Recuperation of the high germinability condition of papaya seed through priming technology and bioregulators

Recuperación de la alta capacidad de germinación de la semilla de papaya mediante la tecnología de preacondicionamiento y biorreguladores

Francisco Bautista–Calles, Guillermo Carrillo–Castañeda* y Ángel Villegas–Monter

Genética. Campus Montecillo. Colegio de Postgraduados. 56230. Montecillo, Estado de México.* Author for correspondence: (carrillo@colpos.mx)

Abstract

Rapid loss of seed viability of Carica papaya L. (papaya) and its high commercial price impairs the availability of high quality seed to growers. Certified papaya seed cv. Maradol was utilized to define practical methodologies to restore the seed germination performance. Seed was submitted to: 1) hydropriming: 4 d of hydropriming rose seed germination up to 84%, 250% more than the untreated seeds; however, the seedlings generated from untreated seeds accumulated more biomass than the other treatments; 2) chemopriming: seeds treated 4 d in a 10^–5 M calcium chloride solution germinated 262% more than did the untreated seeds and in addition, the seedlings generated from treated seeds accumulated more biomass than the control seedlings. When seeds were treated either in the 10^–4 M salicylic acid or in Agromil S® solution high germination was accomplished 81 and 73% higher than the untreated seed respectively. All treated seeds germinated faster than the untreated ones.

Keywords: Carica papaya, acetylsalicylic acid, agromil S®, calcium chloride, chemopriming, hydropriming.

Resumen

La pérdida rápida de la viabilidad de la semilla de Carica papaya L. (papaya) y su elevado precio comercial afectan la disponibilidad de semillas de alta calidad para los cultivadores. Se uso semilla certificada de papaya cv. Maradol para determinar las metodologías prácticas para recuperar la capacidad de germinación de las semillas. Las semillas se sometieron a: a) preacondicionamiento hí d rico: tras 4 d de preacondicionamiento hídrico la germinación de semillas aumentó hasta 84%, 250% más que en aquellas que no se trataron; sin embargo, las plántulas generadas a partir de las semillas no tratadas acumularon más biomasa que las que sí se trataron; 2) preacondicionamiento químico: las semillas tratadas durante 4 d en una solución de cloruro de calcio 10^–5 M germinaron 262% más que las no tratadas y, además, sus plántulas acumularon más biomasa que las plántulas testigo. Cuando las semillas se trataron ya sea en solución de ácido salicílico 10^–4 o en la de Agromil S®, se obtuvo una germinación significativa, 81 y 73% más alta que en las no tratadas. Todas las semillas tratadas germinaron con mayor rapidez que las no tratadas.

Palabras clave: Carica papaya, ácido acetilsalicílico, Agromil S®, cloruro de calcio, preacondicionamiento químico, preacondicionamiento hídrico.

INTRODUCTION

Failure of seed germination and subsequent seedling growth is a major cause of yield decrease for papaya (C. papaya) due to a rapid decay of seed capacities. Papaya growers normally deposit six or up to twelve seeds per germination container, which increases production costs (Vargas, 1996). The fast reduction in seed germinability capacity observed in this seed has been correlated with the presence of inhibitors of germination in both the testa and sarcotesta (Paz and Vázquez, 1998). The activity of seed inhibitors has been reduced by soaking and washing the seed in water (Vargas, 1996) or by sun drying (Wood et al., 2000). Papaya seed conditioned in the presence of gibberellic acid improved its germination, but, after drying, a decrease in seed vigor and germination was shown by the conditioned seed (Marcos and Maleus, 2008).

Methodologies have been developed to add quality to the seeds as well as to identify high quality seed (Artola et al., 2003). The beneficial responses observed in the primed seed are: germination under a broad permissive temperature range, uniformity and hasten speed of germination, as well as improvement of seedling vigor (Welbaum et al., 1998). Commercially available bioregulators are also used to stimulate seed germination; nevertheless, there are not defined criteria for their appropriate utilization in papaya germination. Therefore, the present study was conducted to develop methods, based on the priming technology to restore the germination capacity of papaya seed and to characterize under such conditions the early seedling development. The use of biorregulators was taken into consideration as well.

MATERIALS AND METHODS

Biological material and chemicals

Three seed lots of certified papaya cv. Maradol with slight differences in germination percentages were used in the present research. Chemical reagents, commercial germination enhancers: Agromil S®, Biozyme pp®, Germiboost®, and the fungicide Captan® (50%) were used in this research. The fungicide Captan® (50%) contains N–trichloromethyl–thio–4ciclohexane, 2 dicarboximide and the biorregulators contain citokinins, auxins, gibberellins, vitamins microelements. The chemical composition of Agromil S® in ppm is: citokinins, 208; gibberellins, 31; auxins, 30.5 and in ppb, folic acid, 0.92; pantotenic acid, 12.5; riboflavin, 0.86; nicotinamid, 0.16; coline, niacin, 84.5; biotine, 100. Biozyme pp® contains: gibberellins, 31; indoleacetic acid, 30.2; Zeatin, 83.2 ppm; and the microelements Mn, Zn, Fe, B, and S (1.86%).

Conventional germination method

The method used by papaya growers consist in soaking a sample of 100 seeds in 0.5 L of tap water 72 h, changing the water every 12 h; after 60 h, 0.25 g of Captan® (50%) was added to the water. Then, a lot of 25 seeds was placed in a stripe of a flannel piece (15 X 20 cm) previously boiled and soaked with fresh tap water; each flannel piece was folded in two to cover the seeds, which were kept soaked at 29±3 °C.

Seven lots of 80 seeds were primed at 1 d intervals (days 3 to 6) in 1 L of aerated distilled water (18–20 °C) (Artola et al., 2003). The water was replaced every 24 h and 24 h before the seed completed its priming period, 0.5 g of Captan® (50%) was added to the water. After each priming period two seed lots were taken and only one of them was washed with distilled water. The seeds were then air–dried on the bench 30 min at 20±2°C. In each experiment two sets of 25 seeds per priming period were allowed to germinate (Carrillo–Castañeda et al., 2003).

Chemopriming procedure

Seed lots were subjected 4 d to the procedure described in the hydropriming section but instead of pure water there were used: solutions 10^–3, 10^–4 and 10^–5 M CaCl₂; solutions 10^–5 M of giberellic acid (GA₃), 6–bencylaminopurine (6–BAP), and absicic acid (ABA); solutions 10^–4 M acetylsalicylic acid (ASA), salicylic acid (SA), and sulfosalicylic acid (SSA); CaCl₂ 10^–3 M plus SA 10^–4 M. Agromil S® 1.25 mL 0.5 kg^–1 of seed, Biozyme pp® 10 g 0.5 kg^–1 of seed and Germiboost® 5 g 0.45 kg^–1 of seed. These solutions were adjusted to pH 5.9±0.1, and replaced every 24 h; in the last replacement, 0.5 g of Captán® (50%) was added. After the respective treatment, the seed without washing was air–dried as indicated. In each experiment three lots of 25 seeds per treatment were allowed to germinate. In addition, three lots of 25 untreated seeds were set to germinate (Carrillo–Castañeda et al., 2003).

Percentage of daily germination, final germination after 10 d, and days to 50% of germination (T₅₀), were determined. A seed was considered germinated when the radicle protrusion was > 1 mm. The final germination was expressed as percentage of normal seedlings.

Determination of seedling development

The germinated seeds were placed 0.5 cm from the upper edge of a paper towel sheet moistened with 7 mL of distilled water and rolled up. The rolls were placed into plastic bags in a vertical position with the germinated seeds on top during 14 d at 29±3 °C under 16 h photoperiod. Seedling length, stem length, root length, biomass fresh and dry weight (oven–dried at 70 °C for 72 h) of seedling, stem, and root were determined. Lots of 30 seedlings per treatment were used in these determinations. The determination of seedling development was repeated three times, one per week.

Experimental design

A completely randomized design was used with two and three repetitions for the priming and chemopriming experiments. For the conventional germination method averages were calculated. Data were tested by analysis of variance (ANOVA), and treatments mean were compared by Tukey test (p<0.05). In any set of experiments, the comparisons were done among the different treatments and the control.

Hydropriming

Seed hydroprimed 4 d and unwashed before drying exhibited the highest germination rate (84%), which was 2.5 times higher than that of the corresponding untreated seeds and higher speed of germination since T₅₀ was reduced from 8 (untreated seed) to 4 d (Table 1). Seed from the same lot subjected to the conventional germination method germinated 35%. Thus, our procedure did promote two important functions of papaya seed, surpassing the results of the conventional germination method. The maximum beneficial effects were detected in the 4 d hydroprimed seeds, although exposing seeds to more days of priming or additional washing did not caused reduction of germination (treatments 3, 4, 7 and 8). These are the experimental conditions required for the appropriate removal of substances from seeds that inhibit their germination. Bhattacharya and Khuspe (2001) showed that testa free seeds germinated in vitro 95.5% compared to only 40.2% in intact papaya seeds.

Untreated seed generated seedlings that exhibited the best vigor condition, based on the accumulation of fresh (900 mg) and dry (72 mg) biomass in comparison to the 4 d hydroprimed seeds that accumulated less fresh (700 mg) and dry (40 mg) biomass. Priming allows advancement of germination during which seeds undergo the early physiological process (Heydecker and Coolbear, 1997) and the integration of interlocking metabolic systems (Leubner–Metzger, 2005) that occur before germination takes place. However, along the imbibition process lose of nutritional compounds (imbibitional leakage) takes place due to the lipid–phase transitions occurring in the plasma membrane as a result of dehydration and rehydration of seeds. Subedi and Ma (2005) showed that seed priming does not improve time to seedling emergence, seedling vigor, and growth. It seems that the imbibitional leakage did not affect seed functions involved in the speed of germination (T₅₀) since the results showed (Table 1) that to longer time of imbibition, correspond faster germination.

Despite the fact that all treatments removed inhibitory substances from seeds (2 to 8), exhibited excellent germination and speed of germination, as compared to control, the treatment 5 was selected becaused it assure both high and prompt germination. Thereafter, the seeds were always primed 4 d and never washed before setting them to dry.

Chemopriming

The highest germination (76%) was obtained in seeds exposed to the 10^–5 M CaCl₂ solution (2.6 times higher than the untreated seeds) (Table 2). However, the seedlings, stems and roots generated from seeds that were germinated in the 10^–3 M CaCl₂ solution, exhibited both the highest dry biomass accumulation and the fastest rate of germination (T₅₀). The embryo development can be distorted by poor nutrition, and poor root growth is usually the first sign of calcium deficiency (Burton et al., 2000), which may influence the control of seed germination. A calcium–modulated, serine/threonine protein phosphatase, which may respond to the presence of abscisic acid, could play a role in signal transduction pathway, involving Ca²⁺, protein kinases, and phosphatases (Trewavas and Malhó, 1997). It explains why calcium influences the control of seed germination. Besides, calcium is an essential plant nutriment required for the biosynthesis of membranes and the wall matrix, bridging pectin chains together into expanded, highly hydrated gel networks (White and Broadley, 2003). The seedlings generated from the seed that were germinated in the 10^–3 M CaCl₂ solution exhibited both, the fastest rate of germination (T₅₀) and the best development (highest fresh and dry biomass accumulation).

The highest germinability (87%) was exhibited by the seed exposed to the SA solution (81% more than the corresponding untreated seeds; Table 3); however, abnormal seedlings were generated (6.9%). To our knowledge, this is the first report for this species. Salicylic acid, a natural product member of the phenolic family which is biosynthesized in plants, interact both antagonistically (Mao and Zentgraf, 2007) and synergistically and with another plant growth regulators such as methyl jasmonate and jasmonic acid in regulatory processes (Takahashi et al., 1993; Mur et al., 2005; Beckers and Spoel, 2006), activating different components of signal transduction pathways (Chen et al., 2002) and in the induction mechanisms of stress tolerance in plants. Compounds analogs to SA are ASA and SSA which also improve seed germination; in addition, these two compounds promoted seedling growth (Table 3). Salazar and Carrillo–Castañeda (2001) demonstrated that SSA induces in vitro the process of tuberization in Solanum tuberosum plants.

Speed and promotion of germination were observed when seeds were treated in solutions of either: SA, 10^–4 M (58%); CaCl₂, 10^–5 M (54%); or calcium in conjunction with SA (54%) and additionally, accumulation of similar amounts of biomass by the seedlings was observed (Table 4). The interactive effect of calcium and SA allowed (statistically significantly, p<0.05), 31% increase in stem length as compared to the seeds exposed to the SA solution. No abnormal seedlings were generated in the seeds treated in the solution of calcium in conjunction with SA.

In the earliest in signal transduction, high cellular concentration of SA cause superoxide generation followed by an increase in cytosolic calcium I tobacco cell suspension culture (Kawano et al., 1998). Thus, involvement of Ca²⁺ influx and active oxygen species (partially reduced metabolites of oxygen) generation as components of SA signaling is suggested.

The potential performance of seeds during seed germination and development can be different among lots of seeds according to their physiological quality or deterioration condition, and their ability to withstand a wide variety of experimental conditions (Sung et al., 2008).

Germination was promoted by all the commercial products tested; however, Agromil S® and Biozyme pp® promoted the maxima germination (73 and 70% more than the corresponding untreated seeds). All of them caused reduction by 48 h of T₅₀ (Table 6); but, it is important to recall that the hydroprimed seed germinated 250% more than the corresponding untreated seeds (Table 1).

CONCLUSIONS

Applying our hydropriming technique, the papaya seed germination performance is significantly restored. Conceptually simple, it is based on essential variables involved in the seed functions. It is economical and technically practical. In addition, valuable information was considered concerning seed expressions that take place beyond the germination process, the early seedling development. How the seed expressions can be enhanced by inorganic and organic chemical compounds was shown. It is posible that our procedures could become techniques performed to satisfy the needs of both seed producers and farmers that demand the guaranty to acquire high quality papaya seed.

ACKNOWLEDGMENTS

The authors thank La Semilla del Caribe seed dealers for the papaya seed donated for the development of this research.

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