Vegetal material

The seeds of genotypes "Atred (S)", "Elinia 48 (R)" and "Mirlo 26 (R)" were used. The first characteristic of susceptibility to leaf rust and the remaining two considered resistant. The seeds were provided by the National Institute of Forestry, Agriculture and Livestock (INIFAP) Field Experimental Valley of Mexico (CEVAMEX).

Culture medium and incubation conditions

The basic culture medium used consisted of mineral salts ^{Murashige and Skoog (1962)} (Sigma Aldrich® M5519, 4.4 g L^-1), supplemented with sucrose (30 g L^-1). The pH was 5.7 ± 0.1 adjusted with NaOH or HCl 1N, by a potentiometer (Thermo Scientific® Orion 3 Star meter). The phytagel was added (Sigma^®, 2.5 g L^-1) as a gelling agent. The sterilization of the culture medium was autoclave vertically (AESA®, modelo 300) at 121 C and 1.5 kg cm^-2 pressure for 20 min.

The cultures were incubated with photoperiod of16/8 h light/ dark provided by fluorescent lamps 75 W white light, with photosynthetic irradiation of 45 μmol m^-2 s^-1, temperature of 26 ± 2 °C and relative humidity of 30% .

Establishment of aseptic culture

Of the three wheat genotypes uniform size seeds without biological or mechanical damage were selected. They were washed with detergent powder for15 min with tap water and finally a wash with sterile distilled water. After disinfection treatments were established hydrogen peroxide^® (H₂O₂), colloidal silver, commercial sodium hypochlorite (NaOCl), Tween® 20 and fungicides: Benlate®, Captan® and Vitavax 300® (Table 1).

Table 1 Tested Treatments in the disinfection of seeds of wheat genotypes "Atred" (S), "Elinia 48" (R) and "Mirlo 26" (R). 

The seeds were rinsed with sterile distilled water to remove excess disinfectant. The process of seed disinfection was done on a horizontal laminar hood (VECO®) flow. The planting was done in basic medium (^{Murashige and Skoog 1962}) without hormones with 15 repetitions per treatment. At three weeks of culture contamination and the percentage of germination they were evaluated.

Germination and callus induction

To induce germination in vitro, seeds of three wheat genotypes were planted on basic medium ^{Murashige and Skoog (1962)} supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) and kinetin (KIN) (Table 2). The planting was done with the root meristem region of 3 mm immersed in the culture medium. After three weeks of culture the percentage of germination was evaluated. The treatments were composed of 15 repetitions, and each repetition consisted of a seed planted in a culture flask.

Table 2 Dose of 2,4-D and kinetin spiked to medium ^{Murashige and Skoog (1962)} on germination of wheat genotypes "Atred" (s) "Elinia 48" (R) "Mirlo 26" (R). 

Plant regeneration via direct organogenesis

From seedlings germinated in vitro one month of age, basal stem cross sections were dissected [thin cell layer (thin layers of cells)], 6-9 mm diameter and 1-2 mm thick. The explants were plated on medium staple crop ^{Murashige and Skoog (1962)} supplemented with benzyladenine (BA) and 3-indoleacetic acid (AIA) (Table 3). At 30 days they were evaluated: bud break (%), number ofbuds per explant, shoot length (cm) and number of leaves.

Table 3 Dose of BA andAIA evaluated in direct and indirect organogenesis of wheat genotypes "Atred" (S), "Elinia 48" (R) and "Mirlo 26" (R). 

Regeneration of plants via indirect organogenesis

The callus explants were used of 60 days of age obtained from the germination of the three genotypes. They were seeded in culture medium ^{Murashige and Skoog (1962)}) supplemented with BA and AIA (Table 3). The measurement time and measured variables were the same as in direct organogenesis.

Rooting in vitro

During regeneration of plants via direct and indirect organogenesis induction of roots in hormone levels tested (Table 3) was evaluated. The variables evaluated 30 days after sowing, were rooting percentage and number and root length (cm).

Seedling acclimation

Three batches of rooted plants of the three wheat genotypes according to the height are selected: five floors 5 cm, 10 cm and five of five 15 cm. The plants were removed from the jars and roots were washed with sterile distilled water. It transplanted in styrofoam cups of 237 mL capacity with perlite mixed and peat-moss (1:1). The plants were kept in greenhouse temperature of 24 ± 2 °C for two weeks (^{Mujeeb, 2000}) and were watered every other day with the solution ^{Murashige and Skoog (1962)}.

Inoculation of rust

To evaluate the resistance of the three genotypes, the rust strain used BBG/BPC where the formula avirulence/ virulence was: Lr1, Lr2a, Lr2b, Lr2c, Lr3, Lr3bg, Lr3ka, Lr9, Lr13, Lr14a, Lr15, Lr16, Lr17, Lr18, Lr19, Lr21, Lr24, Lr25, Lr26, Lr28, Lr29, Lr30, Lr32, Lr35, Lr36, Lr61/Lr10, Lr11, Lr12, Lr14b, Lr20, Lr23, Lr27, Lr31, Lr33, Lr72.

Acclimatized plants inoculating

At16 days after acclimatization plants of the three genotypes they were inoculated with a suspension of uredinio spores of rust race BBG/BPC. A concentration of2.3 mg mL^-1 of spores in mineral oil was applied (Soltrol^® 170) (^{Herrera-Foessel et al, 2003}). The inoculation was made when the seedlings showed the first extended sheet. With an atomizer (FELISA®, Modelo FE-1500L) were sprayed evenly; they allowed to dry 20 minutes and placed in a chamber (Hydrofogger®) for 16 h with 100% relative humidity. Then they moved to the greenhouse temperature of24 ± 2 ^oC.At10 days potted plants were transplanted with characteristics of interest for seed. At 12 days after inoculation with were made evaluations of scale ^{Roelfs et al. (1992)}.

Inoculation in vitro plants

Of the three genotypes in vitro plants of 5, 10 and 15 cm were selected and inoculated with leaf rust. Spores were suspended in 200 mL of distilled water and applied at three concentrations (0.01, 0.03 and 0.05 mg mL^-1). The four concentrations of sucrose (10, 15, 20 and 30 g L^-1) were also added to culture medium ^{Murashige and Skoog (1962)} to assess the growth of the spores that will eventually fall to the culture medium. Inoculated plants were kept in the incubation room and at 20 days was evaluated the presence or absence of rust pustules leaf.

Statistic analysis

All experiments were conducted in a completely randomized design. For each variable analysis was applied of variance with SAS Statistical Analysis System (^{SAS Institute, 2003}) and mean comparison was used Tukey test (p ≤ 0.05).

Establishment of aseptic culture

The survival of the seeds in the three wheat genotypes was associated with disinfection treatment (p≤ 0.05). The H₂O₂ (2% v/v) for 22.5 h, colloidal silver stable (10% v/v) + NaClO (50% v/v) + Tween 20 (4% v/v) for 50 minutes and the mixture Benlate (8 g L^-1) + Captan (8 g L^-1) for 50 min was the treatment produced the best results. The average survival rate was 85% in the three genotypes; contamination by bacteria was 5% and 10% by fungi. In the treatment with the fungicide it was included Vitavax^®300 germinated only 10% of the seeds. While controlling fungi and bacteria was efficient; result is likely to be toxic to the seeds and negatively affected the germination. Pollution caused by fungi and bacteria is common in the in vitro establishment of seeds that are present regularly in natural conditions and should define an efficient method of disinfection (^{Das and Pal, 2005}; ^{Rodríguez et al, 2008}; ^{Folguera et al, 2009}).

Germination and callus induction

The statistical differences detected in the five tested in the three wheat genotypes evaluated treatments were not significant; however, the higher rate of germination in the three genotypes was achieved with 20.35 μM of 2,4-D and 5.8 μM kinetin and average was 79%. Regarding the formation of callus from seeds, there were differences in the treatments for each genotype (p≤ 0.05). Most callus formation was promoted by the 2,4-D combined with kinetin. For genotype "Elinia 48" (R) the highest rate (73%) it was obtained with 20.35 μM of 2,4-D and 12.5 μM of kinetin. For "Mirlo 26" (R) was 60% and was achieved only 20.35 μM of 2,4-D without kinetin; while in "Atred" (S) the highest percentage (53%) was achieved with 20.35 μM of 2,4-D and 5.8 μM of kinetin.

In grasses, callus formation has been reported from different explants, as mature and immature embryos, inflorescences segments, young leaves and stem segments (^{Armstrong and Green, 1985}). That is why the response observed in the seeds of the three genotypes of wheat is the product of stimulus of 2,4-D and kinetin (^{George and Debergh, 2008}).

The callus culture is a valuable tool for the spread of wheat plants and for studies of in vitro selection in breeding programs (^{Pérez et al, 2010}). The callus culture has allowed the isolation of cells and protoplasts, the production of secondary metabolites of biological interest, the conservation of germplasm and introducing new genetic variability. Therefore, regeneration of wheat plants may have commercial applications, such as set forth in other grasses including rice, wheat and maize (^{Huang and Shaolin, 2002}).

Plant regeneration via direct organogenesis

Seedlings three weeks after germination of genotypes "Atred" (S), "Mirlo 26" (R) "Elinia 48" (R) were used as explant source for evaluating the induction of direct organogenesis by leaf segments and sections transverse stem.

The sprouting variables were affected by the type of explant in the three wheat genotypes evaluated (p≤ 0.05). The stem cross sections were the only explants showed morphogenic response while leaves no response. In the cross sections of stem sprouting was presented to ten days after planting, and increased production of sprouts and greater length there of was obtained with 11.1 μM of BA and 1.0 μM of AIA in all three genotypes. In "Mirlo 26" (R) and "Atred" (S) eight outbreaks were obtained on average, while in "Elinia 48" (R) was seven. The average length of sprouts was 7 cm (Figure 1, Table 4).

Figure 1 Organogenesis wheat "Atred", "Elinia 48" and "Mirlo 26 " and response and in vitro plants acclimated to rust. (a) regenerated plants via direct organogenesis genotype "Atred"; (b) inoculated seedlings genotype "Atred"; (c and d) freckling of white on the leaves; (e) rust pustules on genotype "Mirlo 26"; (f) leaf death in genotype "Atred"; and (g) pustule at apex of leaf genotype "Elinia 48". p= rust pustule. 

Table 4 Organogenesis wheat "Atred" (S), "Mirlo 26" (R) and "Elinia 48" (R). 

B= brotes; LB= longitud de brotes; H= hojas; * = p≤ 0.05; NS= no significativo.

The BA is widely used for multiplication in vitro cereal because it stimulates the formation and shoot length and the number of sheets (^{Patiño, 2010}); cereals such as rice and barley stimulates proliferation of sprouts (^{Patiño et al., 2007}). The response of the three genotypes of wheat to the cytokinin was different; 11.1 μM of BA with the number of buds, leaf length and number of leaves increased considerably. The addition of AIA affected the number of outbreaks, since the combination with BA produced a greater response variables organogenesis. The effect cytokinins and auxins in of morphogenesis in vitro is different between species and varieties, it has been suggested that endogenous levels in the plant also influence morphogenic responses in vitro (^{Britto et al., 2003}).

Regeneration of plants via indirect organogenesis

At this stage, the callus derived from seeds of three wheat genotypes were established in medium culture MS (1962) with different concentrations of BA and AIA. After 60 days of culture was obtained regeneration of plantlets with differences between genotypes (p≤ 0.05). The maximum response number and length of sprouts was achieved with 11.1 μM of BA and 1.0 μM of AIA in the three genotypes. To "Elinia 48" (R) 4 outbreaks 3 cm in length were obtained; for "Atred" (S) 3 outbreaks of 3.5 cm and for "Mirlo 26" (R) 5 outbreaks of4.5 cm. The average germination percentage was 45% in the three genotypes (Table 4). Most outbreaks and regenerated plantlets with direct organogenesis was the best route morphogenic in the three genotypes.

Rooting in vitro

The in vitro rooting was achieved in the same treatments used to induce regeneration of plantlets via direct and indirect organogenesis in the three genotypes. However, the number and length of roots used for each treatment and for each genotype was different (p≤ 0.05). The highest values of number and length of roots were obtained in the treatment consisting of11.1 μM of BA and 1.0 μM of AIA in the three genotypes; the rooting was 60%.

Seedling acclimation

The survival rate in the acclimatization of plants of the three genotypes of wheat was 90% under greenhouse conditions. Although there were no significant differences between plant height and percentage of survival (p≤ 0.05), the highest percentage of naturalized plants indicates that the employee acclimation process (^{Mujeeb, 2000}) was optimal. The mixture of peat moss + perlite in proportion 1:1 was efficient; its high content of organic matter favors the adaptation of plants; also provides good water retention and excellent aeration, properties benefited survival at 10 days after transplantation (^{Pospisilova et al, 1999}).

Inoculation of rust in plantlets in vitro

The significant differences were detected in the inoculation dose of rust on the seedlings, but not in spiked sucrose concentrations to the culture medium on the growth of spores (p≤ 0.05). In susceptible wheat genotypes spores start germination at 30 min after contact with the sheet and a dewdrop. The period between spore germination, penetration, establishment of the colony and sporulation time in the field can range from 8 to 10 days under constant temperatures of 20 to 24 °C (^{Singh and Dubin 1997}). Therefore, in this study evaluations at different times they were made to observe the behavior of the pathogen in the three genotypes in vitro wheat.

The in vitro seedlings of the three genotypes of wheat cultured with 30 g L^-1 sucrose, pustules on the leaves was observed when inoculated with 0.03 mg L^-1 of rust spores. The first symptoms of leaf rust are the appearance of freckles small or tiny white spots between 7-10 days (^{McIntosh et al., 1995}; ^{Singh et al., 2004}) after infection. Genotypes inoculated in vitro, signs appeared 20 days after inoculation, disease therefore had a slower growth compared to the field or greenhouse plants (Figure 1). The maximum rate of pustules on the three genotypes in the dose of 0.01 mg mL^-1, spore was 10%, while a dose of 0.03 mg mL^-1 was 70% for "Atred", 40% for "Mirlo 26 "and 30% in" Elinia 48 ". In the dose of 0.05 mg mL^-1 pustules percentage was 25% in the three genotypes.

The formation of pustules under greenhouse conditions is favored by temperatures of 20 to 24 °C; is delayed if less than 16 °C. These aspects coincide with the response obtained in vitro inoculation where the temperature of 26 ± 2 °C was constant 24 hours a day; relative humidity 30% and photoperiod 16/8 h light/dark. These conditions facilitated the occurrence of rust (Figure 1).

In the literature there are no reports of inoculation procedures of leaf rust of wheat under in vitro conditions. The methodology developed here is the first study that was done about it, which may be useful in the characterization of the genotypes evaluated so they can be selected and used in breeding programs and the consequent obtaining varieties resistant to this pathogen. Therefore, evaluation of leaf rust under in vitro conditions generates significant for genetic improvement alternatives being Puccinia triticina E. selective agent, making it feasible to make the in vitro selection without regeneration of plants in greenhouses or countryside.

Under the premise that the behavior of plants in vitro wheat to the pathogen is similar to stem explants were extracted where plants, the selection process would be greatly facilitated in resistance to leaf rust. This in vitro selection was carried out using different concentrations of the selective agent Puccinia triticina E. This matches with ^{Cubero (2003)} who mentions that this method can be simple and efficient since the agent sensitive cells die, allowing growth tolerant and perhaps with a higher level of differentiation genotypes would have been possible to regenerate tolerant plants.

Within five days of the appearance of the pustules plants began to show symptoms that started with necrosis of leaves. This response was due to Puccinia triticina stressed out in vitro plants, assuming that those who survived and showed no pustules are tough, response that matches what obtained in the materials evaluated in vivo. The information obtained could be applied to the breeding of genotypes with tolerance to this disease.

Some of the advantages of inoculating in vitro rust, compared to that made in the field, are that can be applied in any season, can maintain control of the environmental conditions of experimentation, it requires relatively small space, it is feasible evaluate many genotypes simultaneously and can perform multiple evaluations in parallel (^{Gunn and Day, 1986}).

Inoculation of rust in acclimatized plants

The acclimatized plants of the three genotypes of wheat "Atred" (S), "Mirlo 26" (R) and "Elinia 48" (R) kept in a greenhouse at 26 ± 2 °C and inoculated with leaf rust. After four days after inoculation the disease symptoms began to be visible; they started with small chlorotic spots on inoculated leaves, generating an ostensible resistance response (^{Huerta and Singh, 2000}). At seven days after inoculation, it took place germination and sporulation of the fungus; showing the same reproduction characteristics pathogen with the material evaluated in vivo.

At 12 days after inoculation unexpected variation in the response compared to seedlings in vivo and via indirect organogenesis it regenerated observed. Genotypes considered resistant, "Elinia 48" and "Mirlo 26", it was observed that 10% of plants showing resistance lost susceptibility characteristics. In genotype "Atred" susceptible, 21% of the plants showed some degree of resistance to leaf rust; this genotype is used as a spring parent in crosses of new lines of resistance to Puccinia triticina in conventional breeding.

The percentage of crystalline genotypes resistant to leaf rust in a group of winter habit only represent about 4% (^{Cubero, 2003}). The response in genotype "Atred" (S) is an option to generate and increase resistance lines relevant to leaf rust in winter bread wheats features. This response is a new contribution to plant breeders because it can be a variant whose essential characteristics should be retained.

The resistance observed in "Mirlo 26" was conferred by a recessive gene in response to the BBG/BPC race; while the resistance in "Elinia 48" (R) was by a dominant gene and one recessive, in materials obtained under greenhouse conditions. Further studies to corroborate the presence or absence of these genes characteristic of the lines evaluated in vivo in materials obtained in vitro in conditions are required. The plants obtained in vitro may have phenotypic, morphological and biochemical differences compared to the parent plants. ^{Larkin and Scowcroft (1981)} distinguished transient epigenetic variation and the "true" genetic variations, the probable causes; both known as somaclonal variations.

Probably the results obtained in the three genotypes are attributed to epigenetic variations. However, further molecular studies to confirm or refute the answers are needed. The somaclonal variations can sometimes be a limiting factor, but in others, as obtained in this study, can be beneficial. It is an efficient tool when plants showing adaptive advantages and can be applied in breeding (^{Bairu et al, 2011}) are obtained.

The response of susceptibility and resistance genotypes "Atred" (S), "Elinia 48" (R) and "Mirlo 26" (R) would be unstable and would revert with high frequency if an epigenetic change (^{Sánchez and Jiménez , 2009}), so the evaluation of the progeny of these materials is required by inoculating the pathogen Puccinia triticina E. in vivo. Conversely, if the progeny show similar responses would somaclonal response heritable; genetic variability useful for plant breeding with superior agronomic characteristics.

The results obtained in this research are the first report where the techniques of plant tissue culture in vitro are applied to evaluate the susceptibility and resistance of wheat genotypes to leaf rust. It can be a valuable tool feasible incorporated into breeding programs of wheat and other cereals.