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Production of Callus From Coffea Arabica

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Materials and Methods

Somatic embryogenesis

There are two types of somatic embryogenesis: direct embryogenesis or indirect embryogenesis. According to Carneiro (1999), direct embryogenesis or known as low frequency of somatic embryogenesis (LFSE) uses only one medium without producing callus and there will be less number of somatic embryos obtained. However, the indirect embryogenesis or high frequency of somatic embryogenesis (HFSE) uses two media (Carneiro, 1999). The first medium involves induction of callus and second medium is responsible for regenerating few hundred thousand somatic embryos per gram of callus (Etienne, 2005). According to Etienne (2005), it takes only 9 to 10 months for Coffea arabica from leaf explants to somatic embryos. Therefore, it is more practicable to use indirect somatic embryogenesis for the effective propagation of superior Coffea arabica planting materials (Ahmed et al., 2013).

Collection of explants

Leaf explants are most widely used in micropropagation. The selected leaves must be obtained from superior Coffea arabica plant in order to supply the high demand of superior planting material. Superior young leaves are collected from perfectly healthy Coffea arabica with resistant to most fungal diseases and susceptibility of other diseases and pests Ahmed, Feyissa & Disasa, 2013). In order to get high quality explant material, particular attention has to be given to the mother plant to maintain the physiological and sterility status of the plant and limit the possible contamination sources (Etienne, 2005). Etienne (2005) also states that young leaves will usually be selected for their tenderness and reactiveness.

Surface sterilization

The chosen explants are first washed with running tap water and disinfected by immersing the explants into 10% calcium hypochlorite solution containing 1% Tween 80 for 20 minutes followed by 8% calcium hypochlorite solution for 10 minutes and rinse 4 times with sterilize water (Etienne, 2005). The fully sterilized Coffea arabica leaves are cut at size measuring at 1 square cm and place underside upwards on the culture medium with all the essential grow factors (Etienne, 2005). All these actions are performed under laminar air-flow carbinet to avoid any contaminations.

Culture initiation

The callus culture is necessary to initiate the production of callus from Coffea arabica. The leaf explants are placed in 50 ml sample tubes containing 15 ml medium, with the abaxial surface in contact with modified Murashige and Skoog (MS) medium that use the following salts

Chemical

Amount (mg per liter)

Chemical

Amount (mg per liter)

NH4NO3

412.5

ZnSO4.7H2O

4.3

KNO3

475

Na2MoO4•2H2O

0.125

MgSO4

92.5

CuSO4•5H2O

0.05

KH2PO4

85

Fe.Na.EDTA

21

CaCl2.2H2O

110

 

H3BO3

3.1

MnSO4.7H2O

11.2

Table 1. Salts used for formation of callus tissue (Mari, Takeshi, Naotsugu, & Tadashi, 1994)

The MS medium also contain Gamborg’s B5 vitamins (Gamborg Miller, & Ojima, 1968) with exactly 3% (w/v) sucrose and 5 μM N-(phenylmethyl)-1H-purine-6-amine (BA) as the only plant growth regulator (Yasuda, Fuji, & Yamaguchi., 1985). The pH of the MS medium must be adjusted to 5.7 before any sterilization process occur and it must be consistent throughout all the experiments (Mari et al., 2007). All cultures must be incubated at temperature approximately 25ºC with a fourteen hour of light strength at of 30 µmolm-2s-1 supplied from cool white fluorescent lamps.

Shoot multiplication

By using about 150 ml solid MS media, microshoots of about 10 mm are induced. Accrding to Naji, Rida, Ibrahim, Mohamad, & Abdallah (2007), in order to maximizing microshoot production, the effects of plant growth regulator are determined. Different types of plant growth regulator such as BA (6-Benzylaminopurine), TDZ (Thidiazuron), 2ip (6-γ-γ-[Dimethylallylamino] purine) or Zeatin are separately prepared at different concentrations of 0.0, 2.0, 4.0, 6.0 or 8.0 mg/L (Naji et al., 2007). For each of the experiment volume, 0.5 mg/L IAA (Indole-3-acetic acid) has been added ((Naji et al., 2007). Then, the microshoot cultures are incubated under 26±2 °C and exposure to 16 hours light and 8 hours darkness (Naji et al., 2007). Data are collected based on the number of shoots and height of the shoots after three months. A completely randomized design (CRD) with at least 3 replicates will be able to summarize the effects of BA, TDZ, 2ip or Zeatin on the multiplication of shoots. The most effective plant hormone regulator is used to mass produce the shoots.

Root formation

Same for the root formation, the plant growth regulators are adjusted to produce roots. The microshoots are cultured with 25 ml solid half-strength MS media and 15 g/L sucrose (Naji et al., 2007). Different concentrations (0.0, 1.0, 2.0 or 3.0 mg/L) of IAA, NAA (napthyl acetic acid), or IBA (indole-3-butyric acid) are prepared separately (Naji et al., 2007). As stated in Naji et al. (2007), the experiments are maintained at under 26±2 °C and exposure to 16 hours light and 8 hours darkness. Data of the number of roots, root length are recorded after 2 months. Completely randomized design (CRD) with 3 replicates summarizes the best plant growth regulator to be used to form root in the Coffea arabica microshoots.

Acclimatization

The ex vitro acclimatization was used for plantlet hardening process. Rooted plantlet in culture disk is left exposed to surrounding clean air for 3 days (Naji et al., 2007) before transferring plantlets outside of the growth chamber. The plantlets are extracted from culture disk and the agar from MS medium was removed by washing with warm distill or sterile water. The plantlets are then planted in 1 peat: 1 perlitemixture in 84 cell polystyrene trays covered with glass beakers for 3 weeks (Naji et al., 2007). Plantlets were exposed to 16 hours supplementary light of 40-45 µmolm-2s-1 along with 8-hours of darkness at 26±2 ºC for acclimatization process (Naji et al., 2007). Naji et al. (2007) states that acclimatized plants is then transferred to 12 x 23 cm polyethylene bags containing 1 soil:1 perlite mixture and grown at temperature 24±2 ºC during the day, 20±2 ºC during the night with overhead irrigation after three weeks.

Experimental design and data analysis

The use completely randomized design (CRD) and General Linear Model model in Statistical Analysis System (SAS) help to analyze the various variability and effects of one factor on another, and thus able to set up standard and constant variables for making Coffea arabica plantlets of the same quality in large quantities (Ahmed et al., 2013).

Expected result

The micropropagation method is used to produce Coffee arabica that is identical to its parent genome; hence the quality of the fruits produce is guarantee if the parents have favorable characteristics. Expected germination percentage of seedlings is 100% (plant using seeds) whereas survivability of plantlets from both that originates from both seed and callus tissues of Coffee arabica are more than 90%. This method will take less than 3 years to produce fully mature Coffee arabica tree from plantlets (The life cycle from seed to mature is 3-5 years). This method shortens time taken to mature, increase survivability and enable mass production of healthy plantlets compare to natural germination.

Reference

Ahmed, W., Feyissa, T., & Disasa, T. (2013). Journal of horticultural science & biotechnology. Somatic Embryogenesis of A Coffee Hybrid Using Leaf Explants, 88(4), 469-475.

Carneiro, M. F. (1999). AgBiotechNet. Advances In Coffee Biotechnology, 1, ABN006.

Etienne, H. (2005). Somatic embryogenesis protocol: Coffee. In S. Mohan Jain & Pramod K. Gupta (Eds), Protocol for somatic embryogenesis in woody plants (pp. 167-180). Netherlands: Springer.

Gamborg, O. L., Miller, R.A., & Ojima K. (1968). Nutrient requirements of suspension cultures of soybean root cells. Experimental Cell Research 50, 148–151.

Mari, T., Takeshi, Y., Naotsugu, U., & Tadashi, Y. (1994) Formation of Somatic Embryos from ?????

Naji, E., Rida. S., Ibrahim. M., Mohamad. S., & Abdallah A.E. (2007). In vitro Propagation and In vivo Acclimatization of Three Coffee Cultivars (Coffea arabica L.) From Yemen. World Applied Sciences Journal 2(2), 142-150.

Protoplasts of Coffea arabica L. Hortscience 29(3), 172–174.

Yasuda, T., Fuji, Y., & Yamaguchi, T. (1985). Embryogenic callus induction from Coffee arabica leaf explants by benzyladenine, Plant and Cell Physiology: Oxford Journals. 26, 595-597.


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