some regulation of in vitro growth of bush mango- irvingia wombolu mildbr (irvingiaceae)

Some regulation of In Vitro growth of bush Mango- Irvingia wombolu Mildbr (Irvingiaceae)

WRJAS

Some regulation of In Vitro growth of bush Mango- Irvingia wombolu Mildbr (Irvingiaceae) 1*Etukudo M Mbosowo, 2Roberts MI Eneni and 3Ilesanmi B Omotayo 1*,2,3

Department of Biological Sciences, Federal University Otuoke, P.M.B. 126, Yenagoa, Bayelsa State, Nigeria.

Studies were conducted to regulate in vitro protocols for the establishment of Irvingia wombolu MILDBR. Axillary bud explants of Irvingia wombolu were cultured on various strengths, full (MS + KIN or BAP), half (

1/2 MS + KIN or BAP) and one quarter (

1/4 MS + KIN or BAP) of MS (Murashige

and Skoog) medium supplemented with different levels (0, 1, 2, 3, 4 and 5mg/l) of kinetin (Kin) or benzyl-amino purine (BAP) under aseptic conditions. Subsequently, one quarter strength growth medium with 3mg/l of kinetin (Kin) and 4mg/l of benzyl-amino purine (BAP), which gave the best growth performance was used to study the effects of culture passage (frequency of subculture - 0, 2, 4, 6, and 8 times, subcultured every week) and disinfection time of 0.1% mercuric chloride solution (0, 1, 2, 3, 4, 5, and 6 min) on in vitro establishment of the species. A culture passage of 2 times a week was optimum for regeneration frequency of the species. In addition, a disinfection time of 5 minutes was optimum in terms of reduced contamination and best regeneration frequency for establishment of the species. This study suggests that establishment of Irvingia wombolu explants can be achieved if the basic culture conditions are maintained at optimum levels.

Key Words: Some, Regulation, In vitro, Growth, Irvingia wombolu (Bush mango) INTRODUCTION Irvingia wombolu Mildbr is one of the important economic trees grown either in the wild or domesticated for timber and non- timber products (Etukudo et al., 2010). It belongs to the family Irvingiaceae and is largely widespread in Africa with its geographical distribution extending from Nigeria to Angola (Antagana et al., 2001; Okafor and Ujor, 1994; Silbou et al., 2004). Irvingia species have been rated as the highest priority multi-purpose food trees that need improvement research in West and Central Africa (Asaah et al., 2003; Vihotogbe et al., 2013). It belongs to the most important multipurpose indigenous food tree species widely cultivated and intensively exploited for their seeds in traditional agro-forestry systems, and for industrial purposes (Asaah et al., 2003; Ude et al., 2006). The seeds, which are valued for their fat and protein rich as well as mucilage contents, are commercialized at local, regional and international levels (Degrande et al., 2006; Vihotogbe et al., 2012).

The seeds and seedlings of this crop have become highly prohibitive in cost because of the very high demand for them by individuals and organizations (Omokaro et al., 1999). The reasons underlining this development are that most Irvingia trees are not cultivated but grow in the wild where the fruits are collected periodically from the high rain forest floors mostly by women who trek great distances between widely scattered trees. Similarly, the seedlings, which could be used for farm planting, are not easy to obtain because of hard seed coat resulting in poor germination (Nya et al., 2000). *Corresponding author: Dr. Mbosowo Monday Etukudo, Department of Biological Sciences, Federal University Otuoke, P.M.B. 126, Yenagoa, Bayelsa State, Nigeria. Email: [email protected], Tel.: +2347032448796

World Research Journal of Agricultural Sciences Vol. 1(2), pp. 007-011, February, 2014. © www.premierpublishers.org, ISSN: 2326-7266x

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Etukudo et al. 007 In addition, the seeds (cotyledon) are the main parts consumed. Thus, the current effort is to domesticate Irvingia species through in vitro technology, which could enhance mass propagation of the plants in order to meet the increasing demand for the teaming population of Africa (Etukudo et al., 2010). Micropropagation has recorded notable successes in the plant breeder's repertoire of techniques in the plant propagation industry (Guo et al., 2007; Jain, 2002). In vitro culture is a biotechnological tool that exploits the natural totipotency of plant cells and produces many disease-free plants which helps preserve the genetic resources of trees and crops (Larraburu et al., 2012; Sulusoglu and Cavusoglu, 2013). Techniques that could provide vegetative propagated material for commercial use and for possible reintroduction are therefore of tremendous interest (Abrie and Staden, 2001; Ahloowalia and Maluszynski, 2001; Carrasco et al., 2013; Faize et al., 2013). Similarly, these aseptic propagation methods are reliable and present a significant tool in the plant propagation industry (Bhalla-Sarin et al., 2003; Chen et al., 2001). Basic explants establishment techniques are, however, required for successful micropropagation of Irvingia wombolu. In addition, scanty reports exist on micropropagation of Irvingia wombolu. This present study was, therefore, conducted to develop an effective method of explants establishment of Irvingia wombolu in vitro. MATERIALS AND METHODS Axillary bud explants were collected from Irvingia wombolu grown in the field in Igbere, Bende Local Government Area of Abia State, Nigeria. Explants of (0.5-1.5cm) Irvingia wombolu were pretreated for 5min in 70% (v/v) ethanol solution, and surface disinfected for 5min in 0.1% mercuric chloride solution and rinsed 3-4 times in sterile distilled water. Single explants was placed on various strengths, full (MS + KIN or BAP), half (

1/2 MS +

KIN or BAP) and one quarter (1/4 MS + KIN or BAP) of MS

(Murashige and Skoog) medium supplemented with different levels (0, 1, 2, 3, 4 and 5mg/l) of kinetin (Kin) or benzyl-amino purine (BAP). The pH of all media was adjusted to 5.8±0.1 prior to the addition of 8g agar and dispensing (10ml) into culture tubes (test tubes) and then autoclaving. All cultures were maintained in the culture room at 28± 1

oC and 80% relative humidity with 16-h

photo period under white fluorescent light for shoot initiation for 12 weeks. Subsequently, one quarter strength growth medium with 3mg/l of kinetin (Kin) and 4mg/l of benzyl amino purine (BAP), which gave the best growth performance was used to study, the effects of culture passage (frequency of subculture, 0, 2, 4, 6, and 8 times, subcultured every week) on establishment of the species. Similarly, the effects of disinfection time of 0, 1, 2, 3, 4, 5, and 6min with 0.1% mercuric chloride solution on in vitro establishment of the species were examined.

Regeneration frequency (%), coefficient of velocity of bud burst and contamination (%) were examined. Each treatment was replicated 10 times, repeated 2 times and the mean value expressed. Standard error of the mean values were calculated for replicate readings and data were subjected to analysis of variance (ANOVA), where the differences in the means were tested using the least significant difference (LSD) at 0.05 level of probability (Obi, 2002; Wahua, 1999). RESULT The regeneration frequency of I. wombolu in the quarter strength medium (

1/4 MS + KIN or BAP) was significantly

(P < 0.05) higher than those grown in the half (1/2 MS +

KIN or BAP) and full strength (MS + KIN or BAP) (TABLE 1). There were relative variations in regeneration frequency of the species among the various treatments, viz; control – 0 (MS without plant growth regulators, full (MS + KIN or BAP), half (

1/2 MS + KIN or BAP), and one

quarter (1/4 MS + KIN or BAP) strength media. Full (MS +

KIN or BAP) did not support shoot initiation of explants of I. wombolu. The regeneration frequency increased with increase in concentration of plant growth regulators, with kinetin treatment having the highest growth response than those obtained with benzyl- amino purine. In addition, one quarter strength media (

1/4 MS) with 3mg/l

kinetin (KIN3) and 4mg/l benzyl-amino purine (BAP4) supported best shoot initiation (Table 1). The coefficient of velocity of bud burst of I. wombolu gave significant (P < 0.05) increase over that of the control at various treatment levels. Similarly, the coefficient of velocity of bud burst increased with increase in the concentration of plant growth regulators with one quarter (1/4 MS + KIN or BAP) strength media giving the highest

response than those of full (MS + KIN or BAP) and half (1/2 MS + KIN or BAP) (Table 2).

Regeneration frequency significantly (P < 0.05) decreased with increase in the number of culture passage. A culture passage of 2 times a week was optimum for regeneration frequency of I. wombolu explants with KIN3 and BAP4 recording 65.00% and 60.00% regeneration frequency, respectively. Treatments with 2, 4, and 6 number of culture passages per week gave significant increases (P < 0.05) in regeneration frequency over that of the control (Table 3). The disinfection time of 5 min with a regeneration frequency of 65.00% for both KIN3 and BAP4 was optimum for sterilization of the explants of the species. The regeneration frequency of I. wombolu significantly (P < 0.05) increased with increase in duration of disinfection time of 0.1% mercuric chloride, except at 6 min where there were reductions in treatments with KIN3 and BAP4. The values of regeneration frequency were significantly (P < 0.05) lower than that of the control treatment at all duration of disinfection time (Table 4). The lowest percentage contamination of 15 was recorded at both 5


World Res. J. Agric. Sci. 008 Table 1. Regeneration frequency (%) of Irvingia wombolu in various strengths of murashige and skoog media (ms) at varying concentrations of plant growth regulators (PGR: Kinetin-kin, Benzyl-amino Purine -bap)

Concentration of 0 1 2 3 4 5 PGR (mg/l) Growth medium

MS +KIN 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 MS + BAP 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 0.00 ± 0.00 1/2 MS+KIN 10.00 ± 0.21 15.00 ± 0.23 20.00 ± 0.42 30.00 ± 0.28 25.00 ± 0.39 20.00 ± 0.28

1/2 MS+BAP 10.00 ± 0.34 20.00 ± 0.47 20.00 ± 0.14 25.00 ± 0.63 30.00 ± 0.41 25.00 ± 0.36

1/4 MS+KIN 20.00 ± 0.13 45.00 ± 0.54 45.00 ± 0.21 65.00 ± 0.43 50.00 ± 0.63 40.00 ± 0.42

1/4 MS+ BAP 20.00 ± 0.32 40.00 ± 0.45 40.00 ± 0.17 55.00 ± 0.34 60.00 ± 0.42 50.00 ± 0.21

Mean 10.00 20.00 20.83 29.17 27.50 22.50 LSD (P< 0.05) 0.21 0.32 0.24 0.16 0.20 0.27

*Mean value ± standard error of 10 replicates from two determinations

Table 2. Coefficient of velocity (bud burst) of Irvingia wombolu in various strengths of murashige and skoog media (ms) at varying concentrations of plant growth regulators (PGR: Kinetin-kin, Benzyl-amino Purine -bap)

Concentration of 0 1 2 3 4 5 PGR (mg/l) Growth medium

MS +KIN 0.14 ± 0.02 0.16 ± 0.04 0.16 ± 0.01 0.17 ± 0.02 0.19 ± 0.06 0.20 ± 0.01 MS +BAP 0.14 ± 0.03 0.15 ± 0.06 0.17 ± 0.02 0.18 ± 0.04 0.20 ± 0.07 0.21 ± 0.03 1/2 MS+KIN 0.18 ± 0.01 0.19 ± 0.03 0.19 ± 0.04 0.20 ± 0.08 0.21 ± 0.09 0.22 ± 0.08

1/2 MS+BAP 0.18 ± 0.04 0.19 ± 0.07 0.20 ± 0.08 0.21 ± 0.03 0.23 ± 0.02 0.21 ± 0.06

1/4 MS+KIN 0.19 ± 0.03 0.21 ± 0.04 0.22 ± 0.03 0.29 ± 0.04 0.29 ± 0.03 0.30 ± 0.02

1/4 MS+BAP 0.19 ± 0.02 0.20 ± 0.06 0.20 ± 0.04 0.24 ± 0.02 0.27 ± 0.07 0.29 ± 0.01

Mean 0.17 0.18 0.19 0.22 0.23 0.24 LSD (P< 0.05) 0.01 0.03 0.06 0.04 0.01 0.02


Table 3. Effects of culture passage on regeneration frequency–RF (%) of Irvingia wombolu sub-cultured ON 1/4 MS with 3mg/l Kinetin (KIN3) and 4mg/l Benzyl-amino Purine (BAP4)

Concentration of KIN3 BAP4 PGR (mg/l) Number of Sub-culture

0 30.00 ± 0.33 25.00 ± 0.18 2 65.00 ± 0.47 60.00 ± 0.26 4 55.00 ± 0.34 55.00 ± 1.07 6 55.00 ± 1.21 50.00 ± 1.03 8 35.00 ± 1.06 30.00 ± 1.19 Mean 40.00 36.67 LSD (P< 0.05) 2.42 2.71


and 6 min of disinfection time (Table 4). The disinfection time of 5 min was, however, optimum in terms of reduced contamination and best regeneration frequency for establishment of the species (Table 4 and 5).

DISCUSSION The marked differences in response of explants of I. wombolu to the various growth media may be attributed


Etukudo et al. 009 Table 4. Effects of disinfection time of 0.1% Mercuric Chloride solution on regeneration frequency–RF (%) of Irvingia wombolu on 1/4 MS with 3mg/l Kinetin (kin3) and 4mg/l Benzyl-amino Purine (BAP4)

0 1 2 3 4 5 6 Time (Min) Concentration of PGR (mg/l)

KIN3 0.00 ± 0.00 25.00 ± 1.02 35.00 ± 0.97 45.00 ± 1.34 55.00 ± 1.28 65.00 ± 0.75 40.00 ± 0.32 BAP4 0.00 ± 0.00 20.00 ± 1.07 45.00 ± 0.54 40.00 ± 1.20 50.00 ± 1.23 65.00 ± 1.32 45.00 ± 0.56 Mean 0.00 22.50 40.00 42.50 52.50 65.00 42.50 LSD (P< 0.05) 0.00 1.17 1.03 1.02 1.12 2.04 2.01


Table 5. Effects of disinfection time 0f 0.1% mercuric chloride solution on percentage contamination (%) of Irvingia wombolu on 1/4 MS with 3mg/l Kinetin (KIN3) and 4mg/l Benzyl-amino Purine (BAP4)

0 1 2 3 4 5 6 Time (Min) Concentration of PGR (mg/l)

KIN3 100.00 ± 0.00 60.00 ± 1.02 50.00 ± 0.97 45.00 ± 1.34 25.00 ± 1.28 15.00 ± 0.75 15.00 ± 0.32 BAP4 100.00 ± 0.00 65.00 ± 1.07 45.00 ± 0.54 40.00 ± 1.20 30.00 ± 1.23 15.00 ± 1.32 15.00 ± 0.56 Mean 100.00 62.50 47.50 42.50 27.50 15.00 15.00 LSD (P< 0.05) 2.02 2.37 2.21 2.07 2.02 2.14 2.13


to differences in the concentration of the constituents of the full, half and one quarter strength growth media. Different mineral salt formulations and different concentrations of the minerals with full, half or one quarter strength media have been shown to bring about different responses in vitro. According to Larraburu et al. (2013), variations in growth responses in vitro could be attributed to the different chemical and osmotic compositions of the media and individual species requirement. Different concentrations of basal media have been shown to influence explants proliferation in vitro (Mitic et al., 2012; Mukhtar et al., 2012). In addition, the levels of types of carbohydrate and reduced nitrogen supplements in the medium can also influence morphogenesis (Ellis et al., 1991; Swartz et al., 1990; Stefano and Rosario, 2003). In this study, the rate of contamination of culture decreased with increase in the duration of disinfection time. Contamination has been shown to be a problem with explants from juvenile trees (Cassells and O’Herlily, 2003), although contamination of sprout explants is less than with the mature branches (Warrag et al., 1990; Etukudo et al., 2010). Enclosed buds and young explants were, however, used to reduced contamination rate and achieve shoot initiation at an optimum duration of disinfection time as shown in this study. Similarly, the

appropriate sterilization agent coupled with disinfection time must be worked out as shown in this study. The most effective way of preventing microbial contamination in vitro is elimination of bacteria from initial plant explants that are introduced into the culture (Mihaljavic et al., 2013). This is because one of the obvious requirements for effective establishment of explants in vitro is that the explants material be thoroughly disinfected in order to prevent contamination and loss of culture (Webster et al., 2003). Necrosis, which caused inhibited growth and death of explants, most especially, in full strength medium, may be attributed to oxidation of phenolic compounds released from the cut end of I. wombolu explants. This problem was, however, regulated by the use of ascorbic acids and regular transfer to fresh medium. Necrosis has been reported to occur in some tissue culture, especially woody plants, and is generally considered to result from the oxidation of phenolic compounds in the explants (Bell and Reed, 2002; Kefeli et al., 2003). This results further corroborates the work of Ozygit et al. (2007) and Etukudo et al. (2010) that woody plants, especially angiosperms, often secrete substances into the medium in response to wounding. In addition, these polyphenols and tannins inhibit development of explants and often cause necrosis. Again, the oxidized products of these phenols, such as


World Res. J. Agric. Sci. 010 quinines have been shown to inhibit enzymes activity leading to the death of explants. The rate of plant regeneration in vitro has been shown to be dependent on genotype, explants type and culture medium composition (Anie and Subba, 2010; Groseva et al., 2012). The overall regeneration process from the starting material (explants) may be greatly influenced by the physiological state of the explants, age of the explants and in vitro environment (both the light and temperature regimes) and the constitution of the medium with the hormone concentrations in particular (Bhalla- Sarin et al., 2003; Sharma et al., 2005). The best growth performance of I. wombolu explants at 3mg/l kinetin (KIN3) and 4mg/l benzyl-amino purine (BAP4) has revealed the effective balance of phytohormones and culture medium used in the regeneration process. CONCLUSION Regulation of in vitro conditions for best growth of Irvingia wombolu explants requires effective manipulation of condition in and outside the culture medium as shown in this study. ACKNOWLEDGEMENT I acknowledge the support from Prof. Ani Nkang and other members of Staff of Botany Department, University of Calabar. I appreciate very highly Prof. A. E. Brisibe of Genetics and Biotechnology Department, University of Calabar, Cross River State, Nigeria, and Mr. Oyalade Kehinde for providing the chemicals, materials and tissue culture base for the work. REFERENCES Abrie AL, Van Staden J. (2001). Micropropagation of the

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Accepted 05 September, 2014. Citation: Etukudo MM, Roberts EMI, Ilesanmi OB (2014). Some regulation of In Vitro growth of bush Mango- Irvingia wombolu Mildbr (Irvingiaceae). World Research Journal of Agricultural Sciences 1(2): 007-011.

Copyright: © 2014 Etukudo et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are cited.

some regulation of in vitro growth of bush mango- irvingia wombolu mildbr (irvingiaceae)

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