Breaking of Seed Dormancy in Morindalucida Benth

¹*AJONGBOLO Febisola Bukola, ²OYETUNJI Olusola J, ³JAMALEDDINE Zainab Olubunmi,

⁴ADEDEJI Abdulkabir Adeyemi

Greener Journal of Biochemistry and Biotechnology, vol. 5, no. 1, pp. 001-008, 2018

¹National Centre for Genetic Resources and Biotechnology, Moor Plantation Apata, Ibadan.

²Department of Botany, University of Ibadan.

³National Centre for Genetic Resources and Biotechnology, Moor Plantation Apata, Ibadan.

⁴National Centre for Genetic Resources and Biotechnology, Moor Plantation Apata, Ibadan.

²solatunji2k2 @yahoo.com, ³glizzy00 @yahoo.com, ⁴kbspecial4real @yahoo.com

INTRODUCTION

Morinda lucida Benth is a tropical tree found in the Africa rainforest and commonly known as Brimstone tree, belonging to the family of  Rubiaceae. In the South and Central Regions of Cameroon, Morinda lucida is commonly known as “akeng” , in Yoruba : “Oruwo”, Hausa; ‘’Marga”, Igbo: “Injisi”. It is one of the most widely used plants in this environment for medicinal purposes (Zapfack and Ngobo, 2002). It is an evergreen shrub of small to medium-sized tree measuring up to 18-25 m tall, with bole and branches often crooked or gnarled; bark smooth to roughly scaly, grey to brown, often with some distinct purple layers. It grows in grassland , exposed hillsides, thickets ,forest, often on termite mounds, sometimes in areas which are regularly flooded, from sea level up to 1300m altitude (Zimudzi and Cardon,2005).. It is medium-weight and hard; it works and finishes well, and it is durable, being resistant to fungi, termites and other insects (Zimudzi and Cardon, 2005).

Plant derived products have been used for medicinal purposes since the creation of man. Approximately about 80% of the world population today, relies on botanical preparations as medicines to meet their health needs (Polasa and Nimala, 2003).  Different parts of the tree have been reported to possess medicinal properties. The leaf extract of the plant possess trypanocidal (Asuzu and Chineme, 1990), antimalaria activities (Makinde and Obih, 1985) and aortic vasorelaxant effect (Ettarh and Emeka, 2004). Oliver-Bever (1986) documented the use of a weak decoction of the stem bark to treat severe jaundice. The leaf extract of Morinda lucida has also been reported to have a strong oral hypoglycemic property (Olajideet al. 1999; Adeneye and Agbaje, 2008) by increasing the utilization of peripheral glucose. It’s leaf extract has also been associated with some reversal toxicity such as the reversible antispermatogenic activities in rats (Rajiet al. 2005). The bark of M. lucida is widely used in Cameroon as a decoction for treating Diabetes mellitus.

Seed dormancy is a great problem for cultivating M. lucida and relatively little information is available on the  seed dormancy and germination requirements of this plant.

Seeds scarification method is used to soften hard seeds (Kimura and Islam, 2012).However, effectiveness of the methods varies, depending on the duration of imposed treatments and species of cultivar to be used.(Taia, 2004). Researchers have been using different scarification methods  since early 20^th century and have reported variable results. Heat, freeze-thaw, mechanical and acid scarification are among the most popular methods.(Kimura and Islam, 2012).

In many seeds, embryos are in genuine (endogenous) dormancy conditions which must either disappear spontaneously or must be broken by the action of certain environmental conditions before germination can take place.(Barton, 1965). To accelerate breaking of seed dormancy, hormones have been applied in several studies (Zigas and Coombe, 1977; Chang and Sung, 2000). Iglesias and Babiano, 1997, reported that Gibberellic acid (GA₃) is one of the hormones proposed to control primary dormancy by inducing germination. Cavieres and Arroyo (2000) from a dormancy-breaking experiment with Phacelia secunda seeds showed that a long period of prechilling treatment resulted in increased germination level.

Aliero (2004) reported that the use of hot water, Sulphuric acid and sand paper scarification affected Parkia biglobosa seeds dormancy. El-Siddiq et al. (2001) found that scarification of Tamarindus indica with hot water and Sulphuric acid decreased mean emergence time and days to 50% emergence compared to control.

There are limited literatures concerning the potential seed dormancy problems of M. lucida. This study therefore aims at investigating the effect of different scarification methods in breaking seed dormancy in M. citrifolia

MATERIALS AND METHODS

Seed collection and Disinfection

Morinda lucida seeds were collected from the field (medicinal garden) of the National Center for Genetic Resources and Biotechnology (NACGRAB). Matured Pods of the seeds were harvested and brought to the Laboratory .Seeds were removed from pods and washed under running tap water with few drops of tween twenty.

Four hundred (400) seeds were surface sterilized by soaking in 70% ethanol for 5 minutes inside the laminar flow cabinet followed by disinfection with 4% Sodium hypochloride (NaOCl) for twenty minutes. Seeds were then rinsed in sterile water three times and left in the third rinse.

Seed Scarification

Seeds were divided into eight batches and subjected to different scarification methods. Four scarification methods were used. This included Mechanical (nicking at the micropylar region), Chemical (soakingten seeds  each in conc. H₂SO₄ for 10,20,30,40 and50 mins) Physical (soaking ten seeds each in 50⁰C water for 10-50mins ), and hormone treatment ( soaking ten seeds  each in GA3 at concentrations 100mg/L,300mg/L, 500ng/L, 700mg/L and 900mg/L)

            The three latter scarification methods were combined with mechanical scarification giving a total of seven treatments, while the eighth batch of seeds were not scarified and used as control.

Aseptic Isolation and  Plant Growth Medium  

All materials used were autoclaved at 121^oC for 30minutes. Surface sterilization of the work area was done by wiping with 70% ethanol. Proper flaming of material during inoculation of seeds and handling of explants was done under the Laminar flow cabinet.

Seeds were  aseptically inoculated with the aid of sterile forceps and scalpel and cultured on full strength Murashige and Skoog’s medium (1962) containing 3% (w/v) sucrose, B5 vitamins, and 0.1mg/L inositol. The pH of the medium was adjusted to 5.7 prior to the addition of 0.7% (w/v) agar and autoclaved at 121oC at 15 lb pressure for 15 mins. The cultures were maintained at 25±2 0C under a 16 h light and 8 h dark regimes with a light Intensity of 3000 lux provided by cool-white fluorescent lamps and 50-55% relative humidity.

Cultures were studied on daily basis and data were recorded weekly.The experiment was concluded at 9^th week.

STATISTICAL ANALYSIS

The experimental design was a Completely Randomized Design (CRD) with five replicates and the data generated were subjected to analysis of variance (ANOVA) using the statistical software (SPSS).

RESULT

The germination of M. lucida seeds in this study was highly stimulated by warm water with nicking method which varied with time, compared with other scarification methods, while conc. sulphuric acid indicated no germination. Scarification methods used with timings (ranging from 10minutes to 50minute) shown significant effects on the germination percentage. Many of the germinated seeds (plantlets) varied according to treatments in combination with timing which affected the morphological characteristics in different ways (Table i).

Highest germination percentage was recorded in warm water for 10minutes which was followed by a gradual increase in germination rate but no uniformity in the growth of plantlets (Plate i). Warm water with or without nicking at 10 and 20 minutes also has a significant differences of higher germination and uniformity in growth, compared to other treatments (Figure i).

The hormone(GA₃) with nicking at different concentrations (100mg^-L, 300mg^-L, 500mg^-L700mg^-L,  900mg^-L), showed no significant difference for the formation of shoot number, plant height, plumule length and leaf number. Only the radicle length had little significant difference (Table ii) .  It was observed from this study that the application of hormone (GA₃) in breaking of Morinda lucida seeds dormancy enhanced elongation of the germinated seeds and as well promote shoot formation.

 Germination percentage in GA₃ and GA₃ with nicking  had no  significant difference based on the level of  concentrations, (100mg^-L, 700mg^-L, 900mg^-L) (Fig. ii),  while significant difference were only recorded in 300mg^-L_{and 500mg}^-L GA₃ and GA₃ with nicking. Highest germination percentage were  recorded in300mg^-L GA₃ with nicking. (Plate ii).

Table1:  Effect of different scarification methods on germination of M. lucida seeds

Values are means and standard errors of three replicates. Values with the same letter are not significant difference at P≤ 0.05 using Duncan Multiple Range Test.

Table2: Effect of different concentrations of Gibberellic acid (GA₃),  GA₃ with nicking on germination of Morinda lucida seeds

Values are means and standard errors of three replicates. Values with the same letter are not significant difference at P≤ 0.05 using Duncan Multiple Range Test.

FIGURE 1: Germination percentage after dormancy breaking of Morinda lucida seeds, using different scarification methods

Abbreviations: C.H₂SO₄= Concentrated Sulphuric acid, N only =Nicking only, WMH₂O =Warm water, N+WMH₂O = Nicking + Warm water, N+CH₂SO₄ =Nicking + Concentrated sulphuric acid, CNTRL = Control.

FIGURE 2: Germination percentage for dormancy breaking of Morindalucida  seeds, using GA_3, GA₃ with nicking methods, with different concentrations

Abbreviations:

GA₃= Gibberellic acid, NIK= Nicking

PLATES 1:Germinated  M. lucida seeds  treated with nicking and warm water at 20 minutes compared with control

PLATES 2: Germinated  M. lucida seeds  treated with Nicking and GA₃ at 300mg^-L compared with control

Effect of scarification methods ,GA₃and GA₃ with nicking treatments on acclimatization of M. lucida plantlets ( from in vitro to ex vitro condition).

Acclimatization of plantlets is the process of hardening and weaning of plantlets from in vitro to ex vitro condition. It is also a stepwise process of adapting plantlets to their own habitat. After 9weeks of the study , the fully grown plantlets which were germinated from the seeds through various methods of scarification, hormone were acclimatized under the acclimatization chamber using sterile soil components of coconut fiber, top soil, river sand in various proportions, for proper growth and development ( Plates iii).

The plantlets acclimatized showed no significant effect with regards to morphological characteristics at the first week of acclimatization. Only the plant height were significantly difference at second week of acclimatization. There was no mortality rate all through the period of acclimatization, this showed that all the treatments had no effect on the adaptability of the plantlets to their natural habitat.

PLATES 3: Acclimatization of Morindalucida plantlets treated  with different scarification methods and hormone

DISCUSSION AND CONCLUSION

Various effective and practical treatments have been developed to break seeds dormancy Edilma et al. (2011). Nicking, hot water, physical and acid scarifications have all been used to good effect with the seeds of many tropical and subtropical species (Edilma et al., 2011).

The present findings from seeds dormancy breaking demonstrate the possibility of breaking seeds dormancy in M. lucida , using scarification and GA₃ treatments. Germination of seeds was reported in M. citrifolia using scarification techniques (Nelson S.C., 2005). Francis J.K (2003), also reported that Morinda citrifolia seeds scarified with nicking, usually germinate in about four weeks and have tap roots that emerge uniformly and easily through the slit.

Generally, the inability of treated seeds with conc. Sulphuric acid method to germinate did not agree with the report of El-siddig et al.(2001) which stated that concentrated sulphuric acid accelerated water uptake and resulted in earlier and faster germination in Tamarindus indica.

Also, the study of Babashpour et al. (2011)  reaffirms the earlier report, which stated that acid scarification is known to be highly effective in improving germination of species with hard seed coats. Seeds that has been kept for a long period in store may require a longer period in the acid than fresh seeds, which could be severely damaged by the same length of treatment ( Bonner et al.1987).

Seeds treated with warm water gave optimum germination percentage. This study support the observation gave by Bonner et al.(1987) in his report which stated that Parkinsonia aculata responded better to warm water (20⁰_C) treatment by given good results.

Moreover, hot/warm water treatments have yielded beneficial results with a number of leguminous seeds. It has been shown that the initial water temperature has a larger effect on germination rate than the periods of soaking and cooling of the seeds (Willan, 1985).

Permeability of seed to water and oxygen is improved by nicking the seed coat and this is in agreement with the findings of Ayisire et al.(2009).

Nicking has been found to be extremely effective for most species but it requires care, for instance it has to be done on one side away from the micropyle to prevent destruction of the embryo. The results got from this study are also supported by the findings of Likoswe et al.(2008) who reported nicking and soaking in hot water to have enhanced the highest germination and growth in Terminalia sericea. However, nicking treatment is laborious and slow such that its applicability is only limited to small seed samples.

In this study, it was observed that germination response of M. lucida to increasing concentrations of GA₃ treatments did not follow a defined pattern of growth. This observation is in tandem with the study of Tigabu et al. (2001), which stated that some of the studies on effect of scarification, gibberellic acid and temperature on seed germination of multiple species of Albizia species, revealed that exogenous application of GA₃ on the breaking of seed dormancy and seed germination differed widely among species and within species.

However, based on the observation from this study , it can be averred that the application of GA₃ with nicking in breaking of M. lucida seeds dormancy supports the observation by Afrasyab et al.(2007) which was reported that GA₃ with scarification methods are more effective for breaking of seed dormancy in Ferula gummosa.

Successful establishment of fully grown in vitro M. lucida plantlets on sterile soil compositions ex vitro, involved a stepwise process. Also in support was a similar effort by Pospisilova et al.(1999) who reported that the threats for survival in ex vitro can be overcome by acclimatizing the plantlets with gradual lowering air humidity , temperature, airflow and irradiation level.

This corroborate the observation by researchers like  Ayelign et al.(2013) , on a similar work, who reported that the ultimate success of in vitro plantlets depends upon the successful transfer and establishment of plantlets in ex vitro conditions. Nevertheless, high loss or damage of in vitro raised plantlets can occurred when transferred to ex vitro conditions because of the transfer shock, Pospisilova et al., (1999). This is due to the exposure of plantlets to many new ex vitro conditions such as low humidity, high level of irradiation, water deficit because of the poor hydraulic conductivity of the root and low root stem connection, Fila et al., (1998).

In conclusion, these results suggest that since GA₃ coupled with nicking, Warm water with nicking both induced seeds germination of  Morinda lucida with uniformity, therefore the ability of  seed germination in this plant is said to be controlled by both exogenous and endogenous regulating factors. It was observed that the seed coat cannot be the only problem in germinating M. lucida seeds, since the application of scarification methods or GA₃ alone cannot enhance germination to its maximum level. Therefore the use of GA₃ and nicking together can be more efficient. The highest germination percentage can be achieved when GA₃ treatment in lower concentrations (between 300mg^-L to 500mg^-L) accomplished with nicking. Accordingly it is recommended that for breaking of M. lucida seeds dormancy and increasing seeds germination percentage for mass production and uniformity within a short time, GA₃ treatment with nicking should be applied together. While nicking coupled with warm water can as well be applied as another good techniques.

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