Disinfection procedure for stem cuttings and in vitro production of axillary buds for the Persian lime sanitation

The research work covered the in vitro establishment of Persian lime nodal segments using a twig disinfection protocol, and their culture on Murashige and Skoog (MS) culture media supplemented with antibiotics (Estrepto-Ler ® and Rifampicin) and fungicide (Chlorothalonil). The axillary buds production was performed cultured the nodal segments without contamination signs on MS culture media enriched with two cytokinins (6-BAP and Kinetin) at different concentrations. Results showed that 52 % of the nodal segments did not show signs of in vitro contamination when the twig submerged in 20% chlorine for 15 min during disinfection process and nodal segments were grown in MS culture medium supplemented with 2 mg L -1 of Rifampicin and fungicide Chlorothalonil. Interestingly, MS culture medium enriched with 6-BAP (0.5 mg L -1 ) + Kinetin (0.5 mg L -1 ) turned out to be the best combination to achieve that 78 % of nodal segments promoted 1 to 2 axillary buds.


Introduction
In Mexico, citrus fruits are an agricultural product that are part of the usual diet, likewise, they represent an important source of vitamin C, pectin, terpenes, flavonoids, among other components, that positively influencing consumer health [1].Regarding to Persian lime (Citrus x latifolia Tan.), is exported to various countries around the world due to the high quality of the harvested fruits, it is also the crop with the highest demand in United States of America, being Mexico the main exporter with 98% of imports.Therefore, in recent years, Mexico has positioned itself as the second largest producer of this crop worldwide [2]; being Veracruz the main producer and exporter state [3].
However, the Persian lime is not exempt from diseases caused by bacteria, viruses, fungi, mycoplasmas and other similar organisms which cause significant economic losses in citrus cultivation worldwide [4].Thereby, citrus fruits are facing new challenges and threats related to phytosanitary problems that incur more risk in the citrus fruit national industry.Consequently, at end 2014, 16 of the 23 citrus producing states in Mexico were affected by presence of Huanglongbing (HLB) and Citrus Tristeza Virus (CTV), devastating vascular diseases that have not cure, as well as of fungal diseases caused by different species of Lasiodiplodia [3], diseases that cause a decrease in yield and productivity, which lead to mortality in Persian lime.
To solve this problem, plant tissue culture represents a very valuable biotechnological tool that allows through plant sanitation, the possibility of massively propagate pathogen-free plants, as well as establish germplasm banks whose purpose is safeguard in vitro plant species that due to natural disasters and/or diseases can be lost [5].In this context, in vitro micrografting shoot tips can be an efficient for shoots sanitation, rejuvenation of plant material and has proven to be effective for eliminating systemic pathogens in citrus [6][7][8][9].Therefore, this technique is effective for obtained pathogen-free plants transmissible by grafting and to guarantee plant health [4].However, to perform the in vitro micrografting shoot tips technique, is necessary to aseptically establish the nodal segments obtained from the stem cuttings and adapt the necessary conditions to successfully perform the micrografting technique [6,8].Unfortunately, to date, not disinfection protocol has been established to obtain nodal segments under controlled in vitro conditions, which crucial step for citrus sanitation.Furthermore, optimal culture media still need to be standardized in order to produce in vitro axillary buds using different cytokinins, for example, 6-Benzylaminopurine (6-BAP) and Kinetin (Kin).
Therefore, the aim of this study was:  To establish a disinfection protocol from Persian lime stem cuttings and establish nodal segments under in vitro conditions. To standardize the culture medium enriched with different cytokinins concentrations (Kin and 6-BAP), to induce axillary buds of the nodal segments introduced in vitro.This research work is the basis for later performing the micrografting shoot tips, which are necessary for Persian lime sanitation.Likewise, with the help of pruning shears (disinfected with 20% commercial chlorine), a minimum of 25 stem cuttings with a length of 10 to 15 cm were cut for each Persian lime tree.To avoid dehydration of the stem cuttings, they were stored in moistened paper inside sterile polyethylene bags and kept in cooler to maintain the temperature close to 4 °C until transfer to laboratory.

Stem cuttings disinfection
Stage 1.Under laboratory conditions, the stem cuttings were defoliated leaving only leaf petiole in order to protect the axillary buds.Disinfection process consist of washing the stem cuttings with water and mild soap in three repetitions, then, are rinsed in sterile distilled water and submerged in Tween® 20 solution (5 %) for 10 min, later are again rinsed with sterile distilled water in three repetitions.Subsequently, the stem cuttings were submerged in Terra-Vet ® 200 (Oxytetracycline) bactericide solution (0.06 g L -1 ) for 1 h and then in Amistar (Azoxystrobin) systemic fungicide solution (2 ml L -1 ) for 1 h.Finally, the stem cuttings were rinsed in sterile distilled water and placed on sterile paper for 5 min to dry.Stage 2. Completed the previous stage, in laminar flow hood, the stem cuttings were disinfected with different concentrations of chlorine (10, 15 and 20 %) for 15 min (Table 1).Then, were rinsed in sterile distilled water for 5 min and submerged in 75% alcohol solution for 10 min.Subsequently, were rinsed again in sterile distilled water for 5 min to remove alcohol traces.To finish, stem cuttings were sectioned into segments of 5 to 8 cm (nodal segments) and left to dry on sterile absorbent paper for 5 min until cultured under in vitro conditions.

Axillary buds production
After 30 days, the contamination percentage of in vitro nodal segments was analyzed.Likewise, nodal segments that did not show signs of contamination were sub-cultured in new culture media (as described above), however, these were supplemented with 6-BAP and Kinetin in order to improve the axillary buds production.The different treatments to produce axillary buds from nodal segments were performed as following: Control Treatment 1 (C1: MS culture medium without phytohormones), Treatment 1 (T1: MS culture medium + 0.5 mg L -1 of 6-BAP), Treatment 2 (T2: MS culture medium + 0.5 mg L -1 of Kinetin), Treatment 3 (T3: MS culture medium + 0.25 mg L -1 of 6-BAP + 0.25 mg L -1 of Kinetin) and Treatment 4 (T4: MS culture medium + 0.5 mg L -1 of 6-BAP + 0.5 mg L -1 of Kinetin), as indicated in Table 2.

Statistical analysis
The nodal segments introduced in vitro were analyzed as completely randomized designs.One-way analysis of variance (ANOVA) was used to determine a significant difference among treatments.Tukey multiple comparison test was used to determine significant differences between the means (p<0.05) using Statgraphics Plus Ver.5.1 Software (Statistical graphics Corp., USA).Graphics were performed using the Sigma Plot ver.11.0 program.

Disinfection protocol
Our results showed that nodal segments from stem cuttings disinfected with 10, 15 and 20 % of chlorine, when were culture in MS medium without antibiotic presence, showed 100 % contamination after 60 days.Similarly, nodal segments disinfected with 10, 15 and 20 % of chlorine, but with the addition of Estrepto-Ler antibiotic (T1 -T3 treatments), showed high contamination percentage (between 74 and 79 %).Interestingly, when to MS medium Rifampicin antibiotic was added, the T4 -T7 treatments showed a lower contamination percentage (between 48 until 69 %).Likewise, the results showed that stem cuttings disinfected with 20% chlorine for 15 min during disinfection process and the subsequent in vitro culture of the nodal segments in MS medium supplemented with 2 mg L -1 of Rifampicin (T6) turned out to be the best treatment reaching a 35 % disinfection (Table 1).In general, although bacterial contamination decreased (T1-T6 treatments), the fungal contamination remained persistent (Fig. 1a).Therefore, it was decided to performed a final treatment, which consisted of carrying out the same steps of Treatment 6 but adding Chlorothalonil fungicide (1 ml L -1 ) to MS medium (T7), the above, to reduce fungal contamination.Thus, the nodal segments showed 52 % disinfection after 60 days of culture in MS medium under in vitro conditions (Table 1).As shown in Fig. 1a, when not bactericide solution was added to the MS medium, the main contamination of nodal segments (Treatments Control) was by bacteria.Likewise, although both bactericides reduced bacterial contamination, the fungal contamination was persistent even when Chlorothalonil fungicide was added (T7).

Axillary buds production
Nodal segments cultured after 60 days in MS medium without cytokinins (Treatment Control) showed a low axillary buds number and only the 16 % of these segments produced buds.When the MS medium was enriched with 0.5 mg L -1 of 6-BAP, 52 % of the segments produced one axillary buds (T1).Similarly, the results showed that the 56 % of nodal segments produced one axillary buds when were culture in MS medium enriched with 0.5 mg L -1 of Kinetin (T2).However, when the MS medium was enriched with 0.25 mg L -1 of 6-BAP and with 0.25 mg L -1 of Kinetin (T3), only the 32 % of nodal segments produced axillary buds, but a increases of axillary buds was observed (1.67 buds per nodal segments).Also, when the MS culture medium was enriched with high concentrations of 6-BAP (0.50 mg L -1 ) + Kinetin (0.50 mg L -1 ) (T3), a best axillary buds production was observed (78 %) (Table 2).These nodal segments showed 2.43 buds per nodal segments at 60 days under in vitro conditions (Fig. 1b).

Discussion
Over the years, several sanitation methods has been described to establish in vitro plants, all with the same goal, optimize the process and propagate pathogen-free plants.CEIXTA currently has a germplasm bank that holds a wide variety of Persian lime trees rescued in the region, as well as introduced from United States of America.Due to their agronomic importance, the producers selected all these Persian lime varieties introduced in the CEIXTA germplasm bank, however, in the last decade, others varieties have been of interest to the producers, therefore, these varieties should be included in the CEIXTA germplasm bank collection and thus have greater varietal diversity.For this reason, the aim of this study was establish in vitro nodal segments for achieve an adequate number of axillary buds per nodal segment.In this way, we introduced new varieties of Persian lime to germplasm bank; so that micrografts shoot tips can be carried out in the medium term with the purpose of massively propagate pathogen-free Persian lime plants.
Different reports have described that the important aspects for achieve the citrus production under controlled in vitro conditions is based on the adequate rootstocks selection [11], as well as an efficient protocol for explants disinfection from field, nursery and greenhouse [12].However, to the day, disinfection protocol for obtaining aseptic explants in citrus fruits is not established.In addition, under natural field conditions, citrus fruits have a high group of parasitic microorganisms that lead to the development of pests and diseases, mainly caused by bacteria, fungi and virus.Therefore, the few in vitro buds that it can be obtained present defoliation problems and although it can be controlled with AgNO3, the buds length not develop adequately to be used as explants source [12].In this context, Varela [13] described the sanitation of 35 different citrus varieties, including Persian lime, their experimental design consisted in stem cuttings cut into nodal segments of approximately 3 cm and then were submerged by immersion in 70% chlorine for 2 min.After, these nodal segments were submerged in different NaClO2 solutions at different exposure times and treated with bactericide (Oxytetracycline; 2.0 g/L) and fungicide (Amistar; 2.0 g/L) solution for 1 h.However, Varela [13] reported nodal segments death under in vitro conditions due to the presence of a large contaminants number and the short time of immersion of the nodal segments in NaCIO2 solution.Similarly, García [14] reported high contamination rate of nodal segments of Persian lime under in vitro conditions, of which 20 % corresponded to fungi and 10 % to bacteria.Furthermore, Herrera-Flores et al. [15] reported a disinfection protocol for the in vitro establishment of Persian lime nodal segments; however, they showed a percentage asepsis close to 34 %.Due to the above, we confirm that the plant material disinfection is a complex process because the stem cuttings brought directly from field have high fungi and bacteria rates, which highlights the importance to establish a disinfection protocol in suitable explants of Persian lime.Although some works present a disinfection protocol to follow, they are not standardized due to the difficulty of in vitro establishment.The results obtained in our research work coincide with those reported because also finding high contamination percentages during the disinfection process in Persian lime nodal segments under in vitro conditions.In this way, we believe that although the use tissue culture techniques is a viable alternative for the sanitation and Persian lime micropropagation, the large number of microorganisms present in explants from field, limits it's in vitro establishment.Therefore, is necessary to continue conducting research that allows us to reduce bacterial and fungal contamination, however, at 60 days, we obtained 52% of nodal segments without contamination signs under in vitro conditions.
Respect to axillary buds production, most reports indicate the use of cytokinins such as 6-BAP and Kinetin, as well as, auxins (AIA and ANA) in order to produce a greater number of axillary buds under in vitro conditions using nodal segments as explants in species different.Thus, Singh et al. [16] reported cultured stem cuttings in MS medium enriched with 1 mg L -1 of 6-BAP and 0.5 mg L -1 of Kinetin to induce buds in Citrus reticulata Blanco and Citrus limon Burm.f.Likewise, García [14] reported that cultured Persian lime nodal segments in MS medium enriched with 6-BAP (1 mg L - 1 ) and Indole-acetic acid (0.5 mg L -1 ) produced 1.19 axillary buds, however, this treatment not showed difference significant in relation to other treatments; this variable was related to cell dedifferentiation caused by cytokinins-auxins interaction.In addition, Herrera-Flores et al. [15] reported that achieved 42% of axillary buds 15 days after in vitro establishment when used a combination of 1 mg L -1 of 6-BAP and 0.5 mg L -1 of Naphthalene-acetic acid, obtained 1.2 axillary buds per explant.Our results showed that culture the nodal segments during 60 days (two sub-cultured of 30 days) in MS medium enriched with 6-BAP (0.5 mg L -1 ) + Kinetin (0.5 mg L -1 ), was sufficient for that 78 % nodal segments induced at least two axillary buds (Fig. 2).Therefore, this MS medium enriched with 6-BAP and Kinetin is promising for use in the in vitro propagation of Persian lime.Finally, our showed the disinfection protocol using Persian lime nodal segments from stem cuttings, as well as, disinfection process of these nodal segments under in vitro conditions, also, is shown the axillary buds obtained when the MS medium was enriched with 6-BAP and Kinetin (Fig. 3).

Conclusion
We must develop new strategies which allow us reduce the contaminants growth in nodal segments of Persian lime during its in vitro establishment.The results of our research showed that 52% of the nodal segments did not show signs of in vitro contamination when the stem cuttings were disinfected with 20% chlorine for 15 min and when the nodal segments were cultured with Rifampicin bactericide (2 mg L -1 ) and Chlorothalonil fungicide (1 ml L -1 ).Furthermore, the use of cytokinins as 6-BAP (0.5 mg L -1 ) and Kinetin (0.5 mg L -1 ) in culture medium promoted that 78% of the nodal segments produced of 1 to 2 axillary buds.The disinfection and production of axillary buds protocols will allow us to medium term perform the sanitation of Persian lime varieties using in vitro micrografting shoot tips technique.

Figure 1 Figure 2
Figure 1 Effect of disinfection treatments on the establishment of nodal segments from Persian lime, after 60 days of culture on MS medium (a).Effect of treatments with 6-BAP and Kinetin, alone or combined, on organogenic response of in vitro nodal segments for axillary bud production, after 60 days of culture in MS medium (b).Letters indicate significant differences at p>0.05, bars indicate mean ± SE.

Figure 3
Figure 3 Disinfection of Persian lime nodal segments obtained from stem cuttings and in vitro axillary buds production.a) Persian lime tree of CEIXTA.b) Stem cuttings from 10-15 cm.c-d) Disinfection process of stem cuttings.e) Stem cuttings treated with bactericide and fungicide.f) Stem cuttings disinfection with different chlorine concentrations.g) MS culture medium enriched with 6-BAP and Kinetin, alone or combined, for axillary buds production.h) Stem cuttings cut by segments and in vitro culture of these nodal segments.i-j) In vitro establishment of nodal segments in culture room.k-l) Axillary buds production from in vitro nodal segments at 60 days.

Table 1
Disinfection protocol in nodal segments of Persian lime.

Table 2
culture medium enriched with different concentrations of cytokinins to induce axillary bud production.