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European Journal of Applied Sciences – Vol. 11, No. 4

Publication Date: August 25, 2023

DOI:10.14738/aivp.114.15147

Levai, L. D., Ngone, M. A., Monono, E. Y., Ngale, J. E., Nsimi, A., Yaya, F. V., Oumar, D., Achidi, E., & Bechem, E. T. (2023). Effect of

Photoperiod and Ascorbic Acid Concentration on Degree of Browning, Shoot Regeneration and Survival of Plantain (Musa ssp.)

Cultivars Growing in vitro. European Journal of Applied Sciences, Vol - 11(4). 175-185.

Services for Science and Education – United Kingdom

Effect of Photoperiod and Ascorbic Acid Concentration on Degree

of Browning, Shoot Regeneration and Survival of Plantain (Musa

ssp.) Cultivars Growing in vitro

Lewis D. Levai

JP Johnson Biotechnology Laboratory, Institute of Agricultural

Research for Development (IRAD) Ekona, PMB 25 Buea, Cameroon

Mercy A. Ngone

Plant Biotechnology Group, Faculty of Agriculture and

Veterinary Medicine, University of Buea, PO Box 63 Buea, Cameroon

Ekwa Y. Monono

JP Johnson Biotechnology Laboratory, Institute of Agricultural

Research for Development (IRAD) Ekona, PMB 25 Buea, Cameroon

and Department of Plant Science, University of Buea, P.O. Box 63

Buea, Cameroon

Jemimah E. Ngale

JP Johnson Biotechnology Laboratory, Institute of Agricultural

Research for Development (IRAD) Ekona, PMB 25 Buea, Cameroon

Armand Nsimi

Center for Research on Oil Palm (CEREPAH), IRAD La Dibamba,

Cameroon

Forkwa Victorine Yaya

International Potato Center, (CIP), PO Box 2008 Messa, Yaoundé,

Cameroon

Doungous Oumar

JP Johnson Biotechnology Laboratory, Institute of Agricultural

Research for Development (IRAD) Ekona, PMB 25 Buea, Cameroon

Eric Achidi

Department of Biochemistry and Molecular Biology,

University of Buea, PO Box 63 Buea, Cameroon

Eneke T. Bechem

Department of Biochemistry and Molecular Biology,

University of Buea, PO Box 63 Buea, Cameroon

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Services for Science and Education – United Kingdom 176

European Journal of Applied Sciences (EJAS) Vol. 11, Issue 4, August-2023

ABSTRACT

Plantain, an important crop in Sub-Saharan Africa, faces challenges such as low

fertility and susceptibility to stresses. Micropropagation techniques offer disease- free planting materials, but success depends on various factors. This study explored

the effects of ascorbic acid (AA) concentration and photoperiod on browning, shoot

regeneration, and survival of explants in in vitro culture. Three plantain cultivars

and four AA concentrations were examined, with explants subjected to darkness

and a light/dark cycle. Browning, shoots/buds per explant, shoot height, and

survival were measured. Results showed that darkness reduced browning

compared to light, particularly in the French Clair and Batard cultivars.

Preservation of AA in darkness prevented browning by inhibiting its decay and

reducing enzyme activity related to phenolic compounds. Photoperiod did not

significantly affect shoot characteristics or survival, while AA concentration had a

significant impact on shoot regeneration. The Batard cultivar exhibited highest

regeneration with 50mg/L AA, while higher concentrations had a negative effect.

Therefore, incubating explants in darkness effectively reduces browning during in

vitro culture. Optimal AA concentration (50mg/L) enhances shoot regeneration,

while higher concentrations should be avoided. These findings have implications

for improving micropropagation techniques, addressing plantain production

challenges, and meeting the demand for high-quality planting materials in Sub- Saharan Africa.

Keywords: Plantain, micropropagation, browning, ascorbic acid, shoot regeneration.

INTRODUCTION

Plantain (Musa spp.) is an important food crop in Sub-Sahara Africa, providing approximately

25% of the food energy needed for people in the region [1]. It is considered a valuable source

of nutrients and serves as a cash crop for rural farmers in Cameroon, contributing to both food

security and economic sustainability [2]. However, plantain production faces challenges such

as low reproductive fertility, slow propagation rate, and susceptibility to biotic and abiotic

stresses [3].

Micropropagation techniques have been developed to overcome these challenges by providing

healthy and disease-free planting materials [4]. However, the success of micropropagation is

influenced by various factors, including explant type, nutrients, plant growth regulators,

additives, temperature, light intensity, and duration [5].

During in vitro culture, plantain explants are prone to browning caused by the oxidation of

phenolic compounds [6]. Browning reactions can lead to high mortality rates and hinder

successful shoot regeneration [7]. Phenolic compounds, such as catechin, chlorogenic acid, and

tannins, are abundant in plant tissues and are produced as a defence mechanism against injury

[8, 9]. The oxidation of these compounds results in tissue browning and the production of toxic

quinones, which negatively affect explant survival and growth [10].

To mitigate browning and promote successful in vitro culture, various approaches have been

employed, including the use of antioxidants like ascorbic acid and pre-treatment of explants

with substances such as potassium citrate and citrate [7, 11]. Activated charcoal has also been

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177

Levai, L. D., Ngone, M. A., Monono, E. Y., Ngale, J. E., Nsimi, A., Yaya, F. V., Oumar, D., Achidi, E., & Bechem, E. T. (2023). Effect of Photoperiod and

Ascorbic Acid Concentration on Degree of Browning, Shoot Regeneration and Survival of Plantain (Musa ssp.) Cultivars Growing in vitro. European

Journal of Applied Sciences, Vol - 11(4). 175-185.

URL: http://dx.doi.org/10.14738/aivp.114.15147

utilized to control browning by adsorbing inhibitory substances, although it can affect the

balance of medium components and influence plant regeneration [12].

Light intensity and duration are critical environmental factors in tissue culture that influence

shoot regeneration, growth, and metabolite biosynthesis [13, 14]. Understanding the impact of

light on phenolic oxidation and the efficacy of plant growth regulators is crucial for successful

in vitro culture [5, 15].

This study aims to investigate the effect of ascorbic acid concentration and photoperiod on

browning, shoot regeneration, and contamination of plantain explants during in vitro culture.

By understanding these factors, improvements can be made to micropropagation techniques,

addressing the challenges associated with plantain production and ensuring a steady supply of

high-quality planting materials in Sub-Sahara Africa.

MATERIALS AND METHODS

Study Location

The experiment took place at the JP Johnson Biotechnology Laboratory, located in the Institute

of Agricultural Research for Development (IRAD) in Ekona, Cameroon (4°16'44'' N and

9°17'50'' E) in the South West Region [16, 17].

Explant Preparation

Sword suckers from three cultivars (Ebanga, Batard, and French Clair) were collected from the

collection plot at the IRAD Ekona Station. The suckers were carefully trimmed to remove roots

and debris and transported to the laboratory's screen-house on the same day. After washing

under running tap water, the suckers were further trimmed to have approximately five leaves

and a base measuring about 5cm x 5cm x 5cm, resulting in explants. These explants were

washed again under running water and then surface sterilized with 70% ethanol [2, 18].

Sterilization

In the laboratory, the explants were trimmed to a size of 3cm x 3cm x 3cm and rinsed with

distilled water. Surface disinfection was performed by briefly flashing the explants with 70%

alcohol for 1 minute, followed by three rinses with sterile distilled water (SDW). Subsequently,

the explants were soaked in a disinfectant solution (30% sodium hypochlorite (NaOCl) mixed

with two drops of Tween 20) for 20 minutes, and then rinsed three times with SDW. In the

laminar flow hood, the explants were further trimmed to a size of approximately 1.5cm x 1.5cm

x 1.5cm before inoculating them on the culture media [19](Figure 1). While in the laminar flow

hood, the explants were submerged in 1L of the disinfectant for 10 minutes. They were then

rinsed in three changes of SDW before initiation [6, 20].

Culture Conditions and Media

The explants were cultured in jars containing 40 mL of Murashige and Skoog (MS) Basal

Medium [21] supplemented with 30 g/L sucrose, 4 mg/L 6-benzylaminopurine (BAP), 0.8 mg/L

indole-3-acetic acid (IAA), 2 g/L Gelrite, and 1 g/L ceftriaxone. The MS medium and all other

components, except ceftriaxone, were weighed into a 1L glass bottle containing 700 mL of

distilled water on a magnetic stirrer. After adding all the components, the water was topped up

to the 1L mark, and the content was preheated while stirring and brought to a boil. The bottle