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Research Article

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J Plant Biotechnol (2024) 51:055-062

Published online March 7, 2024

https://doi.org/10.5010/JPB.2024.51.006.055

© The Korean Society of Plant Biotechnology

Best practices for initiation of banana and plantain (Musa spp.) cultures

Fatma H. Kiruwa ･Emanuel Epathra Mlinga ･Agatha Amnaay Aloyce ･Mpoki Mathew Shimwela

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Tanzania Agricultural Research Institute Tengeru Sub Center, P. O. Box 1253, Arusha, Tanzania
Nelson Mandela African Institution of Science and Technology, Department of Sustainable Agriculture, Biodiversity and Ecosystem Management, P.O. Box 447, Arusha, Tanzania
Tanzania Agricultural Research Institute Maruku Sub Center, Bukoba, Tanzania

Correspondence to : e-mail: fatma.kiruwa@tari.go.tz

Received: 6 December 2023; Revised: 24 January 2024; Accepted: 24 January 2024; Published: 7 March 2024.

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

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Banana is a staple food and income crop worldwide. Here, we identified the best practices for the initiation of banana (Musa spp.) cultures. The cultivars studied included East African highland bananas (Mshare/Mchare and Nshakara) and plantains (Mzuzu), which are highly demanded by farmers. The following factors were assessed: sterilization techniques for rainy and dry seasons, explant size, effect of benzylaminopurine (BAP) concentration on regeneration of cultures, effect of ascorbic acid on culture browning, and culturing conditions. We found that treatment with ethanol alone (70%) or in combination with sodium hypochlorite (1%) yielded a relatively higher proportion (≥ 96%) of aseptic culture establishment in both rainy and dry seasons. Explant size affected the survival and regeneration of cultures where small explants (< 10 mm) like meristem dome showed relatively slower re-growth and high mortality in contrast to large explants (≥ 10 mm). Moreover, culturing in BAP-supplemented Murashige and Skoog medium (MS) significantly enhanced the greening/shoot regeneration percentage at 14 d. However, there was no significant increase in number of green shoots/culture regeneration at BAP concentrations of 5 mg/l, 6 mg/l, and 8 mg/l for Nshakara and 3 mg/l, 5 mg/l, and 6 mg/l for Mzuzu. Browning decreased by 18% when explants were pre-treated with 100 mg/l ascorbic acid for 10 min. Further, growth conditions such as light were a relatively greater determinant for regeneration of initiated banana cultures. Therefore, our findings suggest the use of appropriate sterilization techniques, explant size, growth regulators, and conditions to ensure sufficient production of planting materials.

Keywords Browning, Culture contamination, East African highland banana, Tissue culture, Sterilization

Introduction

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Banana (including plantain; Musa spp.) is a staple food and cash crop to many people worldwide (Chabi et al. 2018). The crop provides food security, nutrition and income for many smallholder farmers in East and Central African countries, including Tanzania, Rwanda, the Democratic Republic of the Congo, Burundi, Kenya, and Uganda. (Kilimo Trust 2012). In Tanzania, it is massively grown in North-western, Southern highlands, and Northern regions. The value attached to bananas is so high in some areas - e.g., North-western Tanzania where any shortage of bananas, even when there are plenty of other staple foods, is considered famine. However, its production has declined over the past decades due to pests and diseases, and other underlined factors causing low yield. Major pests and diseases include nematodes, weevils, banana Xanthomonas wilt (BXW), Fusarium wilt and a recently reported quarantine Banana bunchy top disease (Shimwela et al. 2022; Swennen et al. 2013). The pests and diseases pressure has been identified to be a cumulative constrain due to the use of poor quality planting materials. Farmers usually rely on the naturally produced suckers for the supply of planting materials, which are contaminated by pests and diseases. Consequently, the productivity and lifespan of banana plantations have been drastically reduced (Lefranc et al. 2008). In Tanzania, potential yields are 70 MT/hectare but banana farmers harvest less than 6 MT/hectare annually. Several studies have shown tissue culture to be the best solution to propagate disease

free quality seeds in a large scale within a short period (Anis and Ahmad 2016; Shahzad et al. 2017; Singh 2015). The technique specifically for banana, involves various steps such as selection of explants, sterilization, initiation, shoot proliferation, and rooting. Each step requires best handling practices to produce quality planting materials.

The use of field grown plants as a direct source of explant for the propagation of in vitro plantlets presents major challenges (Webster et al. 2003). Microbial contaminations are the major hurdle to initiating and maintaining viable in vitro cultures. Explant contamination occurs due to several plant and environmental related factors such as plant species, age of the plant, explant source, and prevailing weather conditions (Webster et al. 2003). Despite the best timing and selection efforts it is challenging to eliminate contamination from in vitro grown plants. Some contaminants are in latent form in mother stocks which requires indexing to prevent the spread of pathogens to plantlets and hence, disease free planting materials to farmers. Losses due to contamination in in-vitro condition range from 3-15% at every subculture in the majority of commercial and scientific plant tissue culture laboratories (Leifert et al. 1994). The rate may be even higher in the initiation phase and is assumed to vary based on seasons, the majority of which is caused by fungal, yeast, and bacterial contaminants. The best season for explants collection and initiation needs to be established to minimize losses.

Various sterilization procedures have been proposed by several researchers, sodium hypochlorite and ethanol being the most commonly used disinfectants for surface sterilization of banana explants (Ali and Mehmood 2017; Keshari et al. 2016; Reza et al. 2013). Some other investigators have replaced sodium hypochlorite (NaOCl) with low concentration of mercuric chloride (Goswami and Handique 2013). These disinfectants are toxic to plant tissues, hence proper concentration, duration of exposure and the sequences of use have to be standardized to minimize injury to the explant to improve survival rate. Two chemicals i.e. sodium hypochlorite (0.5% and 1.0%) and ethanol (70%) were used for this study to standardize the best sterilization protocol for in-vitro propagation of East African Highland Bananas (Mshare/Mchare, Nshakara) and Plantains (Mzuzu) cultivars in dry and wet seasons. The three cultivars selected in this study are highly demanded by communities and have shown difficulty in propagation, especially in the initial stages (initiation phase). Therefore, the present study aims to improve their propagation at the initiation phase to ensure a sufficient supply of seedlings. Moreover, the study used disease indexing techniques to identify contaminants (i.e viruses) at early stages from mother stocks. Apart from the assessment of different sterilization techniques and their effects based on seasonal variations, other practices in the initiation of cultures were studied. These included appropriate sucker sizes to be used as explants, ways to decrease culture browning and increase survival rates as well as best growth hormone concentrations for initiation of cultures.

Materials and Methods

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Plant materials and study site

Banana suckers from Mshare, Mzuzu (Plantain) and Nshakara (Matooke) cultivars were collected from Arusha, Mbeya and Kagera-Bukoba respectively during dry and rainy seasons in 2021. They were one to three months old sword suckers which were detached from the healthy parent plants. The work was conducted at Tanzania Agricultural Research Institute (TARI) Tengeru Laboratories, Arusha, Tanzania.

Concentration of disinfectants vs exposure time and season of culture

Banana suckers of approximately 30-100 cm were washed with soap under running tap water to remove soil and debris. In addition, suckers of ≥ 100 cm were used to assess whether size affects performance. In the laboratory, the outer leaves were peeled off until the explants were 5 cm in height and 3 cm at the base diameter. These explants were surface-sterilized with the following seven treatments to find out the best sterilization concentration and exposure time; -

  • T1=70% ethanol for 10 min,

  • T2=0.5 Sodium hypochlorite for 15 min,

  • T3=0.5% Sodium hypochlorite for 20 min,

  • T4=1.0% Sodium hypochlorite for 15 min,

  • T5=1.0% Sodium hypochlorite for 20 min,

  • T6=Combination of 0.5% Sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min, and

  • T7=Combination of 1.0% Sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min

In each treatment, a few drops of Tween-20 were added during sterilization, and explants were shaken continuously for uniform sterilization. During sterilization, explants were washed with sterile distilled water thrice. Further trimming was carried out under aseptic environment (in a laminar flow hood) to the required size of 1.5 cm diameter. The explants were cultured in sterilized full-strength MS (Murashige and Skoog 1962) medium with vitamins containing 5 mg/l BAP, 20 mg/l ascorbic acid, and solidified with 2.5 gL-1 phytagel. Explants were cultured on the medium in such a way that the explant’s rhizome was embedded in the medium. Each treatment was conducted with 25-30 explants cultured in MS medium stored in constant conditions (light 16 hours, dark 8 hours, and temperature 25 degrees celsius). The number of contaminated and healthy cultures was recorded within 21 days of culture. The experiment was set similarly for both explants collected during rainy and dry seasons to find the best sterilization technique(s) for cultures based on seasons.

Assessment of ascorbic acid and light on browning of cultures at the initiation stage

Nshakara (Matooke) and Mzuzu (plantain) are known banana cultivars for producing phenolic compounds that cause the browning of cultures resulting in nutrient uptake inhibition. In this study, ascorbic acid at different concentrations was evaluated. After trimming and sterilization of explants under aseptic conditions, they were pre-treated/soaked in ascorbic acid in different treatments for 10 minutes and then cultured in MS medium containing all necessary requirements including 20 mg/l ascorbic acid. All other conditions were kept constant. Number of explants initiated, days taken for browning, and number of browning cultures were recorded. Treatments were; -

  • PT1=Sterile dH2O (used as control without ascorbic acid) exposed for 10 min

  • PT2=Ascorbic acid (50 mg/l) exposed for 10 min

  • PT3=Ascorbic acid (100 mg/l) exposed for 10 min

As well culture condition was evaluated i.e light vs dark in the initiation stage to assess whether light has an effect in the browning of cultures. Treatments were; - T1 = light 16 hrs, dark 8hrs, T2 = dark 24 hrs. These were evaluated for 8 weeks (2 months) where the number and days of browning of cultures and greening were recorded weekly.

Assessment of growth hormone in initiation of cultures

Sterilization procedures for banana explants were carried out under aseptic conditions with 1% sodium hypochlorite for 20 min. The explants were rinsed, trimmed, and cultured in full strength MS medium containing different concentrations of BAP as follows; - 0 mg/l, 3 mg/l, 5 mg/l, 6 mg/l, 8 mg/l. Each treatment was conducted with 25 explants incubated on constant conditions (light/dark and temperature 25 degrees Celsius). The number of explants initiated, and days taken for greening of explants were recorded.

Results and Discussion

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The study presents a full package for the initiation of East African highland bananas (Mshare/Mchare and Nshakara), and Plantains (Mzuzu) including the best sterilization method for effective reduction of losses due to contaminants, ways to reduce browning, appropriate season for explants collection, size of explants to be collected and cultured as well as BAP concentrations per cultivars that can be used during initiation of cultures.

Assessment of sterilization techniques and season of the culture

The present study was conducted to standardize the best sterilization protocol for in vitro propagation of Nshakara, Mzuzu and Mshare. Two different chemicals i.e. sodium hypochlorite (0.5% and 1.0%) and ethanol (70%) were used with treatment duration of 15 and 20 minutes for sodium hypochlorite and 10 minutes for ethanol respectively. The study revealed significant differences in contamination of cultures for both seasons indicating the influence of seasons in contamination of cultures. The rainy season was observed to have a higher contamination of cultures as compared to the dry season. In both seasons, treatments of disinfectants showed significant differences in the elimination of contaminants as shown in Table 1. The treatment combination T7 followed by T1 were found to be the best combination with regard to achieving the highest percentage of contamination free healthy culture in both rainy and dry season.

Table 1 . Effect of disinfectant treatments on seasonal contamination percentage

TreatmentsDry season Mean contamination (%)Rainy season Mean contamination (%)
T117.33cd15.00b
T236.87a40.42a
T330.77ab30.92a
T425.13abc41.58a
T523.90abc41.29a
T622.86bc32.15a
T77.08d12.7b
LSD 0.0512.1611.68
CV (%)29.1921.47

Mean values with the same letter indicate no significant difference. LSD, least significant difference; CV, coefficient of variation; T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min.

During rainy season (Fig. 1), treatment T1, T3 and T7 were found to be the best combination in Mshare with regards to achieving a higher percentage of healthy cultures of above 90%. Whereas in case of Mzuzu and Nshakara, treatment combination T7 followed by T1 for Mzuzu and T1 and T7 followed by T6 for Nshakara respectively gave the highest percentage of healthy cultures following the disinfection procedure described in the materials and methods section. In the dry season (Fig. 2) treatments T5 and T7 gave higher survival rates of cultures in Mshare cultivar while treatments T6 and T7 were the best in Nshakara and Mzuzu followed by T1.

Fig. 1. Effect of surface sterilization on the survival of cultures during rainy season. T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min

Fig. 2. Effect of surface sterilization on the survival of cultures during dry season. T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min

The findings revealed that ethanol alone (70%) or in combination with 1% sodium hypochlorite gives a higher percentage of aseptic culture establishment in in-vitro conditions which is in agreement with several other studies (Jalil et al. 2003; Rahman et al. 2002). However, for cost reasons, still 0.5% or 1% of sodium hypochlorite at high exposure time (20 min) can minimize rate of contamination in a significant amount in Mshare and Nshakara cultivars. A similar finding revealed sodium hypochlorite alone or in combination to have a better effect in controlling contaminants without adverse effects on explants even for a long duration (Goswami and Handique 2013). Apart from using disinfectants, the use of antibiotics or plant preservatives is advised since could drastically reduce contamination rates when in cooperated in initiation media (Nandwani et al. 2000; Wakil and Mbah 2012). Their use in cultures needs to be optimized as excessive use tends to have a negative effect on the growth and proliferation of cultures.

In relation to contamination, the study observed that big sized suckers (above 1 meter) have high rates of contamination compared to when 30 cm to 1 meter suckers are used. Possibilities for accumulation of pests are higher in big suckers as compared to small suckers. Moreover, big sized suckers involved too much work when it comes to the preparation of explants and were found to have traces of pests specifically weevils and burrowing nematodes which provides room for deeper penetration of other pathogens/contaminants to the suckers.

According to various research, the size of explants to be cultured influences survival rates and culture proliferation (Al-Amin et al. 2009; Lassois et al. 2013; Shankar et al. 2014; Youssef et al. 2010). The use of various sizes depends on the purpose of the culture. If the purpose is to eradicate bacteria and viruses, a meristematic dome surrounded by one or two primordia is the best option. As for normal propagation, the size of the explant to be used varies (1 cm or even more). Meristem dome culture leads to slower regrowth and high mortality following rapid explant dehydration, which is not the case for large explants. On the other hand, large explants can have lateral buds that provide shoots for multiplication. However, the risk of loss by contamination and browning is higher with large explants, leading to a final survival rate even lower than when meristem culture is used. Hence, the optimal size must be taken into account for both survival and proliferation of cultures.

Daungban et al. (2017) investigated the effect of explant division by cutting when culturing in a media and found that cutting the explants in 4 sections resulted in an increased number of shoots of up to 11.03 new shoots per explant in the third culture cycle. Moreover, the number of shoots per container increased to 48 and 132 in the second and third cycles, respectively, or 2.08 and 5.74 times more than the number of shoots per container in the first cycle. This shows that not only size of the explant affects the survival and regeneration of cultures but explant division matters.

Effect of ascorbic acid and light on browning of cultures at the initiation stage

Since Matooke is known to be affected by phenolic compounds excreted during wounding causing browning of cultures, the study investigated the effect of ascorbic acid pretreatments and light for in vitro propagation of Nshakara. Different concentrations; (0 mg/l as a control, 50 mg/l and 100 mg/l) of ascorbic acid were tested to prevent the browning of explants after surface sterilization. It was observed that browning of cultures decreased with an increase in ascorbic acid concentration (Fig. 3). Ascorbic acid at the rate of 100 mg/l exposed for 10 min gave the best results with regard to less percentage (38%) and number of days for initial browning of explants. Similar results were observed when ascorbic acid prevented the development of lethal browning in banana (Ko et al. 2009; Ngomuo et al. 2014). Even with ascorbic acid pretreatment and incorporation into media, explants transfer to fresh media should be considered after 2 weeks for matooke cultivars to 3 weeks depending on the level of browning per genotype. Ngomuo et al. (2014) reported a lower survival rate of explants due to lethal browning at 3rd and 4th week of cultures. Cystene, Potassium Citrate and Citrate have also been reported as an alternative in controlling lethal browning which have adverse effects on explants nutrients uptake (Onuoha et al. 2011; Titov et al. 2006).

Fig. 3. Effect of ascorbic acid treatment on the browning of cultures at the initiation stage. PT1, sterile distilled water exposed for 10 min; PT2, ascorbic acid (50 mg/l) exposed for 10 min; PT3, ascorbic acid (100 mg/l) exposed for 10 min

As for the light treatments, cultures incubated at 24 hrs of darkness for 14 days showed less browning as compared to treatments with alternate light (16 hrs) and dark (8 hrs) conditions (Fig. 4). Amente and Chimdessa (2021) reported the same with regards to excessive browning of cultures aided by light conditions. Light enhances the oxidation of phenolic compounds more quickly than when initiated explants are cultured in dark conditions. However, early greening of cultures was observed in cultures with alternate light and dark conditions. Whether multiplication rates of explants exposed in alternate light and dark or complete darkness during the first days of initiation differ has not been investigated in this study.

Fig. 4. Effect of culture conditions and ascorbic acid treatment on browning and regeneration of cultures. (A) Explants exposed to dark treatment show relatively less browning with light-green shoot tips. (B, C) Explants exposed to light treatment show excessive browning with a greenish shoot tip. (D, E) Explant treated with ascorbic acid. (F) Lethal browning of cultures in the absence of ascorbic acid treatment

Effect of growth hormone on initiation of cultures

During the present course of investigation (Table 2) revealed that MS medium supplemented with growth hormone (BAP) significantly enhanced the percentage of greening/regeneration of cultures at 14 days. When basal medium was used alone without BAP, number of green shoots/regeneration of cultures was very minimal in Mzuzu (8%) as compared to Nshakara cultivar (38%). It was also observed that the number of green shoots/regeneration of cultures increased with an increase of BAP concentration. Similar findings were reported by Jafari et al. (2011) in a study of the effect of (BAP) pulsing on in vitro shoot multiplication of banana. Other studies have reported a combination BAP and auxin in low concentration in cultures to improve regrowth and proliferation (Hossain et al. 2016; Rehana et al. 2013; Sipen and Davey 2012)

Table 2 . Effect of benzylaminopurine (BAP) at different concentrations on regeneration of cultures of Nshakara and Mzuzu

BAP concentration (mg/l)NshakaraMzuzu
Initial number of culturesNumber of green shoots at 14 dGreening at 14 d (%)Initial number of culturesNumber of green shoots at 14 dGreening at 14 d (%)
0249382428
3181267251144
5181583251144
6191684251144
825228816850

The study revealed no significant increase in the number of green shoots/ culture regeneration at concentrations 3 mg/l, 5 mg/l and 6 mg/l for Mzuzu, and 5 mg/l, 6 mg/l and 8 mg/l for Nshakara. This entails that either of the concentrations can be used but the lowest optimal concentration is recommended per studied cultivars. The findings also revealed that the optimal concentration of BAP varies with genotypes similar to other studies. Setyowati and Kesumawati (2022) showed 3 mg/l of BAP had the biggest response in revival of Barangan Merah banana cultivar while Ngomuo et al. (2013) reported an optimum BAP concentration for Yangambi cultivar to be 6 mg/l.

Unlike cost reasons for hormones, it should be noted that when BAP is used in high concentrations can result in higher chances of genetic variability such as somaclonal variations as a result of gene mutation or changes in epigenetic marks (Krishna et al. 2016; Sato et al. 2011). The occurrence of subtle somaclonal variation is a drawback for in vitro cloning. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage to produce true-to-type plant materials. Unlike growth regulators, studies have revealed other sources of mutation in tissue culture which are caused by numerous stress factors including wounding;- exposure to sterilants during sterilization, tissue being incomplete (protoplasts as an extreme example), imbalances of media components such as sugar from the nutrient medium as a replacement of photosynthesis in the leaves, lighting conditions and the disturbed relationship between high humidity and transpiration (Joyce et al. 2003; Smulders and de Klerk 2011).

The magnitude of stress that cause variability in tissue culture depends several factors including i) tissue culture technique used i.e production of plants via axillary branching does not normally result in the production of variants, while cultures that go through a callus phase are the ones that theoretically promote a higher mutation rate (Zayova et al. 2010), ii) length of culture period and number of subculture cycles more than 8 subcultures increases somaclonal variants with a simultaneous decrease in the multiplication rate of propagules in banana (Khan et al. 2011), iii) explant source; highly differentiated tissues such as roots, leaves, and stems generally produce more variations than explants with pre-existing meristems, such as axillary buds and shoot tips (Duncan 1997), iv) culture environment; external factors like growth regulators, temperature, light, osmolality and agitation rate of the culture medium are known to influence the cell cycle in vivo in plants considerably which indicates that inadequate control of cell cycle in vitro is one of the causes of somaclonal variation (Nwauzoma and Jaja 2013) and lastly plant genotype is probably the most important determinant of variation (Nwauzoma and Jaja 2013; Tican et al. 2008).

With these notes, appropriate growth conditions, sterilants used and source of explants should be put into consideration to ensure disease free and true-to-type planting materials are produced. In general, to have a healthy and proliferated culture requires proper and close monitoring of lab tidiness, adhering to lab guidelines (wearing masks when working on lamina flow hood, sterilizing each and everything used for culturing including towels, beakers, and forceps to mention a few). Moreover, there is a need to determine and improve the multiplication rates of the studied cultivars to ensure a sufficient supply of planting material to farmers.

Acknowledgement

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The team acknowledges the Government of Tanzania for its continuous support, BMGF through Mennonite Economic Development Associates (MEDA) for the grant, tissue culture companies (Maua Mazuri Tissue Culture laboratory and Crop biosciences solution) and TARI team members for their cooperation.

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Article

Research Article

J Plant Biotechnol 2024; 51(1): 55-62

Published online March 7, 2024 https://doi.org/10.5010/JPB.2024.51.006.055

Copyright © The Korean Society of Plant Biotechnology.

Best practices for initiation of banana and plantain (Musa spp.) cultures

Fatma H. Kiruwa ･Emanuel Epathra Mlinga ･Agatha Amnaay Aloyce ･Mpoki Mathew Shimwela

Tanzania Agricultural Research Institute Tengeru Sub Center, P. O. Box 1253, Arusha, Tanzania
Nelson Mandela African Institution of Science and Technology, Department of Sustainable Agriculture, Biodiversity and Ecosystem Management, P.O. Box 447, Arusha, Tanzania
Tanzania Agricultural Research Institute Maruku Sub Center, Bukoba, Tanzania

Correspondence to:e-mail: fatma.kiruwa@tari.go.tz

Received: 6 December 2023; Revised: 24 January 2024; Accepted: 24 January 2024; Published: 7 March 2024.

This is an Open-Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Abstract

Banana is a staple food and income crop worldwide. Here, we identified the best practices for the initiation of banana (Musa spp.) cultures. The cultivars studied included East African highland bananas (Mshare/Mchare and Nshakara) and plantains (Mzuzu), which are highly demanded by farmers. The following factors were assessed: sterilization techniques for rainy and dry seasons, explant size, effect of benzylaminopurine (BAP) concentration on regeneration of cultures, effect of ascorbic acid on culture browning, and culturing conditions. We found that treatment with ethanol alone (70%) or in combination with sodium hypochlorite (1%) yielded a relatively higher proportion (≥ 96%) of aseptic culture establishment in both rainy and dry seasons. Explant size affected the survival and regeneration of cultures where small explants (< 10 mm) like meristem dome showed relatively slower re-growth and high mortality in contrast to large explants (≥ 10 mm). Moreover, culturing in BAP-supplemented Murashige and Skoog medium (MS) significantly enhanced the greening/shoot regeneration percentage at 14 d. However, there was no significant increase in number of green shoots/culture regeneration at BAP concentrations of 5 mg/l, 6 mg/l, and 8 mg/l for Nshakara and 3 mg/l, 5 mg/l, and 6 mg/l for Mzuzu. Browning decreased by 18% when explants were pre-treated with 100 mg/l ascorbic acid for 10 min. Further, growth conditions such as light were a relatively greater determinant for regeneration of initiated banana cultures. Therefore, our findings suggest the use of appropriate sterilization techniques, explant size, growth regulators, and conditions to ensure sufficient production of planting materials.

Keywords: Browning, Culture contamination, East African highland banana, Tissue culture, Sterilization

Introduction

Banana (including plantain; Musa spp.) is a staple food and cash crop to many people worldwide (Chabi et al. 2018). The crop provides food security, nutrition and income for many smallholder farmers in East and Central African countries, including Tanzania, Rwanda, the Democratic Republic of the Congo, Burundi, Kenya, and Uganda. (Kilimo Trust 2012). In Tanzania, it is massively grown in North-western, Southern highlands, and Northern regions. The value attached to bananas is so high in some areas - e.g., North-western Tanzania where any shortage of bananas, even when there are plenty of other staple foods, is considered famine. However, its production has declined over the past decades due to pests and diseases, and other underlined factors causing low yield. Major pests and diseases include nematodes, weevils, banana Xanthomonas wilt (BXW), Fusarium wilt and a recently reported quarantine Banana bunchy top disease (Shimwela et al. 2022; Swennen et al. 2013). The pests and diseases pressure has been identified to be a cumulative constrain due to the use of poor quality planting materials. Farmers usually rely on the naturally produced suckers for the supply of planting materials, which are contaminated by pests and diseases. Consequently, the productivity and lifespan of banana plantations have been drastically reduced (Lefranc et al. 2008). In Tanzania, potential yields are 70 MT/hectare but banana farmers harvest less than 6 MT/hectare annually. Several studies have shown tissue culture to be the best solution to propagate disease

free quality seeds in a large scale within a short period (Anis and Ahmad 2016; Shahzad et al. 2017; Singh 2015). The technique specifically for banana, involves various steps such as selection of explants, sterilization, initiation, shoot proliferation, and rooting. Each step requires best handling practices to produce quality planting materials.

The use of field grown plants as a direct source of explant for the propagation of in vitro plantlets presents major challenges (Webster et al. 2003). Microbial contaminations are the major hurdle to initiating and maintaining viable in vitro cultures. Explant contamination occurs due to several plant and environmental related factors such as plant species, age of the plant, explant source, and prevailing weather conditions (Webster et al. 2003). Despite the best timing and selection efforts it is challenging to eliminate contamination from in vitro grown plants. Some contaminants are in latent form in mother stocks which requires indexing to prevent the spread of pathogens to plantlets and hence, disease free planting materials to farmers. Losses due to contamination in in-vitro condition range from 3-15% at every subculture in the majority of commercial and scientific plant tissue culture laboratories (Leifert et al. 1994). The rate may be even higher in the initiation phase and is assumed to vary based on seasons, the majority of which is caused by fungal, yeast, and bacterial contaminants. The best season for explants collection and initiation needs to be established to minimize losses.

Various sterilization procedures have been proposed by several researchers, sodium hypochlorite and ethanol being the most commonly used disinfectants for surface sterilization of banana explants (Ali and Mehmood 2017; Keshari et al. 2016; Reza et al. 2013). Some other investigators have replaced sodium hypochlorite (NaOCl) with low concentration of mercuric chloride (Goswami and Handique 2013). These disinfectants are toxic to plant tissues, hence proper concentration, duration of exposure and the sequences of use have to be standardized to minimize injury to the explant to improve survival rate. Two chemicals i.e. sodium hypochlorite (0.5% and 1.0%) and ethanol (70%) were used for this study to standardize the best sterilization protocol for in-vitro propagation of East African Highland Bananas (Mshare/Mchare, Nshakara) and Plantains (Mzuzu) cultivars in dry and wet seasons. The three cultivars selected in this study are highly demanded by communities and have shown difficulty in propagation, especially in the initial stages (initiation phase). Therefore, the present study aims to improve their propagation at the initiation phase to ensure a sufficient supply of seedlings. Moreover, the study used disease indexing techniques to identify contaminants (i.e viruses) at early stages from mother stocks. Apart from the assessment of different sterilization techniques and their effects based on seasonal variations, other practices in the initiation of cultures were studied. These included appropriate sucker sizes to be used as explants, ways to decrease culture browning and increase survival rates as well as best growth hormone concentrations for initiation of cultures.

Materials and Methods

Plant materials and study site

Banana suckers from Mshare, Mzuzu (Plantain) and Nshakara (Matooke) cultivars were collected from Arusha, Mbeya and Kagera-Bukoba respectively during dry and rainy seasons in 2021. They were one to three months old sword suckers which were detached from the healthy parent plants. The work was conducted at Tanzania Agricultural Research Institute (TARI) Tengeru Laboratories, Arusha, Tanzania.

Concentration of disinfectants vs exposure time and season of culture

Banana suckers of approximately 30-100 cm were washed with soap under running tap water to remove soil and debris. In addition, suckers of ≥ 100 cm were used to assess whether size affects performance. In the laboratory, the outer leaves were peeled off until the explants were 5 cm in height and 3 cm at the base diameter. These explants were surface-sterilized with the following seven treatments to find out the best sterilization concentration and exposure time; -

  • T1=70% ethanol for 10 min,

  • T2=0.5 Sodium hypochlorite for 15 min,

  • T3=0.5% Sodium hypochlorite for 20 min,

  • T4=1.0% Sodium hypochlorite for 15 min,

  • T5=1.0% Sodium hypochlorite for 20 min,

  • T6=Combination of 0.5% Sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min, and

  • T7=Combination of 1.0% Sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min

In each treatment, a few drops of Tween-20 were added during sterilization, and explants were shaken continuously for uniform sterilization. During sterilization, explants were washed with sterile distilled water thrice. Further trimming was carried out under aseptic environment (in a laminar flow hood) to the required size of 1.5 cm diameter. The explants were cultured in sterilized full-strength MS (Murashige and Skoog 1962) medium with vitamins containing 5 mg/l BAP, 20 mg/l ascorbic acid, and solidified with 2.5 gL-1 phytagel. Explants were cultured on the medium in such a way that the explant’s rhizome was embedded in the medium. Each treatment was conducted with 25-30 explants cultured in MS medium stored in constant conditions (light 16 hours, dark 8 hours, and temperature 25 degrees celsius). The number of contaminated and healthy cultures was recorded within 21 days of culture. The experiment was set similarly for both explants collected during rainy and dry seasons to find the best sterilization technique(s) for cultures based on seasons.

Assessment of ascorbic acid and light on browning of cultures at the initiation stage

Nshakara (Matooke) and Mzuzu (plantain) are known banana cultivars for producing phenolic compounds that cause the browning of cultures resulting in nutrient uptake inhibition. In this study, ascorbic acid at different concentrations was evaluated. After trimming and sterilization of explants under aseptic conditions, they were pre-treated/soaked in ascorbic acid in different treatments for 10 minutes and then cultured in MS medium containing all necessary requirements including 20 mg/l ascorbic acid. All other conditions were kept constant. Number of explants initiated, days taken for browning, and number of browning cultures were recorded. Treatments were; -

  • PT1=Sterile dH2O (used as control without ascorbic acid) exposed for 10 min

  • PT2=Ascorbic acid (50 mg/l) exposed for 10 min

  • PT3=Ascorbic acid (100 mg/l) exposed for 10 min

As well culture condition was evaluated i.e light vs dark in the initiation stage to assess whether light has an effect in the browning of cultures. Treatments were; - T1 = light 16 hrs, dark 8hrs, T2 = dark 24 hrs. These were evaluated for 8 weeks (2 months) where the number and days of browning of cultures and greening were recorded weekly.

Assessment of growth hormone in initiation of cultures

Sterilization procedures for banana explants were carried out under aseptic conditions with 1% sodium hypochlorite for 20 min. The explants were rinsed, trimmed, and cultured in full strength MS medium containing different concentrations of BAP as follows; - 0 mg/l, 3 mg/l, 5 mg/l, 6 mg/l, 8 mg/l. Each treatment was conducted with 25 explants incubated on constant conditions (light/dark and temperature 25 degrees Celsius). The number of explants initiated, and days taken for greening of explants were recorded.

Results and Discussion

The study presents a full package for the initiation of East African highland bananas (Mshare/Mchare and Nshakara), and Plantains (Mzuzu) including the best sterilization method for effective reduction of losses due to contaminants, ways to reduce browning, appropriate season for explants collection, size of explants to be collected and cultured as well as BAP concentrations per cultivars that can be used during initiation of cultures.

Assessment of sterilization techniques and season of the culture

The present study was conducted to standardize the best sterilization protocol for in vitro propagation of Nshakara, Mzuzu and Mshare. Two different chemicals i.e. sodium hypochlorite (0.5% and 1.0%) and ethanol (70%) were used with treatment duration of 15 and 20 minutes for sodium hypochlorite and 10 minutes for ethanol respectively. The study revealed significant differences in contamination of cultures for both seasons indicating the influence of seasons in contamination of cultures. The rainy season was observed to have a higher contamination of cultures as compared to the dry season. In both seasons, treatments of disinfectants showed significant differences in the elimination of contaminants as shown in Table 1. The treatment combination T7 followed by T1 were found to be the best combination with regard to achieving the highest percentage of contamination free healthy culture in both rainy and dry season.

Table 1 . Effect of disinfectant treatments on seasonal contamination percentage.

TreatmentsDry season Mean contamination (%)Rainy season Mean contamination (%)
T117.33cd15.00b
T236.87a40.42a
T330.77ab30.92a
T425.13abc41.58a
T523.90abc41.29a
T622.86bc32.15a
T77.08d12.7b
LSD 0.0512.1611.68
CV (%)29.1921.47

Mean values with the same letter indicate no significant difference. LSD, least significant difference; CV, coefficient of variation; T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min..

During rainy season (Fig. 1), treatment T1, T3 and T7 were found to be the best combination in Mshare with regards to achieving a higher percentage of healthy cultures of above 90%. Whereas in case of Mzuzu and Nshakara, treatment combination T7 followed by T1 for Mzuzu and T1 and T7 followed by T6 for Nshakara respectively gave the highest percentage of healthy cultures following the disinfection procedure described in the materials and methods section. In the dry season (Fig. 2) treatments T5 and T7 gave higher survival rates of cultures in Mshare cultivar while treatments T6 and T7 were the best in Nshakara and Mzuzu followed by T1.

Figure 1. Effect of surface sterilization on the survival of cultures during rainy season. T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min

Figure 2. Effect of surface sterilization on the survival of cultures during dry season. T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min

The findings revealed that ethanol alone (70%) or in combination with 1% sodium hypochlorite gives a higher percentage of aseptic culture establishment in in-vitro conditions which is in agreement with several other studies (Jalil et al. 2003; Rahman et al. 2002). However, for cost reasons, still 0.5% or 1% of sodium hypochlorite at high exposure time (20 min) can minimize rate of contamination in a significant amount in Mshare and Nshakara cultivars. A similar finding revealed sodium hypochlorite alone or in combination to have a better effect in controlling contaminants without adverse effects on explants even for a long duration (Goswami and Handique 2013). Apart from using disinfectants, the use of antibiotics or plant preservatives is advised since could drastically reduce contamination rates when in cooperated in initiation media (Nandwani et al. 2000; Wakil and Mbah 2012). Their use in cultures needs to be optimized as excessive use tends to have a negative effect on the growth and proliferation of cultures.

In relation to contamination, the study observed that big sized suckers (above 1 meter) have high rates of contamination compared to when 30 cm to 1 meter suckers are used. Possibilities for accumulation of pests are higher in big suckers as compared to small suckers. Moreover, big sized suckers involved too much work when it comes to the preparation of explants and were found to have traces of pests specifically weevils and burrowing nematodes which provides room for deeper penetration of other pathogens/contaminants to the suckers.

According to various research, the size of explants to be cultured influences survival rates and culture proliferation (Al-Amin et al. 2009; Lassois et al. 2013; Shankar et al. 2014; Youssef et al. 2010). The use of various sizes depends on the purpose of the culture. If the purpose is to eradicate bacteria and viruses, a meristematic dome surrounded by one or two primordia is the best option. As for normal propagation, the size of the explant to be used varies (1 cm or even more). Meristem dome culture leads to slower regrowth and high mortality following rapid explant dehydration, which is not the case for large explants. On the other hand, large explants can have lateral buds that provide shoots for multiplication. However, the risk of loss by contamination and browning is higher with large explants, leading to a final survival rate even lower than when meristem culture is used. Hence, the optimal size must be taken into account for both survival and proliferation of cultures.

Daungban et al. (2017) investigated the effect of explant division by cutting when culturing in a media and found that cutting the explants in 4 sections resulted in an increased number of shoots of up to 11.03 new shoots per explant in the third culture cycle. Moreover, the number of shoots per container increased to 48 and 132 in the second and third cycles, respectively, or 2.08 and 5.74 times more than the number of shoots per container in the first cycle. This shows that not only size of the explant affects the survival and regeneration of cultures but explant division matters.

Effect of ascorbic acid and light on browning of cultures at the initiation stage

Since Matooke is known to be affected by phenolic compounds excreted during wounding causing browning of cultures, the study investigated the effect of ascorbic acid pretreatments and light for in vitro propagation of Nshakara. Different concentrations; (0 mg/l as a control, 50 mg/l and 100 mg/l) of ascorbic acid were tested to prevent the browning of explants after surface sterilization. It was observed that browning of cultures decreased with an increase in ascorbic acid concentration (Fig. 3). Ascorbic acid at the rate of 100 mg/l exposed for 10 min gave the best results with regard to less percentage (38%) and number of days for initial browning of explants. Similar results were observed when ascorbic acid prevented the development of lethal browning in banana (Ko et al. 2009; Ngomuo et al. 2014). Even with ascorbic acid pretreatment and incorporation into media, explants transfer to fresh media should be considered after 2 weeks for matooke cultivars to 3 weeks depending on the level of browning per genotype. Ngomuo et al. (2014) reported a lower survival rate of explants due to lethal browning at 3rd and 4th week of cultures. Cystene, Potassium Citrate and Citrate have also been reported as an alternative in controlling lethal browning which have adverse effects on explants nutrients uptake (Onuoha et al. 2011; Titov et al. 2006).

Figure 3. Effect of ascorbic acid treatment on the browning of cultures at the initiation stage. PT1, sterile distilled water exposed for 10 min; PT2, ascorbic acid (50 mg/l) exposed for 10 min; PT3, ascorbic acid (100 mg/l) exposed for 10 min

As for the light treatments, cultures incubated at 24 hrs of darkness for 14 days showed less browning as compared to treatments with alternate light (16 hrs) and dark (8 hrs) conditions (Fig. 4). Amente and Chimdessa (2021) reported the same with regards to excessive browning of cultures aided by light conditions. Light enhances the oxidation of phenolic compounds more quickly than when initiated explants are cultured in dark conditions. However, early greening of cultures was observed in cultures with alternate light and dark conditions. Whether multiplication rates of explants exposed in alternate light and dark or complete darkness during the first days of initiation differ has not been investigated in this study.

Figure 4. Effect of culture conditions and ascorbic acid treatment on browning and regeneration of cultures. (A) Explants exposed to dark treatment show relatively less browning with light-green shoot tips. (B, C) Explants exposed to light treatment show excessive browning with a greenish shoot tip. (D, E) Explant treated with ascorbic acid. (F) Lethal browning of cultures in the absence of ascorbic acid treatment

Effect of growth hormone on initiation of cultures

During the present course of investigation (Table 2) revealed that MS medium supplemented with growth hormone (BAP) significantly enhanced the percentage of greening/regeneration of cultures at 14 days. When basal medium was used alone without BAP, number of green shoots/regeneration of cultures was very minimal in Mzuzu (8%) as compared to Nshakara cultivar (38%). It was also observed that the number of green shoots/regeneration of cultures increased with an increase of BAP concentration. Similar findings were reported by Jafari et al. (2011) in a study of the effect of (BAP) pulsing on in vitro shoot multiplication of banana. Other studies have reported a combination BAP and auxin in low concentration in cultures to improve regrowth and proliferation (Hossain et al. 2016; Rehana et al. 2013; Sipen and Davey 2012)

Table 2 . Effect of benzylaminopurine (BAP) at different concentrations on regeneration of cultures of Nshakara and Mzuzu.

BAP concentration (mg/l)NshakaraMzuzu
Initial number of culturesNumber of green shoots at 14 dGreening at 14 d (%)Initial number of culturesNumber of green shoots at 14 dGreening at 14 d (%)
0249382428
3181267251144
5181583251144
6191684251144
825228816850

The study revealed no significant increase in the number of green shoots/ culture regeneration at concentrations 3 mg/l, 5 mg/l and 6 mg/l for Mzuzu, and 5 mg/l, 6 mg/l and 8 mg/l for Nshakara. This entails that either of the concentrations can be used but the lowest optimal concentration is recommended per studied cultivars. The findings also revealed that the optimal concentration of BAP varies with genotypes similar to other studies. Setyowati and Kesumawati (2022) showed 3 mg/l of BAP had the biggest response in revival of Barangan Merah banana cultivar while Ngomuo et al. (2013) reported an optimum BAP concentration for Yangambi cultivar to be 6 mg/l.

Unlike cost reasons for hormones, it should be noted that when BAP is used in high concentrations can result in higher chances of genetic variability such as somaclonal variations as a result of gene mutation or changes in epigenetic marks (Krishna et al. 2016; Sato et al. 2011). The occurrence of subtle somaclonal variation is a drawback for in vitro cloning. Therefore, it is of immense significance to assure the genetic uniformity of in vitro raised plants at an early stage to produce true-to-type plant materials. Unlike growth regulators, studies have revealed other sources of mutation in tissue culture which are caused by numerous stress factors including wounding;- exposure to sterilants during sterilization, tissue being incomplete (protoplasts as an extreme example), imbalances of media components such as sugar from the nutrient medium as a replacement of photosynthesis in the leaves, lighting conditions and the disturbed relationship between high humidity and transpiration (Joyce et al. 2003; Smulders and de Klerk 2011).

The magnitude of stress that cause variability in tissue culture depends several factors including i) tissue culture technique used i.e production of plants via axillary branching does not normally result in the production of variants, while cultures that go through a callus phase are the ones that theoretically promote a higher mutation rate (Zayova et al. 2010), ii) length of culture period and number of subculture cycles more than 8 subcultures increases somaclonal variants with a simultaneous decrease in the multiplication rate of propagules in banana (Khan et al. 2011), iii) explant source; highly differentiated tissues such as roots, leaves, and stems generally produce more variations than explants with pre-existing meristems, such as axillary buds and shoot tips (Duncan 1997), iv) culture environment; external factors like growth regulators, temperature, light, osmolality and agitation rate of the culture medium are known to influence the cell cycle in vivo in plants considerably which indicates that inadequate control of cell cycle in vitro is one of the causes of somaclonal variation (Nwauzoma and Jaja 2013) and lastly plant genotype is probably the most important determinant of variation (Nwauzoma and Jaja 2013; Tican et al. 2008).

With these notes, appropriate growth conditions, sterilants used and source of explants should be put into consideration to ensure disease free and true-to-type planting materials are produced. In general, to have a healthy and proliferated culture requires proper and close monitoring of lab tidiness, adhering to lab guidelines (wearing masks when working on lamina flow hood, sterilizing each and everything used for culturing including towels, beakers, and forceps to mention a few). Moreover, there is a need to determine and improve the multiplication rates of the studied cultivars to ensure a sufficient supply of planting material to farmers.

Acknowledgement

The team acknowledges the Government of Tanzania for its continuous support, BMGF through Mennonite Economic Development Associates (MEDA) for the grant, tissue culture companies (Maua Mazuri Tissue Culture laboratory and Crop biosciences solution) and TARI team members for their cooperation.

Fig 1.

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Figure 1.Effect of surface sterilization on the survival of cultures during rainy season. T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min
Journal of Plant Biotechnology 2024; 51: 55-62https://doi.org/10.5010/JPB.2024.51.006.055

Fig 2.

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Figure 2.Effect of surface sterilization on the survival of cultures during dry season. T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min
Journal of Plant Biotechnology 2024; 51: 55-62https://doi.org/10.5010/JPB.2024.51.006.055

Fig 3.

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Figure 3.Effect of ascorbic acid treatment on the browning of cultures at the initiation stage. PT1, sterile distilled water exposed for 10 min; PT2, ascorbic acid (50 mg/l) exposed for 10 min; PT3, ascorbic acid (100 mg/l) exposed for 10 min
Journal of Plant Biotechnology 2024; 51: 55-62https://doi.org/10.5010/JPB.2024.51.006.055

Fig 4.

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Figure 4.Effect of culture conditions and ascorbic acid treatment on browning and regeneration of cultures. (A) Explants exposed to dark treatment show relatively less browning with light-green shoot tips. (B, C) Explants exposed to light treatment show excessive browning with a greenish shoot tip. (D, E) Explant treated with ascorbic acid. (F) Lethal browning of cultures in the absence of ascorbic acid treatment
Journal of Plant Biotechnology 2024; 51: 55-62https://doi.org/10.5010/JPB.2024.51.006.055

Table 1 . Effect of disinfectant treatments on seasonal contamination percentage.

TreatmentsDry season Mean contamination (%)Rainy season Mean contamination (%)
T117.33cd15.00b
T236.87a40.42a
T330.77ab30.92a
T425.13abc41.58a
T523.90abc41.29a
T622.86bc32.15a
T77.08d12.7b
LSD 0.0512.1611.68
CV (%)29.1921.47

Mean values with the same letter indicate no significant difference. LSD, least significant difference; CV, coefficient of variation; T1, 70% ethanol for 10 min; T2, 0.5 sodium hypochlorite for 15 min; T3, 0.5% sodium hypochlorite for 20 min; T4, 1.0% sodium hypochlorite for 15 min; T5, 1.0% sodium hypochlorite for 20 min; T6, combination of 0.5% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min; T7, combination of 1.0% sodium hypochlorite for 15 min followed by trimming then 70% ethanol for 10 min..

Table 2 . Effect of benzylaminopurine (BAP) at different concentrations on regeneration of cultures of Nshakara and Mzuzu.

BAP concentration (mg/l)NshakaraMzuzu
Initial number of culturesNumber of green shoots at 14 dGreening at 14 d (%)Initial number of culturesNumber of green shoots at 14 dGreening at 14 d (%)
0249382428
3181267251144
5181583251144
6191684251144
825228816850

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