The aim of this study was to evaluate the effects of silver nitrate in the in vitro development and conservation of passion fruit plants. Experiments were carried out at the Laboratory of Culture and Tissues, in the National Cassava and Fruits Research Center (EMBRAPA), using microcuttings of Passiflora gibertii N. E. Br., previously cultivated in vitro. The microcuttings were placed in MS media supplemented with silver nitrate at concentrations of 0, 1, 2, 4 and 8 mg·L<sup>-1</sup>, and, during 30, 60 and 90 days, the following variables were identified: Shooting length (cm), number of roots, number and coloration of leaves. A completely randomized design with 30 replications in a split-plot scheme was employed. Polynomial regression equations were adjusted in analysis of variance to compare averages of treatments. The obtained results support the assumption of high sensitivity of passion fruit tissues to ethylene, which reflects in the lost of vigor and in the induction of culture senescence by its accumulation. Based on obtained results, and in order to mitigate this problem, it is possible to indicate an addition of 2 mg·L<sup>-1</sup> of silver nitrate in culture media, for the micropropagation of passion fruit plant, allowing the controlling of the atmosphere inside the culture test tubes, during the establishment of explants and other steps of the process.
The genus Passiflora includes over 600 species, it is widely distributed throughout the Americas and has high genetic variability to be exploited in breeding programs [
The species Passiflora gibertii N. E. Br., presents resistance to premature death, cladosporiose, bacterial blight and anthracnose, which makes it a strong candidate to be use as rootstock and also in breeding programs [
This technique has been used by several authors and most of the publications focus on Passiflora edulis f. edulis (purple passion fruit), Passiflora edulis f. flavicarpa (yellow passion fruit) and Passiflora alata (sweet passion fruit) [
The need for repeated subcultures, the requirement of infrastructure and labor-specialized work and the frequent phytosanitary contamination, have been hindering the in vitro cultivation [
For the success of this technique, some factors should be considered, such as the explant type, genotype, nutrient media and the environmental conditions to maintain the culture. In general, nodal segments and steam apices are the most widely used type of explant micropropagation for passion fruit.
According to [
The ethylene accumulation has an adverse effect on plant development, affecting differentiation, development, morphology and plant growth, reducing foliar expansion and shoots length, inhibiting the regeneration of new shoots and causing apical necrosis, besides promoting foliar abscission in in vitro cultures [
There are available in the literature reports of several cultures that are influenced by the presence of ethylene in in vitro cultivation. The decrease in the rate of regeneration has been observed in chili pepper, orange and potato [
Several studies have been conducted aiming at decreasing the rate of ethylene in culture flasks, being, the addition of “ethylene inhibitors compounds”, such as silver nitrate, cobalt chloride and silver thiosulfate considered as possible alternatives [
This work aimed at evaluating the effect of silver nitrate as an inhibitor of ethylene action in the in vitro development and conservation of passion fruit.
Experiments were conducted in the Laboratory of Culture and Tissues, in the National Cassava and Fruits Research Center (EMBRAPA, Cruz das Almas, Bahia, Brazil), using the accesses characterized by Passiflora gibertii N. E. Br., from the Active Germplasm Bank of Passion Fruit from the same institution.
In the vegetation house, seeds from controlled pollination were sown in polyethylene plastic bags of 10 × 25 cm, using a mixture of soil and cattle manure, in a proportion of 3:1, considering the necessary phytosanitary care with the seed quality, preparation of substratum, sowing and conduction of seedlings.
Microcuttings of P. gibertii, with 1 cm length, were collected from the vegetation house, and cultivated in test tube of 20 × 150 mm, in 20 mL MS medium [
A completely randomized design was used with 30 replications per treatment. The experimental plot was represented by a test tube containing one explant. Evaluations were performed at 30, 60 and 90 days after incubation of explants in culture media without subculture. We evaluated: 1) shoots length (cm): considered shoots above 1 cm; 2) roots number: considered developed roots; 3) leaves number: Considered fully expanded leaves and 4) coloring of leaves: The following rating scale was assigned: 1―totally green leaves, 2―light green leaves and 3―yellowish leaves (early senescence). Data were subjected to analysis of variance considering a completely randomized statistical model in a split-plot design subdivided in time. The averages obtained were submitted to the Dunnet test at a 5% probability, using SAS® and to analysis regression using SISVAR 5.6® [
The results revealed considerable variation in the shoots length, roots number, as well as the number and color of leaves. Statistically significant differences (p ≤ 0.01) were observed among the treatments for all sources of analyzed variation. The variables shoots length and leaves color showed the lowest coefficient of variation, 2% and 7%, respectively, while the variables leaves number and roots number presented the highest values of 18% and 25%, respectively (
In general, for all variables, the control was lower than the treatments containing silver nitrate as an inhibitor of ethylene action. Concerning the variable length of shoot, at 30 days of evaluation, there was a significant difference and superiority of silver nitrate treatments 1 and 2 compared to control. For the assessment at 60 and 90 days, only concentration of 1 mg・L−1 AgNO3 were statistically significant higher than the control (P < 0.05) (
The addition of silver nitrate favored the increase in the number of leaves in all evaluation periods compared to its absence in the culture media. Lower scores were recorded at 30 and 60 days of evaluation, with concentrations of AgNO3 at 2 and 4 mg・L−1. After 90 days, only concentration of 1 mg・L−1 of silver nitrate did not differ statistically from the control. The other concentrations promoted a decrease in the scores and, consequently, major vigor (
After 30 days of evaluation, there was a significant increase in the number of roots, utilizing only 2 mg・L−1 of silver nitrate. For the evaluation periods of 60
Sources of variation | DF* | Mean Squares | |||
---|---|---|---|---|---|
SL (cm)1 | LN2 | RN2 | LC2 | ||
Treatment | 4 | 0.7732** | 64.8997** | 2.3843** | 0.5549** |
Evaluation | 2 | 0.2512** | 15.5325** | 0.5235** | 0.0123ns |
Treatment x Evaluation | 8 | 0.0937** | 1.4373** | 0.1377** | 0.0095ns |
Coefficient of variation (%) | 2.00 | 18.00 | 25.00 | 7.00 | |
Overall Average | 2.1592 | 5.6059 | 0.3423 | 1.3374 |
*DF: degrees of freedom; **Significant statistically at 1% probability by F’s test; nsNon-significant statistically. 1Transformed to ln(x + 10); 2Transformed to ( x + 0.5 ) .
Treatments | Evaluation (Days) | ||
---|---|---|---|
30 | 60 | 90 | |
Shoots length (cm) | |||
Control | 1.14 | 1.34 | 1.72 |
1 | 2.27** | 4.44** | 6.78** |
2 | 1.71** | 2.09ns | 2.51ns |
4 | 1.24ns | 1.36ns | 1.43ns |
8 | 1.32ns | 1.44ns | 1.53ns |
Leaves number | |||
Control | 0.47 | 0.90 | 1.20 |
1 | 6.00** | 9.50** | 13.73** |
2 | 5.79** | 9.90** | 10.83** |
4 | 2.23** | 4.97** | 4.97** |
8 | 2.93** | 5.52** | 5.38** |
Roots number | |||
Control | 0.00 | 0.00 | 0.17 |
1 | 0.23ns | 0.83** | 1.10** |
2 | 0.45** | 1.00** | 1.34** |
4 | 0.00ns | 0.00ns | 0.00ns |
8 | 0.00ns | 0.00ns | 0.03ns |
Leaves coloration | |||
Controle | 1.67 | 1.67 | 1.86 |
1 | 1.45ns | 1.69ns | 1.67ns |
2 | 1.14** | 1.14** | 1.14** |
4 | 1.09** | 1.13** | 1.17** |
8 | 1.43ns | 1.33ns | 1.27** |
**Significant statistically at 5% probability by Dunnett’s test; nsNon-significant statistically.
and 90 days, 1 and 2 mg・L−1 of silver nitrate showed higher and significant values when compared to the control (
In the evaluations of 30 and 60 days the use of 2 and 4 mg・L−1 of AgNO3 affected the color of the leaves, reducing the values obtained for this variable and differing from the control treatment. At 90 days the highest concentrations (2, 4 and 8 mg・L−1) also decreased the means of leaves colorations, differing from the control (
higher growth of shoots when compared to the other concentrations in all evaluation periods (
By mean values of number of leaves, depending on the treatments and evaluation (
The results recorded in this investigation revealed considerable variation in the length of shoots, number of roots, as well as the number and color of leaves, in microplants cultivated with silver nitrate, when compared to those cultivated without this compound. The results confirm previous findings on the inhibitory effect of ethylene on in vitro morphogenesis of passion fruit. Assuming that the rate of accumulation of ethylene in the test tubes is relatively low but still enough to cause an inhibitory effect, it is possible to affirm that there is a high sensitivity of passion fruit tissue to ethylene.
Overall, for all variables, the control was lower than the treatments containing silver nitrate as an inhibitor of ethylene action (
For adventitious bud induction, [
Reference [
In our investigation, we observed that, regarding the variable length of shoot, significant differences and superiorities of treatments up to 2 mg・L−1 of silver nitrate (
Related to the coloration of leaves, considering that lower scores would reflect in green leaves with more intense coloration, the recommended treatments would be concentrations of 2 (
The quality of microplants is defined by the number of leaves expanded throughout the period of cultivation, as well as by coloration. In this study, when considering the growth of sproots and number of leaves, the treatments with 1 (
The addition of silver nitrate was also positive to the increase in the number of roots of the microplants, especially for doses of 2 mg・L−1. Obtaining microplants with a well-developed root system is of great importance for their survival and growth, particularly related to the acclimatization and transplantation to the field.
Reference [
Similar results were obtained by [
Reference [
The in vitro growth rate of cassava confirmed the efficiency of silver nitrate as an ethylene inhibitor [
In in vitro potato culture, verified that in the system sealed with silver nitrate, there was an increase of five folds related to the foliar area, six folds related to the fresh weight of leaves and a significant increase in the size of roots [
These results corroborate that silver nitrate has beneficial effects on regeneration and clonal propagation of several economically important plants [
The species Passiflora presents some problems related to the development of an efficient protocol for micropropagation, and the action of ethylene may be one of the factors associated to this limitation.
The obtained results support the assumption of high sensitivity of passion fruit tissues to ethylene, which reflects in the lost of vigor and in the induction of culture senescence by its accumulation.
This work can surely be an excellent reference for future experiments, and may contribute significantly to the study of tissue culture and consequently to the maintenance of in vitro germplasm banks of passion fruit.
Based on obtained results, and in order to mitigate this problem, it is possible to indicate an addition of 2 mg・L−1 of silver nitrate in culture media, for the micropropagation of passion fruit plant, allowing the controlling of the atmosphere inside the culture test tubes, during the establishment of explants and other steps of the process.
The authors thank FAPESP for the funding of the project that allowed the creation of the Laboratory of Applied Statistics (LEA) (Process: 2015/18225-4) responsible for the statistical analyzes performed in this work.
Faria, G.A., Felizardo, L.M., Ferreira, A.F.A., Rocha, P.S., Suzuki, A.N., Souza, A.S., Junghans, T.G., Costa, M.A.P.C., Peixoto, A.P.B., Morais, A.R., Lopes, B.G. and Oliveira, T.A. (2017) Concentrations of Silver Nitrate in the In Vitro Development and Conservation of Passiflora gibertii N. E. Brown. American Journal of Plant Sciences, 8, 2944-2955. https://doi.org/10.4236/ajps.2017.812199