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Effect of Physical, Chemical and Light Treatments on Germination and Growth of Tissue-cultured Coconuts

Pablito M. Magdalita1, Olivia P. Damasco1, Joseph C. Beredo1 and Stephen W. Adkins2

Institute of Plant Breeding, College of Agriculture, University of the Philippines Los Ba隳s, College, Laguna 4031, Philippines. Email pmmagdalita@ipb-uplb.org.ph, pablitomagdalita@yahoo.com
2
School of Land and Food Sciences, University of Queensland, Brisbane 4072, Australia. Email s.adkins@uq.edu.au

Abstract

The acclimatization and ex vitro establishment of tissue cultured coconut plantlets regenerated either from zygotic or somatic embryos could result to serious losses. Although high germination rates can be achieved in vitro, the survival of zygotic embryo derived plantlets in soil is very low (0-30%). Hence, treatments that could promote development of good quality seedlings having well-developed shoot and root is needed to increase seedling survival ex vitro. The effect of physical, chemical and light quality treatments on germination and growth of coconut embryos and tissue-cultured seedlings respectively, was investigated. The germination of coconut embryos was promoted when placed in a liquid Euwens (Y3) medium and incubated using a roller drum. Gibberellic acid (GA3) significantly affected growth of seedlings as it promoted shoot elongation, shoot and root expansion, and fresh and dry weight increase. However, GA3 did not significantly affect germination. In addition, the blue, red and yellow light significantly affected growth of seedlings as it promoted leaf and shoot elongation, fresh and dry weight increase, and root and leaf production. These conditions could be used to improve the growth and survival ex vitro of tissue cultured coconuts.

Media summary

The germination and growth of coconut embryos was promoted by incubation in a roller drum, addition of gibberellic acid into the medium and, use of blue, red or yellow light during incubation.

Key Words

incubation, shaker, culture vessels, plant growth regulators, palm

Introduction

The coconut palm (Cocos nucifera L.) is an important oil-crop on an industrial scale, and a good subsistence and cash crop for smallholders. However, the coconut industry is constrained with a number of problems that affect its productivity. In vitro vegetative multiplication via somatic embryogenesis of superior individuals offers the only short and medium term solution for the production of homogenous planting material that could result to plantation productivity. Cloning by tissue culture could also allow rapid multiplication of selected individuals with resistance or tolerance to serious diseases and adverse growing conditions (Hocher et al. 1998). However, the acclimatization and ex vitro establishment of tissue-cultured coconut plantlets regenerated either from zygotic or somatic embryos can result in serious losses. Although high germination rate can be achieved on a culture medium (ca. 80%, Ashburner et al. 1994), the survival of zygotic embryo-derived plantlets in soil is very low. Many factors contribute to the failure of tissue-cultured coconuts to acclimatize properly. In coconut transplantation, the major problem is associated with the development of poor root system and the small size of shoot produced (Ashburner et al. 1993). Therefore, in order to obtain viable whole plants in soil, the quality of the root and shoot systems of coconut plantlets need to be improved prior to ex vitro establishment. This could be done by manipulation of the tissue medium, the culture conditions and physical modification of explants or combination of these factors, hence this study.

Methods

Coconut tissue culture

Ten to eleven months old coconuts were dehusked and split, and the endosperm cylinders were extracted using a cork borer. Initially, the endosperm cylinders were washed with tap water three times followed by 95% ethanol for 3 minutes and then rinsed with sterile tap water twice. Secondly, they were disinfected with 100% commercial bleach for 20 minutes then they were rinsed with sterilized tap water for 5 times. The endosperm cylinders were excised inside a laminar flow cabinet to obtain the embryos. They were washed with 10% bleach for 5 minutes, then rinsed with sterile distilled water for 5 times. They were inoculated into a liquid Y3 medium. The cultures were incubated under 16hrs light and 8hrs darkness at 25蚓 for 6-8 weeks. They were subcultured into a fresh growth medium on a monthly interval.

Effect of physical treatments on embryo germination

This experiment was done to study the effects of physical movements such as shaking and rolling on embryo germination. The coconut zygotic embryos placed in liquid medium contained in a test tube were incubated in a roller drum, horizontal shaker, and stationary position (control). Percentage germination was obtained 60 days after inoculation.

Effect of different concentrations of gibberellic acid on embryo germination

The effect of gibberellic acid (GA3) on germination of embryos was re-investigated by increasing the concentration used in this study. This was done by the addition of four concentrations (0然, 10然, 20然 and 30然) of GA3 on the Y3 liquid medium. Percentage germination was obtained 60 days after inoculation.

Effect of gibberellic acid on growth of tissue-cultured coconut seedlings

This experiment was done to investigate the effect of GA3 on growth of germinated coconut seedlings. This was done by the addition of four concentrations (0然, 10然, 20然 and 30然) of GA3 on the liquid Y3 medium. Data parameters such as shoot length, shoot width, root length, root width, fresh weight and oven dry weight were obtained 90 days after inoculation.

Effect of light qualities on the growth of tissue-cultured coconut seedlings

This experiment was conducted to study the effects of different light qualities on plantlet growth. This was done using cellophane plastics of different colors wrapped around the culture vessels to concentrate selected wavelength of light that will be used by the plantlet. Five colors (blue, red, green, yellow and normal) were used. The data parameters gathered were: leaf length, leaf width, shoot length, girth width, root length, root width, fresh weight, oven dry weight, number of roots, and number of leaves.

Statistical Analysis

The experiment was conducted in a completely randomized design (CRD) with two to three replications for each treatment and 6-15 zygotic embryos/seedlings for each replicate. The data were analyzed using the Statistical Analysis System (SAS) software and significant differences among treatment means were detected using LSD.

Results

Effect of physical treatments on embryo germination

The germination of coconut embryos is highest (51%) using a roller drum but this is not significantly different from the control (36%). The good germination obtained using a roller drum is most likely due to the aeration brought about by the constant rolling of the tissues on the liquid medium and rapid absorption of nutrients by the embryos. It has been observed that the effect of roller drum is beneficial to germination and growth of weak papaya tissues and hybrid embryos (Magdalita et al. 1996).

Effect of different concentrations of gibberellic acid on embryo germination

Gibberellic acid (10然 GA3) marginally enhanced germination but not significantly different to the control. The shoots of germinated embryos are much longer than the control. In a following experiment, the different concentrations of GA3 (0, 10, 20, 30然) did not significantly affected germination. However, 10-20然 GA3 appeared to promote shoot elongation.

Effect of gibberellic acid on growth of tissue-cultured coconut seedlings

Gibberellic acid (20然) significantly (P>0.05) affected the growth of seedlings including: shoot length, shoot width, root width, fresh and oven dry weights (Fig. 1) but not root length. At this concentration, the growing seedlings had more elongated shoot than the control. The promotive effect of GA3 on growth has been observed previously in coconut (Pech y Ake et al. 2002) and other crops like papaya (Magdalita et al. 1996), and avocado (Skene and Barlass, 1983). This result suggests that GA3 could be used to enhance growth of growing coconut seedlings in vitro.

Effect of light qualities on the growth of tissue-cultured coconut seedlings

Light quality significantly (P>0.05) affected the growth of coconut seedlings including: leaf length, shoot length, fresh and oven dry weights, number of roots and number of leaves. Specifically the blue, red and yellow light (400-700 nm) significantly promoted growth such as: leaf length, shoot length, fresh and oven dry weights, number of roots and number of leaves in comparison with the control. This result suggests that at this spectrum (400-700nm), the excitation of electrons needed for photosynthesis could be higher than the others, thereby promoting growth. This is the first report on the beneficial effects of blue, red, and yellow light on growth of in vitro cultured coconut seedlings. However, the same observation was previously noted for date palm, Phalaenopsis (Islam et al. 2001), and seaweed (Folefack and Cosson, 1995).

Figure 1. Growth response of tissue-cultured coconut seedlings to different concentrations of GA3. The growth parameters are shoot length, shoot width, root width and oven dry weights. Bars having the same letters are not significantly different.

Conclusion

The germination of coconut zygotic embryos could be enhanced marginally by incubation in liquid Y3 medium in a roller drum. Gibberellic acid (20μM) significantly affected growth of coconut seedlings as it promoted shoot elongation, shoot and root expansion and fresh and dry weights increase. In addition, light quality significantly affected growth of the tissue-cultured seedlings. The blue, red and yellow light spectrum promoted leaf and shoot elongation, fresh and dry weight increase and root and leaf production.

Acknowledgement

The Australian Centre for International Agricultural Research provides the funds for this project. The University of Queensland and the University of the Philippines Los Ba隳s jointly facilitates this project.

References

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Ashburner GR Thompson WK and Burch JM (1993). Effect of α-naphthalene acetic acid and sucrose levels on the development of cultured embryos of coconut. Plant Cell, Tissue and Organ Culture 35, 157-163.

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Magdalita PM Adkins SW Godwin ID and Drew RA (1996). An improved embryo rescue protocol for a Carica interspecific hybrid. Australian Journal of Botany 44, 343-353.

Pech y Ake A Santamaria J Souza R Talavera C Maust B and Oropeza C (2002). Changes in culture conditions and medium formulation to improve efficiency of in vitro culture of coconut embryos in Mexico. In: Engelmann F Batugal P Oliver J (eds) Coconut Embryo In Vitro Culture Part II: (pp. 122-137). IPGRI-APO, Serdang, Malaysia.

Skene KGM and Barlass M (1983). In vitro of abscissed avocado embryos. Annals of Botany 52, 667-672.

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