Evaluation of the effect of different zinc concentrations in grapevine canes grown hydroponically Cláudia Castro (Ref: BI/INTERACT/VW/183/2016)

Transcription

1 Project 2 - Activity 2.4 Evaluation of the effect of different zinc concentrations in grapevine canes grown hydroponically Cláudia Castro (Ref: BI/INTERACT/VW/183/2016)

2 Brief Introduction Zinc (Zn) is an essential micronutrient for plants since it affects several metabolic processes. However, high levels of Zn in soil inhibit many plant metabolic functions, resulting in retarded growth and senescence. In acid soils Zn availability is generally high. Zn phytotoxicity has been studied in various plant species such as Brassica juncea (brown mustard), Beta vulgaris (sugar beet), Glycine max (soybean), Phaseolus vulgaris (beans), Spinacia oleracea (spinach) and Vitis vinifera cv Combier.

3 Previous studies * Protocols optimization for mitotic chromosome spreads of Vitis vinifera L.: Different protocols were tested aiming the increasing of chromosome size; Nucleoli counts in different varieties, in both root and leaf tissues after silver nitrate staining were performed; Physical location of rdna loci on mitotic metaphase chromosomes and nuclei after FISH experiments was achieved. * - Presented in the 1 as Jornadas INTERACT (16 Nov. 2016, UTAD) - Paper in preparation: Optimization of mitotic chromosome spreads of Vitis vinifera L. for silver staining and sequential fluorescence in situ hybridization

4 Plant material Shoots of ten V. vinifera varieties were collected at UTAD: a) White varieties: 1. Donzelinho Branco 2. Malvasia Fina 3. Malvasia Rei 4. Verdelho Madeira 5. Viosinho b) Red varieties: 6. Bastardo 7. Tinta Amarela 8. Tinta Barroca 9. Tinta Roriz 10. Tinto Cão Shoots were collected in 23 th January and 6 th February and keep at 4 C until use

5 Methodology In 7 th February, shoots were divided in canes with 2 nodes which were placed in distilled water in a heat chamber under forcing conditions (25 C, 60% relative humidity and photoperiod of 12h). The dormancy of shoots was forced for 2 weeks after a natural dormancy of 11 weeks 30 days Canes with leaves and roots were placed in 20% Hoagland solution for acclimation 14 days Canes were distributed by different Zn 2+ concentrations (prepared with 20% of Hoagland s solution): mm - 15 mm - 25 mm - 35 mm Before Zn treatment, 4 canes of each variety were removed and used to measure several plant morphological traits for evaluation of their growth

6 Methodology After 14 days of the beginning of the Zn treatment, the canes placed in 15, 25 and 35 mm of Zn died. The following analyses were performed in control plants and those treated with 5 mm Zn. 30 th March: Additional canes of the varieties Donzelinho Branco, Verdelho Madeira and Bastardo (previously maintained at 4 C, and with a forced dormancy of 7 weeks and 3 days), were submitted to the same procedure and treated with Zn concentrations lower than 5 mm: mm - 3 mm Hence, the following analyses were performed in three varieties - Donzelinho Branco, Verdelho Madeira and Bastardo - treated with the four tested Zn concentrations (control, 1.5 mm, 3 mm and 5 mm).

7 Analyses and sampling Analyses: 1) Morphological traits Leaf number Total leaf area Stem length Main root length Root system area Dry weight of the leaves, stem and roots 2) Membrane permeability (EC%) in leaves using the electrical conductivity method. Sampling for future work: 1) Foliar discs were frozen in liquid nitrogen and conserved at -80 C for biochemical quantifications. 2) Leaves and roots fragments were fixed in FAA for histological observation of tissue sections. 3) Young roots and leaves were fixed in a solution of ethanol: acetic acid (3:1) for the preparation of mitotic chromosome spreads. 4) Adult leaves were frozen in liquid nitrogen and conserved at -80 C for DNA extraction/comet assay.

8 Results ANOVA results: Variety: p = Treatment: p = Variety x Treatment: p = Figure 1: Average main root length (mean ± standard deviation). In the same variety, means with different letters indicate statistically significant differences determined by the Tukey test (p <0.05); n.s.- non significant.

9 Results ANOVA results: Variety: p = Treatment: p = Variety x Treatment: p = Figure 2: Average roots dry weight (mean ± standard deviation).

10 Results ANOVA results: Variety: p = Treatment: p = Variety x Treatment: p = Figure 3: Average leaf dry weight (mean ± standard deviation). In the same variety, means with different letters indicate statistically significant differences determined by the Tukey test (p <0.05); n.s.- non significant.

11 Results ANOVA results: Variety: p = Treatment: p = Variety x Treatment: p = Figure 4: Average leaf area (mean ± standard deviation). In the same variety, means with different letters indicate statistically significant differences determined by the Tukey test (p <0.05); n.s.- non significant.

12 Results ANOVA results: Variety: p = Treatment: p = Variety x Treatment: p = Figure 5: Average percentage of electrical conductivity (mean ± standard deviation). In the same variety, means with different letters indicate statistically significant differences determined by the Tukey test (p <0.05); n.s.- non significant.

13 Conclusions The treatment with Zn was more toxic to the roots than to the leaves, whereas roots growth was impaired at 5 mm and leaves growth only decreased above this concentration. Leaves membrane permeability increased at 15 mm of Zn, suggesting higher membrane damage in this tissue

14 Ongoing work: Statistical analyses of the data achieved in the remaining varieties. Analyses of the data of the different biochemical quantifications performed in the three varieties - Donzelinho Branco, Verdelho Madeira and Bastardo - treated with the four tested Zn concentrations Future work: Study of Zn treatment effects in the leaf and root histology, in cell cycle and DNA using different approaches.

15 Thank you for your attention! Team: Cláudia Castro, MSc Ana Carvalho, PhD Professor José Lima-Brito, PhD Professor Fernanda Leal, PhD Prof José Moutinho-Pereira, PhD Contributors: Professor Eunice Bacelar, PhD Helena Ferreira, MSc Acknowledgements: Projeto NORTE FEDER INTERACT/VitalityWINE (FEDER/ NORTE2020)