for genetic manipulation of this crop. The present study was focused to develop an efficient protocol for high frequency plant regeneration using different growth regulators and silver nitrate via adventitious shoot organogenesis from cotyledonary node explants of commercially important cucumber cultivar ‘Green Long’. In addition, the genetic fidelity of the cucumber regenerants was assessed using RAPD-PCR technology.

Materials and methods

Plant material and seed germination

Matured certified seeds of cucumber (Cucumis sativus L., cv. ‘Green Long’) were collected from the Agricultural farm, Salem, India. The seed coats were removed and soaked overnight in sterile distilled water. Then, the seeds were washed with 10% (v/v) Tween 20 for 10 min, surface sterilized with 0.1% (w/v) aqueous mercuric chloride solution for 5 min and washed with sterile distilled water for 5 times to remove the mercuric chloride traces. Sterilized seeds were placed on half-strength MS medium for germination and kept in the dark condition.

Culture medium and conditions

The cotyledonary node explants were cultured on MS (Murashige and Skoog 1962) medium supplemented with various concentrations and combinations of 6-benzyl amino purine (BAP), kinetin (KIN), indole 3-acetic acid (IAA), indole 3-butyric acid (IBA), α-naphthalene acetic acid (NAA) and silver nitrate (AgNO3). The pH of the medium was adjusted to 5.7 prior to adding 0.7% (w/v) agar and medium (15 ml) was aliquoted into each culture tubes (25 × 150 mm). Then, the media were autoclaved at 121 °C for 20 min with 15 lbs pressure. All the cultures were maintained at 25 ± 2 °C with a photoperiod of 16/8 h (light/dark) at 60 µE m−2 s−1 light provided by cool white fluorescent tubes.

Shoot bud initiation

Initially, the cotyledonary node explants from 5-day-old in vitro grown seedlings were excised by removing the epicotyls as well as cotyledons and used for plant regeneration. For shoot bud induction, the cotyledonary node explants were cultured on MS medium containing different concentrations of BAP and KIN (0.5–2.5 mg l−1) alone. The shoot cultures were transferred into same fresh medium at 2 weeks interval. For each experiment, ten cotyledonary node explants were used and at least repeated thrice.

Multiple shoot bud regeneration and elongation

In vitro raised adventitious shoot buds from the cotyledonary node explants grown on MS medium containing 2.0 mg l−1 BAP were selected for multiple shoot bud regeneration. The selected in vitro adventitious shoot clumps were dissected out and transferred onto MS medium supplemented with various concentrations of BAP (0.5–2.5 mg l−1) alone and/or in combination with 0.5 mg l−1 NAA/IBA for multiple shoot bud development. The adventitious shoot cultures were subcultured onto the same media combinations for further growth and development at 2 weeks interval. After 2 subcultures, the regenerated shoot buds were transferred onto MS medium containing different concentrations of AgNO3 (1.5–6.0 mg l−1) alone and/or in combination with 1.5 mg l−1 BAP for enhancement of multiple shoot bud development. The regenerated shoot buds were elongated quickly on the same media. For multiple shoot bud regeneration, minimum seven regenerants were tried per treatment and each experiment was repeated three times.

Rooting and acclimatization

Elongated shoots (> 2 cm in length) obtained after completion of shoot bud multiplication cycles were dissected out individually and placed on half-strength MS medium augmented with various concentrations of IBA and NAA (0.5–2.0 mg l−1) alone and/or in combination with 0.5 mg l−1 KIN for rooting. Seven elongated shoots were placed in each dose and all experiments were repeated thrice. The rooted plantlets were carefully removed from the culture tubes and gently washed under running tap water to remove traces of agar without damaging the root system. Then, the plantlets were transplanted into plastic cups containing sterile sand and soil in the ratio of 1:1 and kept at 25 ± 2°C with 80% relative humidity under 16/8 h of light/dark cycle provided with light intensity at 60 µE m−2 s−1 by cool white fluorescent tubes. After 2 weeks, acclimatized plantlets were subsequently established in the field.

Genomic DNA extraction and PCR amplification

Clonal fidelity of the in vitro raised plantlets that were isolated from 1.5 mg l−1 BAP and 4.5 mg l−1 AgNO3 combination was analyzed using RAPD markers. For PCR analysis, a total of 10 in vitro regenerated plantlets which were acclimatized in the field conditions were selected randomly and the RAPD banding pattern was compared with the control plant (mother plant derived via seedling). Total genomic DNA was extracted from the control as well as in vitro regenerated plantlets by modified CTAB method (Doyle and Doyle 1990). Leaves were weighed (0.1 g) and ground well and homogenized with 1.0 ml of 2× CTAB buffer [(2% hexadecylcetyl trimethyl ammonium bromide), 1.4 M NaCl, 20 mM EDTA (pH 8.0), 0.1 M Tris–HCl (pH 8.0), 1% (w/v) polyvinyl poly pyrolidone (PVPP), 1.0% (v/v) β-mercaptoethanol] and incubated the extract in a water bath at 65 °C for 20 min. Then, the extract was allowed to cool at room temperature and added 400 μl of saturated phenol, 400 μl of chloroform and 20 μl of β-mercaptoethanol, mixed well and centrifuged at 8000 rpm for 10 min. Aqueous phase was transferred into the fresh tube and the DNA was precipitated with 0.6 volume of ice-cold isopropanol. The DNA pellet was washed with 70% (v/v) ethanol and air dried. Then, it was dissolved in sterile distilled water and quantified spectrophotometrically.

For initial screening of DNA samples, 100 random decamer primers from Operon Inc., USA were used. PCR amplification was carried out in a volume of 20 µl containing 2 µl 1× PCR buffer [10 mM Tris–HCl (pH 8.3), 50 mM KCl, 1.5 mM MgCl2], 2 µl 1.5 mM dNTPs (dATP, dGTP, dCTP and dTTP), 250 nM primers (1 µl), 0.5 units of Taq DNA polymerase, 2 µl genomic DNA (15 ng) and 13 µl of sterile water. DNA amplification was performed in a thermal cycler machine (Cyber Lab, USA) using the following PCR conditions with initial denaturation at 94 °C for 4 min, followed by 40 cycles consisted of denaturation at 94 °C for 1 min, annealing at 37 °C for 1.30 min and extension at 72 °C for 2 min and a final extension at 72 °C for 7 min. After completion of PCR cycles, the amplicons were stored at 4 °C until further use.

Electrophoretic analysis of PCR amplicons

The PCR products mixed with appropriate volume of DNA loading buffer (10×) were analyzed on 1.5% (w/v) agarose gel electrophoresis containing 0.5 µg/ml ethidium bromide in 1× TAE buffer. Electrophoresis was performed at 50 V for 2 h until the bromophenol blue dye front migrated to the bottom of the gel. The molecular standard used was the lambda DNA double digested by EcoRI/HindIII. The gels were visualized under UV light and photographed with gel documentation system (Alpha Inotech Gel documentation system, USA) for further analysis.

Statistical analysis

All the experiments were set up in a completely randomized block (CRB) design and each experiment had three replicates. Based on the visual observation of the cultures, the percentage of cultures showing shoot bud induction, multiple shoot bud differentiation and rooting was recorded and used for statistical analysis. The analysis of variance (ANOVA) was performed using SAS (Statistical Analysis Software) programme. The differences among mean values were determined by Student–Newman–Keuls test at 5% (p > 0.05) significance level.

Results

Initiation of adventitious shoot buds

The morphogenetic responses of cotyledonary node explants to different concentrations of BAP and KIN (0.5–2.5 mg l−1) are summarized in Table 1. After 5 days of culture, the adventitious shoot buds initiated directly from the cotyledonary node explants cultured on MS medium fortified with BAP, while no shoot bud development was noticed on medium containing KIN. It is noteworthy to mention that shoot bud initiation (100%) was recorded at all BAP concentrations used (Fig. 1a). Among the different concentrations of BAP tested, the highest percent of shoot bud regeneration (100%) with maximum number of shoots (6.4 shoots/explant) was noticed on MS medium containing 2.0 mg l−1 BAP followed by 1.5 mg l−1 BAP. It is interesting to note that no shoot bud regeneration was noticed on medium containing KIN, but induced roots from the cultured cotyledonary node explants. The number of shoot buds was increased with increasing the concentrations of BAP up to 2.0 mg l−1; thereafter, it was decreased with further increase in the BAP concentration in the medium (Table 1).

Table 1 Effect of different concentrations of BAP and KIN on induction of adventitious shoot buds from cotyledonary node explants of cucumber
Full size table
Fig. 1
figure 1

Efficient plant regeneration from cotyledonary node explants of Cucumis sativus L., cv. ‘Green Long’). Cotyledonary node explants cultured on MS medium for shoot bud initiation (a), adventitious shoot organogenesis from the initiated shoot buds (4 weeks old culture) (b, c), in vitro rooting (3-week-old plantlet) (d, e) and hardened plant growing in plastic cup with sand and soil (f)

Full size image

Development of multiple shoot buds

The results on percent of multiple shoot bud regeneration are presented in Table 2. Among the different concentrations and combinations used, BAP at 1.5 mg l−1 was found to be the best concentration for maximum number of multiple shoot bud development (19.85 shoots/culture) (Fig. 1b). The highest percent of multiple shoot bud regeneration obtained was 98.4% on MS medium fortified with 1.5 mg l−1 BAP alone and it was statistically significant at 5% level (Table 2). Of the two auxin combinations tested, maximum percent of multiple shoot bud development (97.6%) with 10.6 shoots/culture was noticed on MS medium augmented with 1.5 mg l−1 BAP and 0.5 mg l−1 NAA combination, followed by 1.5 mg l−1 BAP and 0.5 mg l−1 IBA combination in which the percent of shoot bud regeneration noticed was 96.4% with 11.4 shoots/culture. Increased rate of multiple shoot bud induction as well as the number of shoot buds was noticed with increasing the concentrations of BAP up to 1.5 mg l−1, while the multiple shoot regeneration frequency was declined when the BAP dose was increased beyond the optimum level. Results suggest that if auxin (IBA/NAA) was added into the medium along with BAP, the number of multiple shoot bud development was significantly decreased due to the formation of callus at the basal part of the differentiated shoot buds. The present study clearly revealed that multiple shoot bud development was suppressed at higher BAP concentrations.

Table 2 Effect of different concentrations of BAP alone and/or in combination with two auxins on development of multiple shoots from cotyledonary node derived adventitious shoot cultures of cucumber
Full size table

To enhance the shoot bud multiplication rate, different concentrations of AgNO3 (1.5–6.0 mg l−1) alone and/or in combination with 1.5 mg l−1 BAP were added into the regeneration medium. It is important to mention that the stunted shoot growth was overcome by addition of AgNO3 in the medium and also promoted the shoot bud elongation in the same media composition (Fig. 1c). The frequency and number of multiple shoot bud regeneration increased with increasing the concentrations of AgNO3 in the medium up to 4.5 mg l−1 (83.4%, with 32.2 shoots/culture), but it was slightly decreased at higher doses. Maximum percent of multiple shoot bud regeneration (100%) with 54.6 shoots/culture was recorded on MS medium fortified with 4.5 mg l−1 AgNO3 in combination with 1.5 mg l−1 BAP (Table 3).

Table 3 Effect of different concentrations of silver nitrate alone and/or in combination with BAP on shoot bud multiplication from cotyledonary node derived adventious shoot cultures of cucumber
Full size table

Rooting and acclimatization

The results on percent of rooting and number of roots are depicted in Table 4. After 2 weeks of culture, the root initiation was noticed directly from the cut portion of the shoots. The percent of rooting was recorded after 4 weeks of culture (Fig. 1d). Interestingly, the percent of root initiation was increased with increasing the concentrations of auxin in the medium up to optimal level (IBA or NAA at 1.5 mg l−1) and the rooting percentage was decreased at higher doses. Among the IBA concentrations tested, the maximum percent of rooting noticed was 91.4% on half-strength MS medium supplemented with 1.5 mg l−1 IBA followed by 2.0 mg l−1. In the case of IBA and KIN combinations used, the highest percent of root induction (96.2%) with maximum number of roots (8.4 roots/shoot) was observed on half-strength MS medium containing 1.5 mg l−1 IBA and 0.5 mg l−1 KIN combination (Table 4). When different concentrations of NAA were used in the medium, the maximum percentage of rooting (89.7%) with 4.2 roots/shoot was obtained on a medium containing 1.5 mg l−1 NAA. If NAA alone was used in the medium for rooting, callus formation was also noticed at the cut end of the shoot. In the case of NAA and KIN combinations tested, 1.5 mg l−1 NAA + 0.5 mg l−1 KIN combination produced the highest percent of rooting (94.4%) with more number of roots (7.2 roots/shoot). It is interesting to note that the number of roots per shoot was positively correlated with auxin concentrations. The rooted plantlets (> 5 cm length) were gently taken out from the culture tubes and washed initially to remove adhered agar traces to avoid contamination. Then, they were transferred to the plastic cups containing sterile sand and soil in the ratio 1:1 and covered with polythene bags to ensure high humidity and placed in the controlled environment (Fig. 1e). After 2 weeks, the polybags were removed and the plantlets were transferred to the greenhouse. Subsequently, the plantlets were established in the field conditions where about 72% of the plantlets were survived. The regenerated plantlets grew normally without showing any morphological variations similar to the control mother plants (Fig. 1f).

Table 4 Effect of different concentrations of two auxins (IBA and NAA) alone and/or in combination with 0.5 mg l−1 KIN on initiation of roots from elongated shoots of cucumber
Full size table

RAPD fingerprinting analysis

To assess the genetic fidelity, DNA from randomly selected 10 in vitro regenerated cucumber plantlets along with a control mother plant was used for RAPD fingerprinting analysis (Fig. 2). A total of 100 random oligonucleotide primers were tested for initial screening. Among these only 15 random primers produced clear and reproducible DNA bands. Each primer produced a unique set of amplification products ranging in size from 300 to 3000 base pairs. The number of bands for each primer varied from 4 to 8, with an average number of 5.9 bands per RAPD primer. The number and size range of amplified scorable bands for each RAPD primer are listed in Table 5. A total of 89 PCR amplicons were recorded and 87 DNA bands showed monomorphism and 2 were found to be polymorphic DNA bands in this study. It is important to mention that the frequency of monomorphism was 98.2% among the regenerated plants and control mother plants investigated. The RAPD fingerprints obtained after PCR amplification of genomic DNA from 10 randomly selected in vitro derived plantlets and the control mother plant of cucumber were scored for appearance as 1 (band present) and disappearance as 0 (band absent) for each plantlet. Results indicate that the RAPD fingerprinting approach is an effective molecular tool for the detection of genetic variants among the in vitro raised plants in cucumber.

Fig. 2
figure 2

RAPD banding patterns generated from the DNA samples of control mother plant and in vitro raised plantlets of Cucumis sativus L., cv. ‘Green Long’ with different primers OPA10 (a), OPB1 (b), OPC11 (c), and OPD06 (d); Lane M: molecular size marker (lambda DNA double digested by EcoRI/HindIII), lane C: control mother plant, lanes (T1–T10): in vitro regenerated plantlets from the cotyledonary node explants

Full size image
Table 5 RAPD decamer primer sequences with G+C contents and number of the RAPD amplicons with monorphic and polymorphic DNA bands generated by PCR for genetic fidelity analysis in cucumber
Full size table

Discussion

The present study describes an efficient protocol for plant regeneration and confirmation of regenerants by genetic fidelity analysis in cucumber. The adventitious shoot bud initiation was achieved in all the concentrations of BAP tested but the number of shoot buds was enhanced up to 2.0 mg l−1 BAP. Further, no shoot bud growth was noticed on medium supplemented with KIN but later produced roots only. Therefore, the initiated adventitious shoot buds were further subcultured onto fresh medium containing the same concentrations of BAP alone and/or in combination with NAA/IBA for enhancement of multiple shoot bud regeneration. The results showed that the number of multiple shoots was increased significantly but the percentage of shoot bud regeneration was not altered when the shoot buds were subcultured onto MS medium containing BAP alone. Vasudevan et al. (2001) reported that BAP produced maximum number of shoots from shoot tip explants of cucumber. The superiority of BAP on shoot bud induction has also been well documented earlier in Citrullus lanatus (Pirinc et al. 2003; Ganesan and Huyop 2010). Ntui et al. (2009) stated that BAP alone has enhanced the percent of shoot bud induction and elongation in Colocynthis citrullus L. Among the BAP concentrations tested, BAP at 1.5 mg l−1 was found to be optimum dose for induction of maximum number of multiple shoot bud regeneration.

Similarly, Mohammadi and Sivritepe (2007) observed enhanced rate of shoot bud multiplication and proliferation on a medium fortified with BAP in Cucumis sativus. It has been well documented that BA has proved as potential cytokinin for induction of multiple shoot organogenesis in different plant species such as Capsicum annuum (Khan et al. 2011), Moringa oleifera (Saini et al. 2012) and Stevia rebaudiana (Thiyagarajan and Venkatachalam 2012). The number of multiple shoots was found to be decreased when a low concentration of auxins was included in the medium. Similar result was also reported earlier by Han et al. (2004) in bottle gourd. In contrast, Vasudevan et al. (2007) reported that the combination of BAP and NAA in MS medium triggered the initiation of adventitious shoot buds from embryonical axis explants of cucumber. In the present study, BAP was found to be the best cytokinin for multiple shoot bud regeneration. Similarly, it has been reported that BAP at lower concentration was found to be optimum for multiple shoot bud induction from nodal explants of Cucumis sativus (Ahmad and Anis 2005). However, the maximum number of multiple shoots in cucumber was obtained on medium containing the combination of BAP (3.0 mg l−1) and IAA (0.5 mg l−1) by Ugandhar et al. (2011), Grozeva and Velkov (2014) and Jesmin and Mian (2016). It has been suggested that lower dose of auxin could trigger the cytokinin activity effectively for production of more number of multiple shoots. Also, plant regeneration from hypocotyl explants of cucumber cv Gergana was reported on medium fortified with kinetin at 1.0 mg l−1 and it was inhibited at higher doses (Grozeva and Velkov 2014). This result further suggested that the cucumber genotype is considered as one of the major factors influencing shoot bud regeneration frequency and different genotypes are required various growth regulator combinations for plantlet development (Wang et al. 2015). Though the number of multiple shoots was enhanced with BAP alone and/or in combination with lower dose of auxins, no shoot bud regeneration was recorded in the cultures grown on medium containing KIN. Recently, Prem Kumar et al. (2016) proved that BAP showed superior effect on multiple shoot bud regeneration as well as shoot elongation in cotton compared to KIN. Similar observations on the superiority of BA over other cytokinins have been reported for Psoralea corylifolia and Withania coagulans shoot regeneration (Siva et al. 2015; Rathore et al. 2016).

To increase the number of multiple shoot buds, the regenerated shoots were further subcultured onto MS medium containing different doses of AgNO3 alone and/or in combination with 1.5 mg l−1 BAP. It is interesting to note that the number of multiple shoot buds was significantly enhanced by addition of AgNO3. The number of multiple shoots was positively correlated with AgNO3 concentration. Ethylene is produced in in vitro plant culture systems and is known to inhibit plant growth and morphogenesis depending upon the species and culture stages. Consequently, inhibitors of ethylene actions such as AgNO3 and polyamines can induce variable in vitro responses in plants (Giridhar et al. 2001). However, both the frequency of multiple shoot bud regeneration and number of shoots were slightly decreased at higher doses of AgNO3. Similar results were also reported earlier by Park et al. (2012). Balkhande et al. (2013) demonstrated that the addition of AgNO3 along with BAP enhanced the number of shoots in Momordica cymbalaria. Mohiuddin et al. (2005) reported that shoot regeneration was inhibited in Cucumis sativus when AgNO3 (100 µM) was added in the shoot regeneration medium. In the present study, an enhanced rate of multiple shoot bud regeneration was noticed up to 4.5 mg l−1 AgNO3 + 1.5 mg l−1 BAP combinations from cotyledonary node explants of cucumber. Similarly, Diao et al. (2009) also reported that addition of AgNO3 in the medium enhanced the rate of somatic embryo formation in cucumber. One of the reasons for increased percent of multiple shoot bud development was due to the action of silver ions as competitive inhibitor of ethylene rather than inhibiting ethylene biosynthesis (Zhang et al. 2001). According to Kumar et al. (2009), the potent ethylene biosynthesis inhibitor, AgNO3 could increase the arginine decarboxylase (ADC) activity that ultimately enhances the endogenous polyamines level in cultured cells. It has been reported that accumulation of polyamines level in cells exhibits improved rate of in vitro shoot bud multiplication in plants (Kumar et al. 2009). It is presumed that silver nitrate might have altered the ethylene action but triggered the polyamine production and thus it might have played a pivotal role in enhanced production of multiple shoots under in vitro. Similarly, Giridhar et al. (2003) also reported a positive role of AgNO3 on enhanced production of in vitro shoot bud multiplication in C. arabica and C. canephora. Recently, Prem Kumar et al. (2016) also recorded the enhanced rate of shoot bud regeneration and multiplication in cotton by the addition of AgNO3. Results altogether suggest that the addition of AgNO3 along with BAP was greatly promoted the multiple shoot bud development as well as shoot bud elongation in cucumber.

Elongated shoots were cultured onto half-strength MS medium augmented with various concentrations of two auxins (IBA and NAA) alone and/or in combination with 0.5 mg l−1 KIN for rooting. Of the two auxins examined, IBA was found to be the superior auxin for induction of maximum percent of rooting over NAA. However, the IBA and KIN combination was found to be best for root initiation compared to the NAA and KIN combination. Both the frequency of rooting and number of roots were found to be enhanced by addition of lower dose of KIN in the medium. In general, IBA was proved as the best auxin for rooting response in a wide range of plant species including Melothria maderaspatana (Baskaran et al. 2009) and Benincasa hispida (Thomas and Sreejesh 2004). It is interesting to note that IBA and KIN combination produced the best rooting response when compared to IBA alone. Therefore, IBA was found to be a potent auxin for rooting in cucumber followed by NAA in the present study. Similar observation was also reported in cultivated Cucumis species (Compton et al. 2001; Selvaraj et al. 2002). Well developed plantlets were successfully transferred into plastic cups containing soil and sand initially and they were subsequently established in the field. The field grown plants appeared to be phenotypically similar to the mother plant and noticed normal flowering as well as seed set.

In the present study, the genetic fidelity of the in vitro derived plantlets of cucumber was assessed through RAPD-PCR technology. DNA molecular markers are being considered to be the most effective tool to assess the variability found with the genomic sequence of the regenerated plants (Kumar et al. 2015). Earlier reports suggest that RAPD technology was effectively used to identify the somaclonal variations (Al-Zahin et al. 1999; DeVerno et al. 1999) and to identify the genetic fidelity of the tissue culture plants (Qin et al. 2006; Devarumath et al. 2007; Thiyagarajan and Venkatachalam 2012). Results show that a total of 100 random decamer primers were screened and only 3% polymorphism was recorded. The occurrence of polymorphism among the regenerated as well as mother plants of cucumber might manifest during cell division or differentiation under in vitro stress response imposed in the form of DNA methylation, chromosome rearrangements, point mutations (Phillips et al. 1994). Similarly, low percent of DNA polymorphism (2%) was reported by analyzing the genetic stability of tissue culture plants of Brassica oleracea using RAPD-PCR (Qin et al. 2006). Recently, Kumar et al. (2015) reported plant regeneration in Brassica oleracea and noticed 10–12% polymorphism after genetic stability analysis of in vitro regenerated plantlets through RAPD analysis. It is reported that some percent of genetic variability might occur during in vitro cellular dedifferentiation and regeneration of plants (Kumar et al. 2015; Werner et al. 2015). The present results show that the plant regeneration from cotyledonary node explant is a highly reliable propagation procedure for multiplication of genetically identical plants (true-to-type). The low percent of genetic variability noticed could be due to the successive medium changes and the possibility of growth regulators causing variations or mutations (Werner et al. 2015). However, in the present study, about 97.1% of the DNA bands showed monomorphism for all the primers tested, describing genetic stability in the Cucumis sativus variety Green Long used for tissue culture. Earlier, RAPD-based DNA markers were successfully applied to prove the genetic fidelity of in vitro raised plants from different species including Chlorophytum borivilianum (Samantaray and Maiti 2010), Drosera anglica and Drosera binata (Kawiak and Lojkowska 2004), Cuphea procumbens (Fatima et al. 2012), and Stevia rebaudiana (Thiyagarajan and Venkatachalam 2012). In contrast, there was a genetic instability detected among the regenerated cucumber plants via somatic embryogenesis (Elmeer et al. 2009). In the present study, one of the reasons for the absence of genetic variability among the regenerated plants might be due to the development of adventitious shoot buds in which the callus phase was bypassed during plant regeneration.

Conclusion

In summary, an efficient plant regeneration protocol via direct adventitious shoot organogenesis from cotyledonary node explants of Cucumis sativus L. was established. Highest frequency of shoot bud development was noticed on MS medium containing 1.5 mg l−1 BAP. Maximum percent of shoot bud multiplication with highest number of shoots was obtained on MS medium supplemented with 1.5 mg l−1 BAP and 4.5 mg l−1 AgNO3 combination. Results strongly suggest that silver nitrate showed positive effect on not only for enhancement of multiple shoot bud regeneration but also promoted rapid shoot bud elongation in cucumber. Further, the elongated shoots were rooted efficiently on half-strength MS medium containing IBA and KIN combination. The rooted plantlets were successfully established in the field conditions and the regenerated plantlets were found to be phenotypically similar to the control mother plant. DNA finger printing analysis indicated that the in vitro raised plants were found to be true to type. Therefore, the cotyledonary node explants could be successfully used for generation of transgenic cucumber plants in the future. To the best of our knowledge, this is the first report on high frequency of plant regeneration and assessment of clonal fidelity of in vitro regenerated plants from cotyledonary node explants of Cucumis sativus L.