DOI: 10.46909/cerce-2020-016 Original Article Available online: www.uaiasi.ro/CERCET_AGROMOLD/ Print ISSN 0379-5837; Electronic ISSN 2067-1865 Cercetări Agronomice în Moldova Vol. LIII , No. 2 (182) / 2020: 185-194

SEED GERMINATION AND SEEDLING ESTABLISHMENT OF CHERIMOYA ( CHERIMOLA MILL.) AT DIFFERENT TEMPERATURES

A. KHELOUFI1*, L.M. MANSOURI1, H. KHETTACHE1

*E-mail: [email protected]

Received: Apr. 16, 2020. Revised: May 20, 2020. Accepted: May 27, 2020. Published online: July 18, 2020

ABSTRACT. Cherimoya (Annona improved germination with only cherimola Mill.) has an exceptional flavor 25% FGP. Furthermore, significant and aroma, which makes it a with decrease in FGP and SL was observed at great potential. However, little is known 25°C and 40°C of temperature in about its propagation by seeds. According comparison to 30°C. The analysis also to the scientific literature, the germination revealed that cherimoya seed germination, of cherimoya seeds is affected much more day 10-15 after seed sowing is suitable for by external conditions than by internal final counts. An overview on the conditions. Germination of cherimoya emergence of cherimoya seedlings, during variety ‘Concha Lisa’ were tested for a 12-week period in pots is presented. germination at constant temperatures of Keywords: agriculture; ; 25, 30, 40°C, and at room temperature, custard apple; exotic fruit; fruit ; seed varying from 20-25°C, coupled with total quality. darkness. Seeds were sown in Petri dishes (0.8% agar water), for 25 days of INTRODUCTION incubation. The kinetics of germination was determined according to five closely related parameters, viz. final germination The cherimoya (Annona percentage (FGP), mean germination time cherimola Mill.) is a subtropical fruit (MGT), coefficient of velocity of tree of the family Annonaceae. germination (CVG), time to Cherimoya has one of the highest 50% germination (T50) and seedling market approval scores among all length (SL). The temperature of 30°C was commercially produced (Kader found optimally suitable with 70.8% FGP, and Yahia, 2011). The flesh is 17.5 days MGT and 3.91 cm SL, while smooth, delicate, sweet and fragrant the room temperature of 20-25°C slightly

1 Department of Ecology and Environment, University of Batna 2, Batna 05078, Algeria

185 A. KHELOUFI, L.M. MANSOURI, H. KHETTACHE with a custard-like texture. Few especially when cultivated in areas people, however, know of this crop with dry or cool seasons and without because it is not produced on their irrigation (Pinto et al., 2005). With the continent. This fruit has a good exception of a few cultivars, clonal potassium content (compared to its propagation of cherimoya and Annona sodium content) and other hybrids by cuttings or marcottage has micronutrients, such as phosphorus, not been very successful (Pinto et al., calcium and magnesium (Eilers et al., 2005). However, variation in 2011). Due to this composition, rootstock yield of seedlings is a major cherimoya concentrate is a perfect cause of scion yield and reduction in health care product (González et al., fruit quality (George and Nissen, 2010). Cherimoya has been shown to 1987). Propagation of cherimoya by have antioxidant benefits, reduce the stem cuttings was not effective either risk of some forms of cancer and (Bourke, 1976). In addition, low and reduce the risk of certain heart and erratic germination of seeds is one of blood pressure diseases the problems in cherimoya cultivation (Guruvayoorappan et al., 2015). (De Smet et al., 1999). Cherimoya The growing region of seeds exhibit irregular germination cherimoya extends over many distribution with germination countries of Central and South occurring over a long period of time, America and it has been planted in nuking problems of generative most subtropical regions of the world propagation. This pattern of (Pinto et al., 2005). Cherimoyas are germination is possibly due to the mostly commercially cultivated in existence of dormancy, natural areas that are congenial to the planting defense to give seedlings a better of avocados (Palma et al., 1993). For chance of survival, by inducing several countries, cherimoya matures germination under ideal environmental for late winter or early spring conditions, and away from the mother (Grossberger, 1999). Spain is the , thereby preventing competition world’s major producer of cherimoya, (Lambers and Oliveira, 2019). and ‘Fino de Jete’ is the leading Germination of freshly harvested cherimoya cultivar (Padilla and cherimoya seed may be highly Encina, 2003). There are usually two variable and has been reported to commercial varieties generally exist, a range from 30 to 80% (George and smooth skin variety (‘Concha Lisa’) Nissen, 1987). Seed scarification and and hillocky skin variety (Bronceada) stratification do not improve

(Palma et al., 1993). germination of this species (Jubes et al., Most Annona species are 1975; Padilla and Encina, 2003). propagated by grafting and budding Gibberellic acid and high temperature (George and Nissen, 1987; Joshi et al., have been shown to enhance 2000). With few exceptions, germination of cherimoya seeds are deciduous, even tropical species, (Padilla and Encina, 2003). The

186 GERMINATION ON ANNONA CHERIMOLA ANNONACEAE particular characteristic of Annona performed in January-February, 2020, at seed is the presence of a primitive, three constant temperatures of 25, 30, slow-growing embryo, which is often 40°C, and at room temperature, varying not yet distinguished when the fruit is from 20-25°C. To determine the effect of temperature during germination, a total of ripe (De Smet et al., 1999). The four replicates of 12 seeds were development of the embryo continues disinfected with 1% sodium hypochlorite in the seed after harvesting the fruits for one minute, rinsed with distilled water (Garwood, 1995). and immediately sown on 0.8% (water The objective of the present agar), in 9 cm Petri dishes, under aseptic study was, firstly, to investigate some conditions. morphological characteristics of the Seeds were incubated simultaneously seeds, secondly, to evaluate the effect for 25 days, in the dark, under three of different temperatures and to continuous temperature regimes (25, 30 observe the germination curves in and 40°C) and room temperature order to determine the optimal (20-25°C). Seed germination was under dark conditions because we were only condition to enhance and homogenize interested in studying the impact of germination. temperature on seed germination, ignoring the influence of other factors, MATERIALS AND METHODS such as light. Germination counts were performed daily, to determine Seed characteristics and sampling germination kinetics. Seeds were This study focused on cherimoya, considering germinating only when 2 mm Annona cherimola var. ‘Concha Lisa’. radicles emerged. The parameters Ten fruits of approximately 400 g weight evaluated were as follows: were collected from mature commercial Σni FGP(%) = ×100, orchards in Granada (Spain), on N November 2019. Each fruit contained 60- where, FGP is the final germination 70 seeds. The seeds were extracted by percentage, ni is the number of seeds opening the fruits and removing the pulp. germinated on the last day of testing, and Seeds were then cleaned and air dried for N is the total number of seeds incubated 10 days (Fig. 1). The seed sample for our per test (Côme, 1970). However, FGP experiment was obtained by mixing the only reflects the final percentage of seeds to minimize inter-genetic variation germination attained and provides no (Kheloufi et al., 2017). Seeds of length: picture of the speed or uniformity of 15.1 ± 1.25 mm, width: 9.98 ± 1.59 mm, germination. thickness: 5.73 ± 0.61 mm, weight: 0.46 ± Σ(ti.ni) MGT(days) = , 0.04 g, n = 50) were then stored in a bottle Σni glass at 4°C, for one month. The where, MGT is the mean germination thousand-seed weights 466 g. time, ti is the number of days since the start of the test, ni is the number of Germination test in Petri dishes germinated seeds recorded at time ti, and The research experiment was carried Σni is the total number of germinated out at the Laboratory of the Department seeds (Orchard, 1977). of Ecology and Environment‚ University ΣNi CVG(%) = ×100, of Batna 2, Algeria. The experiment was ΣNiTi

187 A. KHELOUFI, L.M. MANSOURI, H. KHETTACHE where, CVG is the coefficient of velocity germination. It increases when the of germination, Ni is the number of seeds number of germinated seeds increases and germinated each day, Ti is the number of the time required for germination days from sowing corresponding to N decreases. Theoretically, the highest CVG (Jones and Sanders, 1987). The possible is 100, which is the case if all coefficient of velocity of germination seeds germinated on the first day (Jones gives an indication of the rapidity of and Sanders, 1987).

where, T50 (days) is the time up to 50% of seeds, t2 is the time necessary for the germination, n1 is the cumulative germination of n2 of seeds (Côme, 1970). percentage of germinated seeds, which The lengths of eight randomly value is closest to 50% by a lower value, selected seedlings (SL) per thermal n2 is the cumulative percentage of condition were also recorded at the end of germinated seeds, which value is the the experimental period using a digital closer to 50% by higher value, t1 is the caliper. time necessary for the germination of n1

Figure 1 - Cherimoya (Annona cherimola): A – Fruits; B, C – Seeds; D - Seed germination and seedling emergence after 25-day period

188 GERMINATION ON ANNONA CHERIMOLA ANNONACEAE

Statistical analysis maximum seed germination was The experiments were conducted found on 10th day at 30°C, 15th day at with four replicates of 12 seeds (n=4), for 25°C, and on 20th day at 40°C and the trait seed germination, and with eight room temperature (20-25°C). The replicates (n=8), for the seedlings lengths, mean percentage of seed germination and the results were expressed as mean ± standard deviation (SD). All the data were over temperatures ranged from 25% subjected to one-way analysis of variance (room temperature) to 70.8% (30°C) (ANOVA) and Duncan’s multiple range (Fig. 2, Table 1). test (P< 0.05), using SAS Version 9.0 The primary environmental (Statistical Analysis System) (2002) factors that influence germination in software. all species are temperature and water supply, influencing both the rate and RESULTS AND DISCUSSION the final percentage of germination. A quantitative analysis of germination Germination of cherimoya seeds sensitivity to temperature would The effect of temperature on the enable better prediction of the germination kinetic of cherimoya potential impacts of temperature seeds is illustrated on Fig. 2, in which fluctuations on this crucial stage in the three stages are indicated: a first plant’s life cycle (Dürr et al., 2015). period of latency due to the Characterization of the threshold imbibition, a second exponential values for germination can thus define period where germination is the limits to the thermal environment accelerated and a stationary phase a species will tolerate (Lockwood, follows. The differential behavior of 2011; Orrù et al., 2012). the 'Concha Lisa' cherimoya variety The differences in germination indicated that the seed germination speed due to temperature, as shown potential was significantly (p< 0.001) by the position and slope of the curves influenced by temperature (Table 1). in Fig. 2, were more or less as might Temperature may affect the be expected. While the first seeds percentage and rate of germination by germinated at 30°C as rapidly, a affecting dormancy loss and the minor slowing at 25°C and a germination process itself (Khurana pronounced pause at 40°C and room and Singh, 2001; Dresch et al., 2014). temperature was observed. Temperature regimes and Nevertheless, the fact that most of the number of days to count have affected seeds remaining at the end of the cherimoya seed germination (Fig. 2). experiment were still apparently Indeed, temperature fluctuations, sound indicates that some factor was number of days to count and their operating to prevent their interactions were highly important germination. Indeed, at the end of the (p< 0.001). The percentage of experiment, those seeds were germination and days to germination transplanted and a significant varied according to temperature. The percentage could germinate in potting

189 A. KHELOUFI, L.M. MANSOURI, H. KHETTACHE soil after a month under greenhouse effect, which played a very important condition at a temperature of 27 ± role in germination activity induction. 2°C. Indeed, temperature of 30°C According to Table 1, cherimoya improved cherimoya seeds by higher seeds exhibit variable behaviors germination rate as indicated by lower (p< 0.0001) at various temperature MGT (17.5 days) and T50 (15.2 days), degrees studied at several parameters, with higher FGP (70.8%), CVG viz. the final germination percentage (57.1%) and SL (3.91 cm) (Table 1). (FGP), and the mean germination time On the other hand, the lowest (MGT), the coefficient of velocity of germination was recorded in the seed germination (CVG) and time to 50% batch with a 20-25°C incubation, germination (T50). In Petri dishes, reporting a too long MGT (20 days) these parameters were measured over and T50 (17.6 days) value with a low an incubation period of 25 days. CVG of 49.8% and 25% FGP (Table 1). These results showed temperature

Table 1 - Final germination percentage (FGP), mean germination time (MGT), coefficient of velocity of germination (CVG), time to 50% germination (T50) (n=4) and seedling length (SL) (n=8) of Annona cherimola seeds exposed to different temperature regimes (n=4)

Temperatures FGP (%) MGT (days) CVG (%) T50 (days) SL (cm) 25°C 35,4 ± 9,4b 17,6 ± 0,25c 56,7 ± 0,08a 15,1 ± 0,31c 1.90 ± 0.45c 30°C 70,8 ± 4,8a 17,5 ± 0,03c 57,1 ± 0,05a 15,2 ± 0,01c 3.91 ± 0.39a 40°C 33,3 ± 7,7b 18,2 ± 0,48b 54,9 ± 0,15c 16,2 ± 0,79b 1.41 ± 0.18c 20-25°C 25,0 ± 8,7b 20,1 ± 0,18a 49,8 ± 0,04c 17,6 ± 0,25a 2.58 ± 0.46b F of Fisher 16.04 70.77 60.87 29.81 39.94 P 0.0002 <0.0001 <0.0001 <0.0001 <0.0001 The same alphabet along the column indicates no significance difference (Duncan Multiple Range Test).

Cherimoya embryo has been decreases (George and Nissen, 1986). found to be small (3-4 mm) and is Seed of most Annona species rapidly located inside an impermeable testa, loses viability and should be planted with the micropyle being the only as soon as possible after removal from water entry (De Smet et al., 1999). the fruit (George et Nissen, 1987). The optimum temperature for However, scarification of sugar apple germination of cherimoya and Annona and cherimoya seed does not improve hybrid cultivars is between 28 and seed germination or reduce the period 32°C (Sanewski, 1985). of time for germination (Jubes et al., Seed can germinate in three 1975; Padilla and Encina, 2003). The weeks under these conditions. At poor germination rate of some lower temperatures (15-20°C), Annona species or cultivars can also germination is delayed by 3-4 months be caused by a high proportion of and the germination percentage infertile seed (Barnes, 1943). The

190 GERMINATION ON ANNONA CHERIMOLA ANNONACEAE propagation of most Annona species poor fruit quality (Campbell and by seed is not recommended since the Phillips, 1983). seedlings are genetically diverse and The growth of cherimoya most are characterized by a long seedlings, over a 12-week period, is juvenile period, irregular bearing and demonstrated in Fig 3.

Figure 2 - Effect of temperature on seed germination of Annona cherimola in Petri dishes (0.8% Agar water) for 25 days of incubation

Figure 3 - Overview on the emergence of cherimoya seedlings during a 12-week period in pots under greenhouse conditions (27 ± 2°C temperature and 35% relative humidity)

191 A. KHELOUFI, L.M. MANSOURI, H. KHETTACHE

Cherimoya seeds were directly the actual optimum for seed germinated (2 cm depth) in plastic germination and the establishment of pots containing 1.0 kg of mixed seedlings. The seeds of cherimoya are substrate (two volumes of sand mixed non-dormant and do not require any with one volume of potting soil), pre-treatment to germinate. This result under greenhouse conditions (27 ± could be suggested as a contribution 2°C temperature and 35% relative for a crop production protocol in humidity). According to George and nurseries or for other agronomic Nissen (1987), seedlings should be studies. grown in root trainers as the root systems are very susceptible to Acknowledgements. Authors are thankful twisting and strangulation and when to Samir Hocini, Laboratory Engineer in the seedlings are 10-15 cm high, they Plant Biology (University of Oran, can be transplanted into either Algeria), for his great help in providing polythene bags or nursery beds. cherimoya fruit.

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