Germination

The work was conducted at the Universidad Autonoma Agraria Antonio Narro in the Laboratory of Plant Tissue Culture from the Plant Breeding Department.

Hydration stage

Hydration was evaluated by weighing the seed every 4 h until seed weight was constant.

Viability

This variable was evaluated determining initial seed viability by the germination capacity test. Where the methodology to assess viability was conducted in accordance with international rules ISTA (2009), determining viable and non-viable seeds, with three replications of 20 seeds, subjected to imbibition in water for 24 hours in a culture tube 15 X100 mm. The seeds were dissected in half, immersed in tetrazolium in dark and incubated at 30 °C for 2 h; subsequently were observed in stereo microscope to evaluate percentage viability.

Emergency rate index

IVE (emergancey rate index) was obtained by counting the seeds emerged daily for 21 days after planting. Considering testa rupture as seed emerged. To calculate its value the following formula was used ^{Maguire (1962)}.

Where: IVE= emergency rate index; Núm. P= number of emerged plants; D= days after sowing.

Pretreatment

The following pretreatments were formed: gibberellic acid: G1= 10^-2 M, G2= 10^-4 M, G3= 10^-6M; sulfosalicylic acid: ASS1= 10^-2 M, ASS2= 10^-4 M, ASS3= 10^-6 M; salicylic acid: AS1= 10^-2 M, AS2= 10^-4 M, AS3= 10^-6 M; benzoic acid: AB1= 10^-2 M, AB2= 10^-4 M, AB3= 10^-6 M and control (T). Each treatment consisted of three replications with 10 seeds per jar. It was determined in a preliminary study the time in which the seeds reached the maximum absorption of water which was 4 h. With this data the seeds were imbibed in solutions with different regulators and organic acids.

Once waterlogging time has passed, the pretreated seeds are disinfected in alcohol solution 70%for one minute and rinsed with sterile distilled water, then placed in sodium hypochlorite 20% and rinsed with sterile distilled water three times under a laminar flow hood, then planted on Murashige and Skoog (MS) to half its concentration supplemented with 100 mg L^-1 of myo-inositol (SIGMA^®, I-3011), 1 mg L^-1 thiamine-HCl (SIGMA^®, T-3906), 1 mg L^-1 of pyridoxine-HCL (SIGMA^®, P-8 666), 1 mg L^-1 Kinetin, 30 g L^-1 sucrose (SIGMA^®, K-0753 ), 8 g L^-1 of agar (SIGMA^®, A-1 296) adjusting pH to 5.7 and sterilized at 120 °C for 15 min and transferred to an incubation room at temperature of 25±1 °C, with 16 h light and 8 of darkness at 2500 lux.

Establishment

For this stage germinated seedlings in vitro on MS basal medium at half concentration added with 100 mg L^-1 myo-inositol (SIGMA^®, I-3011), 1 mg L^-1 thiamine-HCL (SIGMA^®, T-3906), 1 mg L^-1 pyridoxine-HCL (SIGMA^®, P-8666), 80 mg L^-1 of adenine, 30 g L^-1 of sucrose (SIGMA^®, K-0753), 4 g L^-1 of phytagel (SIGMA^®, A-1296), adjusting pH to 5.7, were used. Seedlings generated after 15 d were sectioned to obtain explants.

Micropropagation

For micropropagation MS medium and PCL2 were used, added with 100 mg L^-1 of myo-inositol (SIGMA^®, I-3011), 1 mg L^-1 thiamine-HCL (SIGMA^®, T-3906), 1 mg L ^-1 pyridoxine-HCL (SIGMA^®, P-8666), 40 mg L^-1 of adenine, 30 g L^-1 of sucrose (SIGMA^®, K-0753), 4 g L^-1 of phytagel (SIGMA^®, A -1 296), adjusting pH to 5.7 and sterilized at 121 °C for 15 min. The seed was transferred to the incubation room at a temperature of 25+1 °C with 16 hours light and 8 of darkness at 2 500 lux. The medium was renewed every 15 days. The combination of plant growth regulators used: BAP (benzylaminopurine) and kinetin at 0, 0.5, 1, 2 and 3 mg L^-1 with naphthalene acetic acid (ANA) at 0 and 0.5 mg L^-1.

As explants the hypocotyls, cotyledons and nodal segments were used. Variables assessed were number of buds per explant and explant length, number of roots and root length.

Hardening stage

For this stage MS medium at half concentration without hormones was used and added with the same supplements from the establishment stage.

Statistical analysis

To the obtained data was performed an analysis of variance under a completely randomized design and a mean comparison test Tukey (p≤ 0.05). R^® statistical tool version 2.8.1 (2008) was used.

Viability

75% of tomato husk seeds were viable and 25% non-viable, indicating that the seed is out of the range of physiological quality for seed marketing or for germination studies, SNICS recommends a minimum of 85%. These data are consistent with a previous study to determine the pollen viability on tetraploid varieties of tomatillo where a decrease in viability was observed compared with pollen from diploid plant (^{Ramírez et al., 2013}), this data confirms the aforementioned by some authors on the decrease in viability and fertility of polyploid seed (^{Bretagnolle et al., 1995}).

The comparison of various treatments (Figure 1 and 2) indicated that control had the lowest values for germination with 13.33% and an IVG of 0.19, initiating emergency on day 21. Treatment with AG showed a significant increase in germination at all concentrations (X= 64.44). The emergence began on the fourth day after planting with a germination percentage for AG1, AG2 and AG3 of 73.33, 56.66, and 63.33% and an IVG of 20.26, 10.44 and 11.59 respectively. It has been reported that AG participates in regulatory processes of plant growth and development.

Figure 1 Percentage of germination in implementing solutions tetraploid tomatillo seeds. 

Figure 2 Performance index of emergency speed tomatillo seeds in tetraploid (Physalis ixocarpa), applying regulators and organic acids. 

Treatment with gibberellic acid is associated with rapid use of amino acids and amides sythesis, which increases the germination rate (^{Gupta and Mukherjee 1982}). It is also related with the increase of phenols by modifying the biosynthetic pathway of flavonoids (^{Gosch et al., 2003}; ^{Halbwirth et al., 2003}). In the seed phenols are produced in the testa and are related to the embryo morphogenetic processes (^{Dixon et al., 2005}; ^{Zhao et al., 2010}; ^{Dean et al., 2011}).

Regarding organic acids the ASS treatment showed the highest average in germination (X= 28.88), followed by AB (X= 26.66), and the lowest average AS (X= 18.88). Note that although IVG (Figure 3) were lower, germination with organic acids initiated the first day after sowing. Organic acids derived from the metabolic pathway of phenylpropanoid compounds such as plant hormones involved in the intracellular signaling cascade and control the production of secondary compounds that work as plant defenses (^{Álvarez and Espinosa, 2004}).

Figure 3 Nodal explants and in vitro flowering tetraploid tomatillo (Physalis ixocarpa). 

Seed imbibition pretreatment and planting the same day increase germination rate and seed emergence.

The results confirm that germination pretreatments are a tool that increases germination rate and percentage.

Micropropagation

This stage aimed to establish a protocol for clonal propagation of the tetraploid cultivar P. ixocarpa. For this, two basal mediums and different combinations and concentrations of regulators were tested. All treatments resulted in regeneration of shoots (Table 1 and 2), but the induction was greater in the treatment with 3 mg L^-1 BAP in combination with ANA with 9.5 shoots per explant, from nodal segments. The highest bud length (8.93 cm) was obtained with 0.5 mg L^-1 BAP with 0.5 ANA (8.02 cm) from PLC2, this medium also showed the highest root number (4.3) and highest length of the same (4.6 mm), was achieved in treatment MS medium with 1 mg L^-1 BAP. The lowest response (2.7 shoots / explant) was recorded in the BAP treatment (0.5 mg L^-1) alone, in the PCL2 medium.

Table 1 Comparison of means of the effect of different concentrations of BAP, kinetin and NAA in the number of shoots, shoot length, number and length of roots in MS basal medium. (H= hypocotyls, cotyledons = C). 

Table 2 Comparison of means of the effect of different concentrations of BAP, kinetin and NAA in the number of shoots, shoot length, number and length of roots in PCL2 basal medium (H= hypocotyl C= cotyledons). 

A differential response between the various explants both cotyledons and hypocotyl did not generate a morphogenetic response contrary to that observed by ^{Contreras and Almeida (2003)}, who mention a greater number of buds (66) with hypocotyl on MS medium with BAP ( 2 mg L^-1), while the cotyledons generated lower response (54) with zeatin. Also ^{Ramírez-Malagón and Ochoa-Alejo (1991)} mention a higher proliferation of shoots from hypocotyl with 2.5 mg L^-1 BAP and with 1.0 mg L^-1 ANA.

An interesting observation was the ability of flower formation in vitro system (Figure 3). Between the two cytokinins, BAP was the most effective in inducing greater number of shoots per explant compared to kinetin. In this regard, other researchers agree that BAP was the cytokinin inducing higher formation of buds (29-32) in the genus Physalis (^{Afroz et al., 2009}).

There are other works on this genus where BAP and ANA combination (Alejo, 1991; ^{Yousry, 2013}) achieved an optimal morphogenetic response.

An interesting observation was the ability of flower formation in vitro system (Figure 3). Also in this case, the root system developed spontaneously eliminating the need of a medium with auxin before acclimatization. According to ^{Zhao et al. (2008)} a high ratio between cytokinin and auxin promotes root regeneration.

^{Andrade-Rodríguez et al. (2005)} observed a wide variability in obtaining shoots in different varieties of Physalis ixocarpa from 1.9 in wild varieties to 21.3 in domesticated. These authors mention that the organogenic capacity is given by the genotype of the variety in use. The organogenic ability of this cultivar is below that reported for domesticated cultivars.

Plant response depends largely from the genotype and some modifications and adjustments must be made when new species or cultivars are considered for this technique. The tetraploid cultivar showed need for high concentration of exogenous cytokinin in the propagation step.