Abstract

The effects of hydrogen cyanamide (CH₂ N₂ ) (0.75-2.5% a.i.), of ethephon (0.6-1.8%), and of a combination of the two on the germination of oil palm seeds were studied under different periods of immersion (24-48 hours). CH₂ N₂ concentrations of 1.5% to 2% for 24 hours stimulated germination 15 to 25 days after treatment had begun, reaching 80% germination within 40 to 50 days.  The plantlets had a compact appearance with a well-developed radicle.  Similar results were obtained with ethephon for 48-hour periods, although the plantlets obtained showed abnormalities and a poorly-developed root system.  Combinations of the substances yielded results comparable or inferior to those observed with CH₂ N₂ use. H₂ SO₄ as an initial scarifying treatment, before application of CH₂ N₂ or ethephon, yielded germination results similar to those obtained in the absence of this acid.  The action of these two first two compounds on seed metabolism during the germination process is also addressed.

Introduction

Oil palm seeds pose difficulties in terms of commercial germination due to a strong dormancy.  In 1936, Beinaert indicated that this dormancy could last several months or years, depending on the environmental conditions before and after harvesting.  In the wild, seeds may remain viable and begin germination even after 25 years in the soil (Rees 1963b). According to Ferwerda (1956), it takes six months after planting to reach 30% germination and nine months to reach 50%. Rees (1963a) noted a high germination rate when trees were cleared from the area, possibly due to a stimulating effect of the high temperature on the seed, although light may have also had some effect.

According to Hartley (1993), the dormant period is affected by various factors such as moisture, light, and the concentration of gases and other substances, which can sometimes be manipulated to effect changes in dormancy.  Hussey (1958) rejected the possibility that oil palm dormancy was due to oxygen-storage limitations in the embryo or the presence of some oxidable inhibitor, as oxygen only stimulated germination after being stored for some time at high temperature.

The above-mentioned observations point to the difficulties in obtaining a uniform plantlet population in the nursery.  Due to these difficulties and to the importance of the crop, methods of accelerating the germination process have been the object of investigation since 1922.  One of the most extensive reviews of methods for achieving uniform oil palm germination was carried out by Galt (1953), who had, at that time, already described the dry heat method for accelerated germination.  In 1959, Rees detailed the dry heat process, a commercial method for large-scale plant production, for interrupting seed dormancy in Deli dura palms on a commercial scale in Malaysia.  This method consists of subjecting the seed to temperatures of between 38ºC and 40ºC for 40 to 60 days, although the authors originally indicated periods of 70 to 80 days (Corrado and Wuidart, 1990;  Kin, 1981;  Addae-Kagyah, 1988).

The possibility of using a faster method in place of dry heat would reduce the high cost of this treatment, as well as the time necessary to stimulate germination.  Attempts to achieve improvements in the oil palm seed germination process through the use of growth-regulating substances have not yielded satisfactory results (Corley, 1976; Wan and Hor, 1983).  However, a better understanding of germination metabolism in general and the workings of the growth-regulating substances has allowed for the consideration of  using chemical treatments to stimulate the germination of dormant seeds (Bewley and Black, 1994).

The objective of this study was to evaluate the effects of different chemical compounds on the germination of recently-harvested oil palm seeds.

Materials and Methods

All the experiments were carried out using tenera-type Elaeis guineensis seeds obtained through free pollination between Deli dura and pisifer as (AVROS) palms.  For the second experiment, Deli dura seeds were used.  All seeds were provided by ASD de Costa Rica.  Seeds were extracted by mechanically depulping the fruit and manually removing the material still attached to the endocarp.  The seeds were air-dried until they reached a 15% level of humidity dry base and were stored in 0.17 mm-thick  polyethylene bags.  They were then moved to the “Centro para Investigaciones en Granos y Semillas de la Universidad de Costa Rica” (Seed and Grain Research Center of the University of Costa Rica), where the experiments were carried out.

The products evaluated were hydrogen cyanamide (CH₂ N₂ commericial formula with 49% active ingredient) and ethephon (commercial formula with 39.56% a.i).

Experiment 1

This was a preliminary trial, in which each experimental group consisted of 100 seeds.  Treatments were made by immersion in approximately 400 ml of solution, with the control group receiving the commercially-recommended thermal treatment for 40 days at 40ºC.  With the aim of facilitating analysis of the results, the treatments were divided into two groups.  In the first group, the base product was hydrogen cyanamide and in the second group it was ethephon.  Prior to the application of either product, half of the seeds were submerged in concentrated sulphuric acid (H₂ SO₄ ) for 10 minutes.  and then the seeds were immersed either in CH₂ N₂ in doses of 0.75% and 1.5% for 24 to 48 hours, or in ethephon, in concentrations of 0.6% and 1.2% for similar periods of time.

Then, for seven days the seeds were subjected to 16-hour periods of submersion in water, followed by 8-hour periods of air exposure, always maintaining surface humidity, until they reached 18% humidity dry base.  Finally, for germination, they were placed in closed plastic containers in a chamber of 30ºC and 100% relative humidity.  Evaluations were carried out on a weekly basis beginning 27 days after treatment.  The same procedures were followed for the rest of the experiments.

Experiment 2

The effects of 24-hour immersion of the seed in CH₂ N₂ (0.5%, 0.75% and 1.5%)  and in ethephon (0.6%, 1.2% and 1.8%) were evaluated.  Also evaluated was the combination of the substances in the above-mentioned doses in two applications:

• Seed immersion in a combination of CH₂ N₂ and ethephon for 24 hours, followed by 24 hours in ethephon only.
• Initial immersion in ethephon, followed by immersion in the combination of ethephon and CH₂ N₂ .

Also evaluated were 10-minute immersion in H₂ SO₄ , the commercial thermal treatment, and a control group submerged in water for 24 hours.

In this case, as in the previous experiments, each experimental group consisted of 50 seeds with four repetitions.  Analysis of the results was carried out according to an unrestricted random design.

Experiment 3

This experiment was divided into three treatment groups, with the aim of facilitating the analysis.  

• For the first group, the effects of CH₂ N₂ immersion in doses of 1.5%, 2% and 2.5% for 24 and 48 hours were evaluated.  
• For the second group, evaluation was based on a combination of CH₂ N₂ at 1.5% and ethephon in concentrations of 0.6%, 1.2% and 1.8% in the two application methods used in experiment two.  
• The third treatment group considered the effects of the combination of CH₂ N₂ at 2% and 2.5% and ethephon at 0.6%, applied as in the previous treatment group, in which a mixture of the two substances was also used.

Experiment 4

The effects of CH₂ N₂ immersion with doses of 0% and 2% followed by periods of zero, one, two, and three weeks at 40ºC were evaluated.  Also evaluated was immersion in a combination of CH₂ N₂ (2%) and ethephon (0.6%, 1.2% and 1.8%) for 24 hours, followed by immersion in the same doses of ethephon for an additional 24 hours.  Effects of exposure to thermal treatment for zero, one, two, and three weeks was also evaluated in this case.

At the end of the evaluation period for this experiment, plumule and radicle length were determined by looking at 10 randomly-chosen plantlets per repetition.

Results

Experiment 1

a. Effects of hydrogen cyanamide

The treatment of initial immersion in H₂ SO₄ followed by CH₂ N₂ immersion at 0.75% for 24 hours had a considerable effect on germination (reaching 70% 25 days after beginning the trials).  These values slowly increased, reaching 88% by the end of the evaluation period ( Fig. 1 ).  CH₂ N₂ at 1.5% for 24 hours without previous H₂ SO₄ treatment also stimulated initial germination, although these effects took longer (beginning at 25 days) and the total number of germinated seeds was much lower than in the above-mentioned treatment.  In the other treatments, germination was slower, beginning only on day 34 and never rising above 50% germination, with the exception of the commercially-used thermal treatment, in which germination levels reached 86%.  With this last treatment, however, germination began 61 days after treatment was initiated, reaching high germination levels at 79 days. Use of CH₂ N₂ in doses of 0.75% yielded 40% germination by the end of the experiment, but with germination beginning only 70 days after treatment.  The use of CH₂ N₂ for periods of more than 24 hours yielded germination values under 20%.

b.  Effects of ethephon

The use of ethephon in concentrations of 0.6% and 1.2% for 48 hours, combined with previous immersion in H₂ SO₄ , yielded an initial germination of 88% 25 days after the first treatment and 70% 43 days after the second ( Fig. 2 ).  However, in neither treatment was there an observable sustained variation in the percentage of seeds germinated.  The other treatments showed later germination with values lower than those expressed above, except for the H 2 SO 4 treatment followed by immersion in ethephon at 0.6% for 24 hours, which yielded 77% germination, but only after 97 days.

The results obtained in this experiment suggest positive effects both for hydrogen cyanamide and for ethephon, for which reason subsequent experiments aimed at determining the feasibility of using these treatments without previous exposure to H₂ SO₄ are needed.

Experiment 2

Initial immersion of the seeds in hydrogen cyanamide at 1.5% for 24 hours followed by immersion in ethephon at 0.6% or 1.8% stimulated an initial germination of more than 30% after the first 30 days, reaching a final value of 53% with the former ethephon concentration and 58% with the latter ( Fig. 3 ).  These values were notably lower than those obtained with the thermal treatment, which yielded an 83% final germination, although germination didn't begin until 65 days after treatment.  The CH₂ N₂ treatment at 1.5% for 24 hours yielded a 46% germination rate, while the other treatments showed values under 40%.  The control group only yielded 7.5% germination.

No results are presented for the other treatments in which the seed was submerged in ethephon only, or in ethephon for 24 hours, followed by an ethephon/ CH₂ N₂ mixture for 24 additional hours, as they did not yield germination rates significantly higher than the control treatment.  The three treatments that yielded the highest values began germination 23 days after treatment, with the rest beginning at 30 days.  The application of concentrated H₂ SO₄ for 10 minutes yielded a germination rate (5%) lower than that of the control group.

Experiment 3

a) Effect of high hydrogen cyanamide concentrations

In this experiment, the use of CH₂ N₂ at 2% for 24 hours yielded germination onset at 15 days, reaching 81.5% at 43 days, and a high of 92% at 58 days.  In the other treatments, germination was only observed after 29 days.  The use of CH₂ N₂ at 1.5% for 24 hours yielded 79.5% germination at 58 days ( Fig. 4 ).  Intermediate results were obtained with treatments of 1.5% for 48 hours (60%) and 2% for 48 hours (45.5%). CH₂ N₂ at 2.5% generally tended to yield very low germination values.  The thermal treatment yielded 95% germination 65 days after the experiment began.

b) Effect of hydrogen cyanamide and ethephon combinations for different immersion periods

b1. Use of cyanamide at 1.5%  

In all the combinations carried out, germination began at 15 days.  Germination did not show much variation, and by 58 days all treatments reached values between 79.5% and 84.5% with no significant difference among them ( Fig. 5 ).

b2. Use of cyanamide in concentrations of 2% and 2.5%

The use of CH₂ N₂ at 2% combined with 0.6% of ethephon for 24 hours, followed by 24-hour immersion in the same concentrations of ethephon yielded a high initial germination, reaching 28% after 15 days ( Fig. 6 ).  The other treatments yielded a germination rate of lower than 18% for the same period.  However, by the end of the evaluation period (58 days), there were no significant differences between treatments, the results of which were between 79% and 86%.  This last value was obtained with a 0.6% ethephon treatment for 24 hours followed by the combination of this treatment with 2% CH₂ N₂ .

Experiment 4

The results obtained in all the previous experiments showed a positive effect for hydrogen cyanamide on palm seed germination. Germination, however, was staggered, in contrast with the thermal treatment, in which germination occurred mainly over a maximum period of three weeks, with the downside being that this treatment requires six to seven weeks of heating prior to the onset of germination.  This experiment was aimed at determining the influence of a combined effect of heat with hydrogen cyanamide and ethephon on germination.

With CH 2 N 2 alone, there was rapid germination, reaching 70% at 23 days and more than 80% after 30 days ( Fig. 7 ).  Heat applied after CH₂ N₂ immersion did not stimulate germination, but, on the contrary, delayed it by two or three weeks.

When a mixture of CH₂ N₂ and ethephon was combined with thermal treatments for one-, two-, and three-week periods, there was a noticeable delay in the onset of germination, which increased with the period of heating ( Fig. 8 ).  With one week of heating, 86% germination was achieved at 23 days, while with three weeks of heating similar results took 58 days.  At the end of the evaluation period, 75% germination was achieved, with the exception of the control group.

It was noted that the seeds treated with ethephon showed a greater number of weak or abnormal plantlets with long, thin plumules, and small radicles.  With the use of CH₂ N₂  the plantlets had smaller, more compact plumules, with a thicker stem base and a well-developed radicle.  Increasing doses of ethephon in combination with hydrogen cyanamide produced a more compact root system than that seen with hydrogen cyanamide alone ( Fig. 9 ).

Discussion

The “dry heat method” has been successfully used to stimulate germination in oil palm seeds (Addae-Kagyah 1988; Kin 1981; Comont and Jacquemard 1977; Hussey 1958), but can be inconvenient in terms of the high cost of maintaining the temperature, as well as the amount of time necessary to break dormancy.

The experiments carried out identified chemical treatments stimulating different levels of germination.  The best results were obtained with treatments of H₂ SO₄ followed by immersion in CH₂ N₂ for 24 hours or in ethephon for 48 hours, as well as with CH₂ N₂ immersion only.  These treatments had significant effects on germination over short, but variable, timeframes (15 to 43 days).  The use of germination stimulants on palm seeds had previously been attempted by Wan and Hor (1983), who used ethephon to interrupt the dormant period.  However, they did not obtain conclusive results, probably as a result of using concentrations of 0.1% and 0.2%, which were probably quite low considering that the concentrations used in this study were 0.6% and 1.8%.  These authors indicated that the chemicals may not have been able to penetrate the mass of oily endosperm covering the embryo.  On the other hand, the results obtained in this study show that although an increase in germination was achieved through initial use of H₂ SO₄ , high germination rates were also achieved without previous scarification, which suggests that CH₂ N₂ and ethephon did penetrate the seed.

The high germination rates obtained 25 days after treatment indicate that these seeds do not need a “postmaturation” period, which is in line with Hussey's (1958) findings that isolated embryos placed, for germination purposes, on a moistened paper filter began their growth 24 hours later, and were, therefore, “non-dormant”.  Similar results were obtained by Nwanko (1981) when he eliminated the seeds' operculum and obtained high germination rates 24 hours later.

The application of H₂ SO₄ yielded high germination values, although use of this chemical involves some difficulties, such as the quantities required to carry out large-scale seed treatments, as well as the harmful effect of the residue and the danger to those handling it, all of which make it necessary to look for alternative solutions.  The results obtained in experiments three and four using hydrogen cyanamide at 2% allow for the consideration of chemical treatments without employing H₂ SO₄ .

In the second experiment, a significant decrease in germination values was seen with the treatments used, despite the fact that concentrations were similar to those in the first experiment.  This can be attributed to the fact that the seeds used in this case were dura seeds, which are generally characterized by being larger with a thicker shell, which may have limited the penetration once H₂ SO₄ was eliminated.  It is worth mentioning that these same treatments were carried out in experiment three, in which germination rates of 80% were obtained ( Fig. 4 and Fig. 5 ). It is also probable that, as is further explained below, the humidity conditions had something to do with the results. In this same experiment, the treatments in which combinations of CH₂ N₂ and ethephon were used showed that initial ethephon immersion produced very low values, while initial immersion in both substances yielded higher germination values. This may be attributable to the greater absorption which occurs during the initial inhibition, for which reason the more significant effect is due to CH₂ N₂ and not to ethephon.

In general terms, chemical treatments, particularly with CH₂ N₂ in 1.5% and 2% commercial formula concentrations for 24-hour immersion periods, stimulated germination.  Although the metabolic action of the product has not been completely explained, it is known to be rapidly absorbed by plant tissues, promoting mitochondrial respiration, which produces an accumulation of peroxide (Amberger 1984).  In grape shoots, this affects a change in the oxidative level of the tissues, which causes the activation of the pentoses of phosphate and the onset of growth (Nir et al. 1993).  These actions cause the activation of peroxidases and related enzymes, which are also responsible for breaking down lipid triglycerides in seeds, thus favoring their germination (Amberger 1984).  One of the advantages of this product is its rapid metabolization, as  within 24 hours, 40% of the compound breaks down into urea and its derivatives (Goldbach et al. 1988).   It is interesting to note that longer periods of exposure produce a negative effect on germination, which may be due to toxicity.  In fact, in high doses, this product could even have herbicidal effects (Amberger 1984).

High seed germination rates are also achieved with ethephon.  This synthetic substance, upon being absorbed in liquid form by the plant, is hydrolyzed, producing ethylene, which breaks the dormancy in many species (Reid 1995).  The action of ethephon can be considered similar to that of cyanamide.  Yang and Hoffman (1984) noted that along with the production of ethylene in plants, there is also HCN formation.  This would cause the activation of the pentoses of phosphate and, consequently, germination (Côme 1987).  The exogenous application of ethylene through ethephon use would compensate for the dormant seeds' low ethylene-synthesis capacity, which is considered by Johnston (1977) to be the reason that the enzymes and regulators necessary for germination are not activated.  It is worthwhile to point out that with this product, a high number of abnormal or weak plants was obtained, a phenomenon which is also seen in another palm, the pejibaye ( Bactrus gasipae ) (Villalobos et al. 1992).  On the other hand, plants originating from seeds treated with CH₂ N₂ showed balanced growth and a better-developed root system.  This last result could be explained by the observations of Yang et al . (1990) in grape plants, with which the application of cyanamide produced an increase in the transport of assimilated substances towards the roots.

The use of hydrogen cyanamide promotes germination rates similar to those obtained through thermal treatment.  However, one of the main differences observed was that the time needed to reach acceptable germination levels (over 80%) with CH₂ N₂ varied across the different experiments from 23 to 50 days, in contrast with the high germination values obtained in 15 days, not including the heating time required for the thermal treatment.  This difference, which was seen in the presence and in the absence of initial H₂ SO₄ treatment, could be due to variations in the amount of hydrogen cyanamide absorbed by the seed, as this factor was already noted with ethephon.  Nevertheless, experiments carried out on pejibaye (Villalobos et al. 1992) and coffee (Guevara et al. 1992) showed that moisture, both in the seed and in the environment, is critical for good germination.  In low moisture conditions, hydrogen cyanamide generally has a toxic effect, probably due to the rapid metabolism activation caused by this product (Amberger 1984; Goldbach et al. 1988), which requires conditions of adequate moisture.  Moisture values used for germination of palm seeds in this study were based on commercial management procedures.   It is possible that the appropriate moisture conditions for thermal seed treatment are not suitable for other types of treatment.  Along those lines, Nwanko (1981) achieved germination rates of 80% in Elaeis guineensis pisiferas at the end of 24 hours, bringing the seeds to a moisture content of 27-30% wet base and then manually removing the operculum.

Acknowledgements

The authors would like to thank ASD de Costa Rica for supplying the oil palm seeds for this experiment.  In addition, they would like to thank Francisco Sterling and Amancio Alvarado for the collaboration they offered during the course of this study, and SKW Trostberg Aktiengesellschaft of Germany for the support provided.

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