Datos de investigación
High temperatures during late floral bud stages decrease fertilization in strawberry (Fragaria ₓ ananassa): pollen-pistil interaction and anatomical evidences
Autores:
Zini, Lucia Melisa
Publicador:
Consejo Nacional de Investigaciones Científicas y Técnicas
Fecha de depósito:
28/07/2022
Fecha de creación:
2019/2020
Clasificación temática:
Resumen
High temperature (HT) effects on pistil tissues and female gametophyte have been scarcely investigated in crops species. During strawberry flowering, HT can induce fruit malformations due to poor pollen performance in pistils, reducing the fertilization level. In this study, the cultivars ‘Earlibrite’ and ‘Fortuna’ were exposed to ambient temperature (AT) or HT (>30 °C for 6-hour day-1) at late flower bud development over the duration of 3 or 5 days. To evaluate the capacity of heated pistils to support and guide pollen tubes, we examined the performance of unheated pollen grains and their path along the pistil, as well as the morpho-anatomy of reproductive tissues, in apical and basal pistils on the receptacle. In both cultivars, HT significantly induced a decrease in the number of adhered and germinated pollen grains, and of pollen tubes in the style and the ovule micropyle. After HT treatment, microscopic observations revealed loss of stigmatic papillae turgidity and fertilization failures in the ovary due to abnormal pollen tube paths. The latter finding was anatomically related to the incidence of high immature female gametophytes in apical pistils and unviable female gametophytes in basal pistils. Ovule examinations also suggested the occurrence of facultative apomixis. This is the first report of impaired pistil functions when strawberry flower buds are exposed to brief episodes of HT at late stages, as revealed by in vivo poor pollen performance, abnormalities in pollen-pistil interaction and fertilization failures within 24 h after pollination.
Métodos
Plant material, experimental design, and pollination procedures
The field experiment was carried out at the Centro Tecnológico de Producción Vegetal (27°28 ʹS, 58°46 ʹW) located in Corrientes province, Argentine. The strawberries used for the experiment were two short-day cultivars: ‘Earlibrite’ (Plant Patent 13061, Florida, USA) and ‘Fortuna’ (Florida Radiance, Plant Patent 20363, Florida, USA). These two cultivars were selected because they are commonly sown across the Argentinean strawberries’ geographic region. Following conventional cropping practices, in autumn (1 May of 2019), 35 fresh rooted runners with leaves of each cultivar were transplanted into 4L pots. The pots (1 plant per pot) were arranged linearly at a distance of 25 cm per plant and in two rows spaced 40 cm apart. The pots were filled with commercial substrate of peat, bark compost, calcite, and dolomite, supplemented with controlled-released fertilizer (14-6-18 plus micronutrients, Basacote®). It was experimented with healthy plants that presented the terminal flower buds of the primary inflorescence. The size ranges of flower buds were 8.3 – 11 cm length in ‘Earlibrite’ and 10.6 – 13.5 cm length in ‘Fortuna’.
Thermal regimes also resulted in two types of treatments: ambient temperature (AT) and high temperature (HT) stress. In the AT treatment, temperature was kept at ambient levels, whereas in the HT treatment, plants were exposed to brief episodes (4 hour day-1) of high temperatures (above 30 ºC) over the duration of 3 or 5 days from late flower bud stages (11b, Ariza et al. 2015). Heat treatment was imposed through transparent polyethylene film made covers (100 μm thickness) fixed to a rigid metal tunnel (2 m long, 2 m wide, and 1.5 m high) opened at the bottom (0.05 m above ground level) to enable gas exchange. These covers remained closed only 4 hours a day (10:00 h – 13:00 h) during the days of treatment, after which the polyethylene film was rolled completely on the middle of the tunnel allowing plants of HT plots to be exposed to ambient conditions. Plants were watered before and after this period. The polyethylene film transmitted 85 % of incoming photosynthetically active radiation, and the photosynthetic photon flux density average was 1415.5 μmol m−2 s−1 under the tunnel. We selected flowers in which anthesis occurred on day 3 and day 5 of heating.
Average daily maximum and minimum air temperature data were obtained from Instituto Correntino del Agua y del Ambiente meteorological station, from both we calculated the average mean air temperature. Characterization of heating treatments (HT) in terms of air temperature and relative humidity were assessed by means of button-type digital loggers (DS1923L- F5, resolution: 0.5 °C, I-buttons data loggers, Digi-Key Co. Ltd., USA) which recorded the data every 30 minutes. These loggers were located in the center of each tunnel at the level of the inflorescence. The degree of heat stress was defined as heat-stressful index (°C day-1). This parameter indicates the accumulated degrees Celsius ≥ 30 °C per effective day of heat stress and computed as in Eq. (1):
"Heat-stressful index" =∑_"i=1" ^"N" ▒("Tmax ≥ 30 °C" ) / ("day ≥ 30 °C") ("1")
where N is the duration of treatment period (in days), Tmax is the maximum air temperature registered per day (°C), Tmax ≥ 30 °C is the cumulative temperature above 30 °C, and day ≥ 30 °C is the number of days with temperatures above 30 °C. We selected this temperature because 30 °C had been shown as a threshold for the development of strawberries (Ledesma et al. 2008). Average vapour pressure deficit (kPa) was calculated during the daily hours of heating treatment, based on the framework developed by Allen et al. (1998), and explained in more details in Ergo et al. (2018).
Flower buds were emasculated at least 1 DBA (when corolla is visible) to prevent self-pollination. At the onset of anthesis (0 DBA), petals unfold under the tunnel, and after heating, the plants were placed under field conditions with the control ones. Previous pollination studies suggest that increased fruit set is obtained by cross-pollination among various cultivars compared to the selfing mechanism (Sharma 2018; Żebrowska 1998). Therefore, plants were immediately pollinated with mixed pollen from a total of 10 plants of both cultivars grown in the field. Stigmas were manually pollinated with a paintbrush, attempting to deposit a low pollen load. Then, flowers were bagged and covered with cotton to prevent the uncontrolled deposition of pollen for 24 h. Preliminary trials indicated that this time was long enough for ovule fertilization, so after 24 h flowers were harvested and fixed with FAA (formaldehyde, acetic acid, ethanol, 5:5:90). Five flowers at each duration and cultivar were sampled, representing 20 flowers and 200 pistils per temperature treatment. With the aim of evaluating the apomictic mechanism, three flowers of each cultivar were emasculated, bagged at anthesis to prevent pollination, and fixed in FAA 15 days after.
Analysis of pollen performance in vivo
The capacity of the pistil (stigma, style, ovary, and ovule) to support and guide pollen tubes growth after temperature treatments was evaluated trough pollen performance at first day of anthesis, under fluorescence microscopy. For this, pistils were transferred to ethanol 70 % solution. Then were immersed in an 8 M NaOH solution for 1 h, washed in distilled water, and clarified with 50 % sodium hypochlorite solution for a minute. After three successive washings, pistils were stained and mounted with a 0.1 % blue aniline solution in 0.1 M K3PO4 (Martin 1959). Pistils and pollen tubes were observed and photographed using a Leica DM 1000 (Leica, Wetzlar, Germany) microscope equipped with a callose filter and a Canon EOS Rebel TDi digital camera.
Pistils on the receptacle were categorized into basal and apical because of macroscopical differences; intermediate pistils were not sampled.
Morpho-anatomical analysis of reproductive tissues
Flower buds (N = 20), unpollinated (N = 10), and pollinated pistils held under AT (N = 10) and HT (N = 10) were also examined through qualitative and anatomical criteria. The material was fixed in FAA and then dehydrated in an ethanol series with a rinse using preimpregnating Biopur® (González and Cristóbal 1997). The infiltration in paraffin followed the technique of Johansen (1940). Anthers and pistils were embedded in Histoplast® (Biopack, Buenos Aires, Argentina) and were transversely and longitudinally sectioned at 8 – 10 μm with a rotary microtome (Microm International, Walldorf, Germany). Sections were stained with a combination of Astra blue and safranin and mounted on slides with synthetic Canada balsam (Biopur, Buenos Aires, Argentina). Anatomical traits were observed and photographed using a light microscope (LM) Leica DM LB2 (Leica, Wetzlar, Germany).
Pollinated and unpollinated pistils developed under AT and HT were observed under SEM. Samples were transferred through an acetone series, critical point dried, mounted on a metal stub, and sputter coated with 20 nm of gold–palladium. Photomicrographs were obtained with a scanning electron microscope (Jeol JSM-5800LV; JEOL USA, Peabody, Massachusetts, USA).
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Datos de Investigación(IBONE)
Datos de Investigación de INST.DE BOTANICA DEL NORDESTE (I)
Datos de Investigación de INST.DE BOTANICA DEL NORDESTE (I)
Citación
Zini, Lucia Melisa; (2022): High temperatures during late floral bud stages decrease fertilization in strawberry (Fragaria ₓ ananassa): pollen-pistil interaction and anatomical evidences. Consejo Nacional de Investigaciones Científicas y Técnicas. (dataset). http://hdl.handle.net/11336/163439
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