Revista Facultad Nacional de Agronomía Medellín

MATERIALS AND METHODS

RESULTS AND DISCUSSION

The variables viability, germination and pollen tube length from different oil palm genotypes were significantly affected by temperature and storage time (Table 1).

Table 1: Analysis of variance. Mean squares for the variables: viability, germination and pollen tube length of improved cultivars of African Palm.

The evaluation of viability of pollen not stored showed the highest percentages in the cultivar Guineensis, with 98.38%, and statistical differences with the rest of the treatments. The cultivar Coari x Lame presented 35.98% of viable pollen, with statistical differences in respect to Amazon and Unipalma materials, which presented percentages of 16.94% and 17.10% respectively (Figure 1). These results coincide with those obtained by Bastidas et al. (2007) and Torres et al. (2004), who corroborated the high pollen viability of Guineensis, much higher than those observed in the pollen of palm hybrids. Significant statistical differences presented between the viability of pollen stored at room temperature (4.98%), and at -13 °C (11.75%).

Figure 1: Behaviour of viability (%) of the pollen of African Palm cultivars.

The number of days stored, and the storage temperature did not significantly influence the percentages of pollen viability, demonstrating that the time and storage temperature evaluated had no influence on the data on viability observed.

Sanchez and Romero (2013), confirm that the grains of pollen of the hybrid present low percentages of viability and germination due to the fact that they are very varied in size and form with regard to E. oleífera y E. guineensis, and during their formation, they undergo frequent divisions of abnormal cells. The same authors concluded that the low viability of pollen is not an obstacle for the commercial exploitation of the hybrids E. oleífera x E. guineensis. On the contrary, Turner and Gillbanks (1982), affirm that the pollen used in artificial pollination systems must have a viability of no less than 75% at the moment of laboratory observation. Based on this observation, only the pollen of E. guineensis presents the adequate viability for assisted pollination programs.

On the other hand, the viability results in terms of storage times and temperature differ from those found by Hardon and Tuner (1967), who point out that under natural field conditions, pollen remains viable for one week after the anthesis of the male inflorescences. In this study, it was established that under laboratory conditions, the viability could be maintained for up to 30 days of storage.

In addition, it is necessary to clarify that the viability test do not ensure that the pollen meets the requirements for fertilization, as a grain of pollen may be viable but also present germinative problems (Lagos et al., 2005).

The variable “germinability” presented significant differences for simple effects, and for all possible interactions (Table 1). When stored the pollen at room temperature (28 °C), germination decreased drastically and after five days, germination was reduced by more than 50%; no pollen germination occurred after 15 days of storage (Figure 2A). When the germination of pollen stored at -13 °C was analyzed, it was established that the storage time did not affect the germination of the genotypes, with the exception of the genotype Coari x Lame, which presented greater germination rates (23.40%) after 15 days of storage (Figure 2B).

Figure 2: Interaction of the genotype* days stored A. room temperature; B. at -13 °C, in the percentages of pollen germinability of oil palm. Bars with the same letter do not differ statistically (P<0.05).

According to Corley and Tinker (2009), when the percentage of germination is greater than 70%, the pollen is good. This condition was only found with Guineensis material, confirming the need for pollen of this genotype in assisted pollination programs, as hybrids do not posses pollen with the minimum germination values demanded. The pollen of the hybrids showed low percentages of germinability in this study, which can be explained, according to Davarynejad et al. (2008), by the fact that fertile pollen does not only depend on environmental factors such as humidity, temperature, atmospheric composition, and partial pressure of oxygen. Other determinant factors include genetic characteristics such as the pollen morphology (shape and size), pollen tube growth, viability and capacity of germinative competence (Lin et al., 2017), all of which are factors that can influence pollen germinability of the materials OxG.

In addition, these hybrids come from the parental Elaeis oleífera, which possess germination values between 20 and 60%, and 6.2% in the OxG hybrid, values that contrast with those of E. guineensis, which can surpass 70% (Romero et al., 2013).

Reasons for low pollen germination in interspecific hybrid are not well known. However, it is known that an incomplete mating of chromosomes occasionally occurs, and that the pollen can be released with difficulty, due to anther malformation. The existence of pollen degeneration has also been suggested, which occurs between the stages of meiosis and dispersion in anthesis (Sanchezet al., 2008). Due to the above, pollen storage is one of the activities carried out in order to longer preserve viability, since adverse environmental conditions can result in the reduction of pollen functionality. This characteristic is indispensable for the completion of the germination process in the pollination of feminine inflorescences, resulting in good cluster formation (Sánchez and Romero, 2013).

Knowledge of the characteristics associated with pollen quality is of great importance in order to ensure fertilization, yield, and quality (Andrés et al., 1999).

The pollen tube length presented highly significant differences between genotypes, days and temperature of storage and genotype interactions*days stored, genotype*storage temperature, and between storage days and temperature (Table 1). In the Guinensis, Amazon and Unipalma genotypes, the length of the pollen tube was similar in different periods of pollen storage at -13 °C (Figure 3B).

Pollen stored in environmental condition suffered a strong deterioration after the first five days, significantly reducing its germinability (Figure 3A). The grains of pollen of the genotypes evaluated under room temperature fail to emerge after 5 days, in accordance with Alburquerque et al. (2007), who affirms that the pollen viability decreased rapidly according to storage conditions.

Figure 3: Final longitude of pollen tube reached for each genotype of oil palm, in pollen with different storage periods. A. Room temperature B. temperature of -13 °C. The bars with the same letter do not differ statistically (P<0.05).

Growth Dynamic of Pollen Tube. It was observed that the non-stored pollen presented an average initial length (at 30 minutes) of 20.23 µm for the genotypes evaluated. Guineensis was the fastest to emit the pollen tube, with a length of 28.57 µm in the first 30 min. The genotypes Amazon, Coari x Lame, y Unipalma presented lengths of 22, 12, and 18.33 µm, respectively. According to Tadon et al. (2001), the pollen grains germinate within the following two hours of pollination, and the formation of fruit begins.

The growth of the pollen tubes of non-stored pollen grains finished between 4.5 and 5 hours, with the genotype Coari x Lame reaching the highest longitude, with 631.82 µm. The genotypes Amazon, Unipalma y Guineensis, reached longitudes of 613.89, 571.43 and 503.21 µm, respectively. All of the genotypes showed lineal growth, proving the vigor of fresh, non-stored pollen (Figure 4A).

Figure 4: Growth of pollen tube of genotypes Guinensis, Amazon, Coari x Lame and Unipalma, with different cold-storage times (-13 °C). A. Non-stored, B. Stored for 5 days, C. Stored for 15 days and D. Stored for 30 days.

This behaviour was similar in grains in cold storage during a period of five days (Figure 4B). In 15 days of cold storage (-13 °C), a reduction of growth speed was observed (Figure 4C), but values superior to all the previous ones were recuperated when stored under the same conditions during 30 days (Figure 4D). These results coincide with Kearns and Inouye (1993), who affirm that the pollen quality is maximum at the time of anther dehiscence, decreasing with time, according to pollen storage conditions.

The emergence of the pollen tubes at room temperature was similar, although delayed compared to that reported in non-stored pollen. Guineensis was the fastest to emit the pollen tube, with a longitude of 31.25 µm in the first sixty minutes. For the genotypes Amazon, Coari x Lame, and Unipalma, longitudes of 77.50, 52.50 and 32.50 µm, were presented in two and two and a half hours, respectively. The average final longitude for the genotypes was 484 µm. Coari x Lame presented the greatest average, with 578.88 µm, and Unipalma the lowest, with 307.22 µm.

The non-stored pollen grains presented an average growth rate of 2 µm per minute. During the first minutes of incubation, the genotypes Amazon, Coari x Lame and Unipalma presented a slow growth speed, which increased the 100-120 minute point, in which stabilized. On the contrary, the genotype Guineensis, presented a constant speed from the beginning of incubation (Figure 5A). A high growth speed of the pollen is a determinant of successful masculine reproduction; the pollen tube is the fastest growing plant cell and can breathe up to 10 times more than other tissues (Okusaka and Hiratsuca, 2009).

Figure 5: Growth rate of pollen tube in non-stored A and stored pollen grains during five days at room temperature B.

The emergence of the pollen tubes stored during five days at room temperature was similar, although delayed compared to that reported in non-stored pollen. Guineensis was the fastest to emit the pollen tube, in the first sixty minutes. For the genotypes Amazon, Coari x Lame, and Unipalma, the emission were presented in two and two and a half hours, respectively (Figure 5B).

The growth of the pollen tubes stored during 5 days allowed for the observation of the initial loss of germination speed, considering that the non-stored pollen began its germination and growth of the pollen tube in the initial 30 minutes, while the stored ones began to grow at one hour, two hours, and two and a half hours (Guinensis, Amazon and CoarixLame, respectively) (Figure 5B).

The growth rate began at 60 minutes in the case of Guinensis, at 120 minutes for Amazon and Coari x Lame, at 150 minutes for Unipalma, with accelerated increase up to 300 minutes in Guinensis, Amazon, Unipalma, and Coari x Lame.

The progressive loss observed in growth speed of the pollen tube is a continuous process that involves enzymes that degrade quickly due to environmental factors, and internal factors of each genotype (Dafni and Firmage, 2000).

The results obtained with the pollen stored during 5 days at a temperature of -13 °C are similar to those obtained from the non-stored pollen, showing, at thirty minutes incubation time, average longitudes of 67 µm, with values of 137.7 µm in Guineensis and 66.43, 34.29 and 32.58 µm in Amazon, Unipalma and Coari x Lame, respectively. The maximum average longitude (604.9 µm) was reached in 4 and a half hours. Coari x Lame reached 628.18 µm, Amazon 613,9 µm Unipalma 593.9, and Guineensis 572.6 µm (Figure 4B). In this sense, Pham et al. (2015) affirm that germination and growth of the pollen tube require the interaction of different components, and that it is necessary to evaluate and calibrate according to the specie and genotype.

The growth rate contrasted between the Guineensis genotype with a high initial velocity that decreased to stabilize in 160 minutes, and the genotypes Unipalma and CoariXLame, which presented a low initial velocity and growth to stabilize in 150 minutes. The growth speed of Amazon was constant during the evaluation time. (Figure 6A). These differences in growth speed of the pollen tubes of the genotypes is found in other species, indicating that genotypic differences affect germination capacity and pollen tube growth rate (Pfahler et al., 1997).

Figure 6: Growth rate of pollen tube of the genotypes, stored for five (A) and fifteen days (B) at a temperature of -13 °C.

With 15 and 30 days of storage at room temperature no expression was presented, considering that after 5 days of storage, the percentages of germination decrease until the point of not showing any results. These effects show that the storage temperature is an important factor in the expression of germinability, showing that from the first day of anthesis, the pollen shows decreasing percentages proportional to time.

Nonetheless, when the pollen is stored at a temperature of -13 °C, for thirty days, it was observed that the increase in the length of the pollen tubes of the genotypes remains the same, as observed in non-stored pollen, as shown in Figure 4D.

The average growth rate of the pollen grains stored during 15 days, at -13 °C, showed mean increments of 1.7 µm per minute, with the highest values shown in the Guineensis material, with 2.4 µm per minute. For Amazon, Coari x Lame, and Unipalma materials, the average rates observed were 1.7, 1.6 y 1.4 µm per minute (Figure 6B).

When the pollen was stored during 30 days, at a temperature of -13 °C, the average growth rate was 2.9 µm per minute, with Guineesis presenting the highest rate of 3.1 µm per minute. The genotypes Amazon, Coari x Lame, and Unipalma reached averages of 2.0, 2.7, and 3.2 µm per minute, respectively (Figure 7).

Figure 7: Growth rate of pollen tube of pollen grains stored during 30 days at -13 °C.

With 30 days of storage, the genotype Guineensis began with a rate of 2.0 µm per minute which tended to remain constant up to 75 minutes, with an increase rate of 2.9 µm per minute. This indicates that the conservation of pollen in low temperatures (-13 °C), could serve as a way to lengthen the life of the pollen, improving its vigor in subsequent pollination and allowing the production of pollen in areas other than its application. The same results are reported by Pham et al. (2015), in Dimocarpus and by Wang et al. (2015) in Litchi, with pollen conserved at -86 °C and by Sukhvibul et al. (2000) in four cultivars of mango.

CONCLUSIONS

The viability of the pollen grains was determined by genotype: Guinensis presented better viability than the other genotypes, while Coari x Lame was superior to Unipalma and Amazon.

The number of storage days did not affect the germinability of stored pollen at -13 °C but decreased from the first day of collection to 5 days of storage at room temperature. Germination time of the pollen grains began after 30 minutes of incubation, and ended around four and a half hours, considering work conditions. Growth rate of the pollen tube of stored grains at room temperature during 5 days presented an initial delay when compared to the pollen tube of the non-stored grains. At -13 °C, the rate increased in Guineensis, Unipalma and Coari x Lame genotypes.