Plant material

In this study, powder seeds from six fruit obtained via manual autofecundation from L. autumnalis plants, from the germplasm bank of the National Plant Genetic Resources System Collection, were used.

Culture medium

Culture medium with the ^{Murashige and Skoog (1962)} (MS) mineral salts was used, without phytohormones and supplemented with sucrose (30 g L^-1), myoinositol (100 mg L^-1), thiamine (0.4 mg L^-1) and agar (6 g L^-1). The pH was adjusted to 5.7±0.1 with NaOH 1N. 20 mL of this medium were served in 100 mL glass bottles and autoclaved for 15 min at 1.2 kg cm^-2 pressure and 121 °C.

Aseptic culture establishment

For planting, 33 sample seeds (20 mg each) were placed in 10 mL syringes, and disinfection with commercial sodium hypochlorite 15 % v/v (6 % active ingredient) for 15 min was performed. In a laminar flow hood disinfectant solution was removed and three rinses with sterile water were performed. The powder seeds of each sample were suspended in 0.5 mL of sterile water and placed in a vial with culture medium. Their incubation was carried out in a 16/8 h light/dark photoperiod and photosynthetically active radiation of 45 μE m^-2 s^-1 provided by 75 W white fluorescent light lamps.

Irradiation with ⁶⁰Co gamma rays

Forty-five days after sowing (das), the obtained protocorms were treated with ten irradiation doses (5, 10, 15, 20, 25, 30, 35, 40, 45 and 50 Gy) plus a control treatment without irradiation. The experimental design was completely randomized with 11 treatments and three repetitions, the experimental unit was a vial with protocorms obtained from germination of 20 mg of seeds. The amount fluctuated between 500 and 700 protocorms. One day after irradiation (46 das), the protocorms of each bottle were transferred to fresh culture medium. At 90 das, these were transferred to Petri dishes in order to facilitate their evaluation. Throughout all this time, the development of the protocorms was divided into three phenological stages: photosynthetic protocorms, protocorms with pro-meristems and protocorms with leaves. These phases were observed simultaneously in all the experimental units because the seedlings development in L. autumnalis is an asynchronous process.

At 135 das, protocorms were subcultured in 250 mL plastic containers with 50 mL of fresh basal medium and incubated in the already described environmental conditions.

Variables evaluated in protocorms

In the protocorms, we assessed the percentages of survival, photosynthetically active, and with pro-meristems and leaves. Each experimental unit (bottle) was divided into quadrants to facilitate counts. In each quadrant, the number of living, dead, photosynthetic, with pro-meristems and leafs protocorms were counted. With the addition of protocorms at each phenological stage, the total protocorm number per experimental unit was obtained. This was used as denominator to obtain the survival percentage, percentage of photosintetic protocorms, with pro-meristems and leaves as follows: survival percentage per experimental unit = (number of living protocorms per vial/total number of protocorms per flask) x 100. The same procedure was used for each variable.

Protocorm fresh matter. The total protocorms per experimental unit were weighed on an analytical balance (OHAUS^®) with 0.01 mg readability.

These variables were assessed 45 d after irradiation (90 das).

Variables evaluated in the seedlings

We assessed the seedling, root and pseudobulbs length. The distance between the base and the apex of the seedling, pseudobulbs and root with digital vernier was measured.

Leaves and pseudobulb width. It was measured in the intermediate area of each organ with a digital vernier.

Number of leaves and roots. It was quantitated directly on each seedling.

Seedlings fresh mass (mg). Each seedling was weighed on a digital analytical balance (OHAUS^®) with 0.01 mg readability.

Seedling biomass. Each seedling was dried in an oven (FELISA), at 70 °C for 72 h. The dry seedlings were then weighed into digital analytical balance (OHAUS^®) with a 0.01 mg readability.

The assessment of these variables was performed in five seedlings per repetition, a total of 15 per treatment, at 214 d after irradiation (259 das).

Statistical analysis

An ANOVA was performed on the data obtained at 45 and 214 days after radiation, the Pearson correlation coefficient was calculated and the Tukey test was carried out for means comparison between treatments. These were performed with the SAS software version 9.0 (^{Statistical Analysis System, 2002}).

In order to assess the magnitude of the response of each variable to the radiation doses a linear regression model was evaluated. However, only the survival and leaves emergence variables adjusted to this model, thus, the LD₅₀ and GR₅₀ determination was performed only for these variables.

Radiation effect on protocorm development

The analysis of variance revealed that the radiation affected the protocorm’s survival, the presence of photosynthetic protocorms, protocorm with pro-meristems, with leaves and fresh protocorm matter (Table 1).

Table 1 Analysis of variance for the ⁶⁰Co gamma radiation effect on survival and development of Laelia autumnalis protocorms 

**p≤0.01; ns = not significant.

Radiation at low doses (5 to 20 Gy) did not affect the survival of protocorms compared to the control treatment, although there was a reduction of live protocorms between 17.6 % and 46.4 % in the tissues treated with 25 to 50 Gy (Figure 1A). Doses between 20 and 30 Gy stimulated chlorophyll presence in the protocorms, which was noted as a change from the light yellow coloration to green (Figure 1B), statistically not differents to those of 45 Gy. The fresh material accumulation was similar between the protocorms of the control treatment and those treated with radiation levels, but different between those irradiated at 5 and 10 Gy regard those treated with 35 Gy, the first with 69 % and 66 % more accumulation of fresh matter (Figure 1C). A similar response was observed for the promeristems formation, which was statistically similar in irradiated treatments and the control, but almost twice in the irradiated protocorms at 20, 30 and 35 Gy, compared to the 50 Gy (Figure 1D).

Figure 1 Effect of ⁶⁰Co gamma radiation on the survival and development of Laelia autumnalis protocorms. A) Protocorms survival, B) photosynthetic protocorms, C) fresh matter protocorm, D) protocorms with promeristems and E) protocorms with leaves. LSD: Least Significant Difference. In each figure, different letters show significant differences (Tukey; p≤0.05). 

The formation of protocorms with leaves did not differ (p>0.05) between the non-radiated seedlings and those treated with 5 and 10 Gy. From 15 Gy on, protocorms with leaves formation was reduced (p≤0.05), as the irradiation dose increased (Figure 1E). The explants development accelerated at low radiation doses (radio-stimulation), but decreased with the increasing gamma radiation doses, as in Chrysanthemum morifolium in vitro callus (^{Soliman et al., 2014}).

Although our investigation did not carried out assessments at molecular level, it is known that tissue damage with increasing gamma radiation dose occurred, even though the cells have a DNA-repair mechanism that acts when exposed to any source of radiation. Nevertheless, if the intensity of the radiation source is too high, this mechanism cannot repair any damaged cells, which die as is the case in ginger plants (Zingiber officinale) (^{Yoon et al., 2014}). Free radicals that cause metabolic disorders are produced in surviving cells (^{Pavan et al., 2013}), the gene expression pattern is modified (^{Corthals et al., 2000}), which regulates certain metabolic pathways and defense systems (^{Zolla et al., 2003}), causing quantitative and qualitative changes in the total soluble protein content (^{Corthals et al., 2000}). These proteins have an important role in signal transduction in antioxidant defense, antifreeze, heat shock, anti-pathogenesis, and osmolytes synthesis, which are essential for the functions and growth of plants (^{El-Fiki et al., 2015}). These alterations at cellular level, caused either by physiological or physical malfunctions, which include chromosome damage and reduced survival as the radiation dose increases, are reported in Jatropha curcas L. (^{Dhakshanamoorthy et al., 2011}), as well as in four commercial varieties of Lycopersicon esculentum (^{Ramirez et al., 2006}), Withania somnifera (^{Bhosale and More, 2014}), Orthosiphon stamineus (^{Kiong et al., 2008}) and Chrisanthemun (^{Yamaguchi et al., 2009}).

The correlation coefficients were negative and highly significant between the levels of applied radiation and protocorms survival (-0.91), protocorms with leaves (-0.90) and fresh protocorm matter (-0.69). There was also a positive correlation between survival and protocorms with leaves (0.89) and between fresh protocorm matter (0.71) and the formation of protocorms with leaves and protocorm fresh matter (0.65) (Table 2).

Table 2 Correlation of the effect of ⁶⁰Co gamma radiation on the survival and development of protocorms of Laelia autumnalis. 

**p≤0.01; *≤0.05; ns: not significant.

Radiation effect on seedling development

The ANOVA revealed that the radiation influenced fresh matter, biomass and length seedling, roots and leaves width, but did not affect the number of leaves, pseudobulbs length and width and number of roots (Table 3).

Table 3 Analysis of variance for the ⁶⁰Co gamma radiation effect on Laelia autumnalis seedling development. 

**p≤0.01; ns: not significant.

The length of the seedlings increased 32 % relative to the unirradiated treatment when radiation at k5 Gy was used. This suggests a radio-stimulant growth effect at low radiation doses; at 35, 40 and 50 Gy seedlings were 31, 50 and 42 % smaller than the unirradiated seedlings (Figures 2A and 3). The greatest fresh material accumulation (189 and 176 mg) was recorded in seedlings treated with 5 and 10 Gy. This was statistically similar to the control and treated with 15 Gy, but superior to other treatments (Figure 2B). The width of the leaves decreased by 25 and 29 % with doses of 40 and 50 Gy as compared to control: the roots number decreased by 37% with 45 Gy (Figure 2C and D). Biomass accumulation was similar in all seedlings treatments, except in those treated with 5 and 15 Gy, which accumulated almost twice the biomass than those irradiated with 45 Gy (Figure 2E). The biomass decreased by the increase in the radiation dose was observed in Arabidopsis seedlings that inhibit carbohydrate transport when treated with radiation dosages greater than 50 Gy (^{Bondada and Oosterhuis, 2003}), because of damage and disorientation in the grana of the thylakoids, as well as increased accumulation of starch granules in the chloroplasts. These ultrastructural changes were not observed in the chloroplasts of cells irradiated with low doses (^{Wi et al., 2007}).

Figure 2 Effect of ⁶⁰Co gamma radiation on L. autumnalis seedlings from ⁶⁰Co irradiated protocorms. A) Length, B) fresh matter, C) leaf width, D) number of roots and E) seedling biomass. LSD: Least Significant Difference. In each figure, different letters show significant differences (Tukey; p≤0.05). 

Figure 3 Seedlings of Laelia autumnalis from protocorms irradiated with ⁶⁰Co gamma rays at different radiation doses. 

In contrast to the radioinhibition phenomenon, the hormesis observed at 5 Gy dose on L. autumnalis seedlings growth is described alike to the stimulatory effect of any growth factor on an organism (^{Szarek, 2005}). This is attributed to a change in the hormone signaling network in plant cells or an increase in the antioxidant capacity of the cells in order to overcome stress factors such as fluctuations of light intensity and temperature in growth conditions (^{Kim et al., 2004}). In addition, radiation accelerates the rate of cell division and actives auxin, this phenomenon has been reported in Gypsophilla paniculata in which, when calluses were irradiated with 1 Gy increased shoot length (3.4 cm) was obtained (^{Barakat and El-Sammack, 2011}). In L. esculentum there is a radio-stimulant interval between 5 and 25 Gy for plant height (^{Ramírez et al., 2006}); the gamma radiation on seeds of Triticum durum increased between 32 and 75 % the number and length of roots when irradiated with 20 Gy (^{Melki and Marouani, 2010}). In the regeneration of embryogenic callus of Heliconia psittacorum irradiated with ⁶⁰Co gamma radiation, the 30 Gy dose showed the highest percentage of regenerated callus (80.8 %), 20.8 % more than in the unirradiated treatment and 74.8 % more than in callus treated with 75 Gy (6 %) (^{Urrea and Ceballos, 2005}).

The correlation coefficients were negative and highly significant between the different levels of applied radiation and the seedlings fresh matter (-0.57), seedling length (0.56), leaf width (-0.52), number of roots (-0.40) and length of pseudobulbs (-0.40) (Table 4). This confirms a negative correlation between the energy absorbed per unit mass in the applied dose and the growth and development variables of the plants (^{Giovannini et al., 2015}). In Arabidopsis this inhibition is attributed to the arrest of the cell cycle at the G2/M phase during the division of somatic cells or by genome damage (^{Preuss and Britt, 2003}). On chrysanthemums this is attributed to the generation of chromosomal aberrations and the loss of the cells proliferative capacity (^{Patil et al., 2015}) and damage to the genes involved in the morphogenesis of true leaves, as in Rumex obtusifolius leaves irradiated with 500 Gy (^{Kitano et al., 2015}). In Agave tequilana the absorbed radiation produces mutations in the cell’s DNA and alter their normal development as the radiation dose increases (^{Ángeles-Espino et al., 2013}).

Table 4 Correlation of the ⁶⁰Co gamma radiation effect on seedling development of Laelia autumnalis. 

**p≤0.01; *≤0.05; ns: not significant.

There were positive correlation coefficients between fresh matter and seedling length (0.70), root length (0.66), leaf width (0.64) and roots number (0.55). Significant correlation coefficients were also present between the length of seedling and width leafs (0.65) and pseudobulb length (0.54).

Determination of LD₅₀ and GR₅₀

The linear regression model showed the best adjustment to explain the radiation effect on the protocorms survival (Table 5). The protocorm and seedlings fresh matter showed a tendency to decrease as the radiation dose increased. However, they presented a low R² value (0.48 and 0.35) and, therefore, these variables were not considered for the calculation of the GR_50.

Table 5 Regression analysis for protocorm survival and leaf emergency in Laelia autumnalis protocorms irradiated with ⁶⁰Co gamma radiation. 

The average lethal dose for the protocorms survival was 53 Gy (Figure 4), which is considered as the dose that favors useful mutations development. The average growth reduction dose (GR₅₀) for forming protocorm leaves was 28 Gy (Table 5 and Figure 4).

Figure 4 Effect of ⁶⁰Co gamma radiation on Laelia autumnalis orchid protocorms. A) Protocorms survival rate and B) leaf formation in protocorms. 

The LD₅₀ for L. autumnalis differs to that reported for Alstroemeria aurea G. rhizomes irradiated with gamma rays at doses between 0 and 40 Gy, and LD₅₀ was determined at 40 Gy; this dose reduced survival explants in 50 %, and might be used for breeding purposes (^{Aros et al., 2012}). The GR₅₀ for the percentage of seed germination in two varieties of rice (Oryza sativa L.) irradiated with ⁶⁰Co gamma radiation from 20 to 200 Gy, was 89 and 188 Gy in the JGL and Vijetha varieties (^{Pavan et al., 2013}). In strawberries shoots irradiated with doses between 30 to 325 Gy the survival LD₅₀ was 177 Gy (^{Murti et al., 2013}); long bean seeds (Vigna sesquipedalis) irradiated with 300 to 800 Gy had a LD₅₀ for survival between 600 and 800 Gy, and GR₅₀ for plant height was between 400 and 500 Gy (^{Kon et al., 2007}).

Based on those data, it is confirmed that the DL₅₀ and GR₅₀ are specific to each species, genotype and even size and type of the irradiated tissue, and that the sensitivity to radiation depends on the nature and extent of chromosomal damage (^{Kiong et al., 2008}; ^{Pavan et al., 2013}). Besides, it varies depending on the DNA content, size and number of chromosomes (^{Sparrow et al., 1961}), and upton water content in the irradiated plant material, since the radiation interacts with molecules, particularly water, to produce free radicals in the cells. These radicals modify various important compounds in plants cells, because of which, seeds tolerate higher LD₅₀ due radiation doses compared to succulent tissues (^{Yunus et al., 2013}).

The LD₅₀ determined for L. autumnalis seedlings confirms that this species is highly sensitive to gamma radiation. This sensitivity is also observed for solar radiation, which is attributed to the fact that L. autumnalis is an epiphytic species that lives under tree canopies. In epiphytic orchids high solar radiation intensities reduce photosynthetic capacity and affect gas exchange (^{Stancato et al., 2002}).

The average reductive dose of 28 Gy for L. autumnalis protocorms to form leaves is similar to that of Agave fourcroides, in which, a GR₅₀ of 20 Gy for fresh weight was identified. Besides, weight of fresh callus material was an excellent indicator of radiosensitivity (^{González et al., 2007}). In A. tequilana explants irradiated with gamma rays at doses up to 50 Gy the average reductive dose was between 20 and 25 Gy for seedlings formation and 15 to 25 Gy for callus formation. These doses are likely to induce favorable mutations for selection and increased breeding purposes (^{Ángeles-Espino et al., 2013}). However, the GR₅₀ determined for leaves formation for L. autumnalis protocorms is high when compared with the results in Etlingera elatior explants irradiated with ⁶⁰Co gamma rays from 0 to 140 Gy, where the DL₅₀ for survival explants was determined to be at 10 Gy (^{Yunus et al., 2013}). Also, in nard tubers (Polianthes tuberosa L.) irradiated with gamma ray doses, between 0 to 30 Gy, LD₅₀ was 9.09 Gy for the survival of acclimated seedlings from in vitro cultured shoots; this was not the case for the tubers established in vivo, for which a LD₅₀ of 25.91 Gy for the survival of plants was determined (^{Estrada-Basaldua et al., 2011}).