Research Article

In vitro Culture of Dendrocalamus asper Bamboo in Liquid and Semi-Solid MS Media

Wan Nurfarzana Wan Mohamad Zani1, Norrizah Jaafar Sidik1*, Asmah Awal2, Nurul Izzati Osman3, Lyena Watty Zuraine Ahmad1 and Mohd Khairi Nordin4

1Department of Biology, Faculty of Applied Science, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia; 2Agricultural Biotechnology Research Group, Faculty of Plantation and Agrotechnology, Universiti Teknologi MARA (UiTM) Jasin Campus, Merlimau, Malacca, Malaysia; 3Faculty of Pharmacy, Universiti Teknologi MARA (UiTM) Puncak Alam Campus, Bandar Puncak Alam, Selangor, Malaysia; 4School of Electrical Engineering, College of Engineering, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia.

Received | August 23, 2024; Accepted | September 28, 2024; Published | October 28, 2024

*Correspondence | Norrizah Jaafar Sidik, Department of Biology, Faculty of Applied Science, Universiti Teknologi MARA (UiTM) Shah Alam, Selangor, Malaysia; Email: norri536@uitm.edu.my

Citation | Zani, W.N.W.M., N.J. Sidik, A. Awal, N.I. Osman, L.W.Z. Ahmad and M.K. Nordin. 2024. In vitro culture of Dendrocalamus asper bamboo in liquid and semi-solid MS media. Sarhad Journal of Agriculture, 40(4): 1304-1311.

DOI | https://dx.doi.org/10.17582/journal.sja/2024/40.4.1304.1311

Keywords | Bamboo, Dendrocalamus asper, in vitro culture, Liquid media, Semi-solid MS media

Copyright: 2024 by the authors. Licensee ResearchersLinks Ltd, England, UK.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).

Introduction

Bamboo, a member of the grass family Poaceae, encompasses over 1,600 species with a predominantly tropical and subtropical distribution (Soreng et al., 2017). The economic viability of this plant is relatively high in the global forestry economy due to its silvicultural and morphological features, making it an efficient and environmentally friendly substitute for wood (Gonçalves et al., 2023; Admas, 2024). Apart from that, this plant could sequester more CO2 and lock the carbon source in its fibre and soil where it grows compared to other trees, which eventually could help in mitigating global warming issues (Lou et al., 2010; Patel et al., 2015). Bamboo can also help in reducing soil erosion by having extensive rhizomes and roots which bind to the soil, and the presence of its evergreen canopy and leaf litter helps in intercepting rain as well as reducing its impact on the ground (Kuehl et al., 2011).

One of the prominent tropical bamboo species that is commonly planted in Southeast Asia countries such as Malaysia, Thailand, Indonesia, Vietnam, and the Philippines is Dendrocalamus asper (Mustafa et al., 2021). Its economic significance is multifaceted. It is valued for its edible shoots that are consumed as food, and its mature culms that have been used in furniture, construction, biofuel and handicraft industries (Hartono et al., 2022). Due to this, the demand for bamboo continues to rise, necessitates for efficient and sustainable propagation methods.

Conventional methods such as propagation from rhizomes or culm cuttings are often time-consuming, labour-intensive and have low survival rates (Singh et al., 2013). Propagation using seeds is unreliable since the flowering is irregular and in a long cycle, plus the seeds are mostly sterile and have a short viability period, further hindering propagation efforts (Sandhu et al., 2017). In order to overcome these challenges to fulfil the demand, micropropagation techniques offer a compelling solution to address the limitations of conventional propagation methods. This in vitro approach enables rapid and controlled multiplication of high-quality D. asper plantlets.

Previous studies have been conducted to develop proper protocols for micropropagation of D. asper. During this stage, the frequency of shoot growth is significantly influenced by the physical status of the media, either liquid or semi-solid (Sandhu et al., 2017). In semi-solid media, often solidifying agent such as agar and Gelrite are added, aiming to provide three-dimensional support for the growing shoots and allow for nutrient uptake (Raju et al., 2023). Liquid culture systems, on the other hand, eliminate the need for a solidifying agent and provide a more homogenous distribution of nutrients and growth factors (Sandhu et al., 2017). However, the cons of this system include the lack of physical support for the growing shoots, as well as the risk of hyperhydricity due to continuous immersion in the liquid medium (Polivanova and Bedarev, 2022). Interestingly, despite the possible risk of hyperhydricity, study in the past recorded that culture of some bamboo species in liquid media resulted in better growth compared to semi-solid media (Arshad et al., 2005; Ogita et al., 2008; Rathore et al., 2009). Other than this, multiplication in vitro is also influenced by the number of shoot propagules used during subsequent subculturing stage. During this stage, the shoot clusters were divided into smaller clumps consists of three to ten shoots (Jimenéz et al., 2021). By adopting this method, higher multiplication rate was achieved (Jiménez et al., 2006; Ornellas et al., 2019), but optimum number of shoot clusters that support the highest multiplication rate might vary between different bamboo species.

Hence, in this study, we aim to investigate the growth of D. asper bamboo species in both liquid and semi-solid MS media, along with optimization of number of shoot propagules during multiplication stage. The growth patterns were evaluated and documented over a specified period of time to determine the most effective medium and optimum number of propagules for bamboo micropropagation.

Materials and Methods

Establishment of initial culture

In vitro plantlets of D. asper were being utilized as initial explants, in which the nodal segments without axillary buds were cut into 1.0 – 1.5 cm in size. These nodes were cultured in Murashige and Skoog (MS) medium (Duchefa Biochemies, The Netherlands) with an addition of 100 mgL-1 myoinositol (Duchefa Biochemies, The Netherlands), 30 gL-1 sucrose (Systerm, Malaysia) and 3 gL-1 Gelrite (Sigma, St. Louis, USA). Plant growth regulators (PGRs) were added, 4 mgL-1 6-benzylaminopurine (BAP) and 0.5 mgL-1 indole-3-butyric acid (IBA) (Duchefa Biochemies, The Netherlands) for culture initiation. The cultures were maintained under 16-hour photoperiod under white cool fluorescent lights (Philips, China) in the culture room with the temperature of 27±2℃. After 6 weeks, the shoots were subcultured at different number of propagules for multiplication of shoot.

Optimization of propagules sizes for shoot multiplication

To study the impact of propagules sizes towards shoot multiplication, group of 3-, 4- and 5- shoots were transferred to multiplication medium having the same hormone concentrations from previous culture, which consisted of solidified MS medium with addition of 4.0 mgL-1 BAP and 0.5 mgL-1 IBA hormone. Observation was recorded for 5 weeks period for number of shoots and multiplication rate.

Effect of semi-solid and liquid culture on shoot multiplication

To determine the effect of different culture media towards the growth of explants, propagules containing 3 shoots each were transferred in liquid (without addition of Gelrite) and semi-solid (addition of 3 gL-1 Gelrite) media supplemented with different concentrations of BAP (0.5, 1.0, 2.0 and 4.0 mg L-1). For liquid media, sterile filter papers were used to support the explants, preventing them from fully soaked in the media. Observations for number and length of shoots were recorded after 4 weeks of culture.

Effect of semi-solid and liquid culture on rooting induction

Propagules of three to five shoots were transferred to both semi-solid and liquid MS media supplemented with different IBA concentrations (1.0 – 5.0 mg L-1) for rooting. During this stage, sterile filter paper was not used as support in the liquid media. Observations for rooting response, number and length of shoots were recorded after 5 weeks of culture.

Media preparation

MS media in the present study were prepared at 4.4 g L-1 supplemented with 30 g L-1 sucrose and 100 mg L-1 myoinositol. Solidified MS medium was prepared by adding 3 gL-1 Gelrite. The pH of the media was adjusted to 5.6–5.8 prior to autoclaving at 121℃ at 0.1 kPa for 20 min. All the cultures were maintained in the culture room at temperature of 27±2℃ with 16hr photoperiod under cool white, fluorescent light.

Pre-hardening and acclimatization

The rooted plantlets were then removed from the culture jars and washed thoroughly under running tap water to remove medium traces from the roots. Plantlets were transferred to root trainers filled with mixture of autoclaved vermicompost and vermiculite at 1:1 ratio and maintained in culture room for 2 weeks. Afterwards, the plantlets were acclimatized in mixture of different substrates: soil, cocopeat and compost (1:1:1) for a month in the greenhouse.

Statistical analysis

All collected data were subjected to statistical analysis for Analysis of Variance (ANOVA) at p < 0.05 and standard error using IPM SPSS statistical software Version 28.0.

Results and Discussion

Effect of propagule sizes on shoot multiplication

Propagules of three shoots resulted in the highest multiplication rate with 4.9-fold and shoot number (average 14.8) after 5 weeks of culture (Table 1). As the number of propagules increased, the multiplication rate decreased over time, though the number of shoots produced from each propagule did not differ significantly (p < 0.05). In this study, single or group of 2-shoots were not being utilized since most of them did not manage to multiply and produce new shoots over time. In addition, they showed some browning effects and most of the leaves turned brown (result was not shown).

 

Table 1: The number of shoots and shoot multiplication rate obtained from different sizes of propagules cultured in MS + 4.0 mg L-1 BAP + 0.5 mg L-1 IBA.

Size of propagules used	2 weeks		3 weeks		5 weeks
	No of shoots	Multiplication rate	No of shoots	Multiplication rate	No of shoots	Multiplication rate
Three shoots	6.8 ± 0.2a	2.3	11.0 ± 2.1a	3.7	14.8 ± 3.9a	4.9
Four shoots	6.2 ± 0.7a	1.5	12.0 ± 1.5a	3.0	14.0 ± 1.7a	3.5
Five shoots	8.0 ± 1.0a	1.6	9.5 ± 0.9a	1.9	13.0 ± 1.6a	2.6

Values = Mean ± SE.

 

Previous study on the same species revealed that propagules with 8 shoots cultured in MS medium fortified with 10 µM BAP and 75 µM adenine sulphate (Ads) produced the highest shoot multiplication fold at 3.9 (Singh et al., 2012a). However, most of the studies in the past had utilized propagules of 3 shoots, maximum was 4 shoots, during shoot multiplication stage in different bamboo species such as P. stocksii (Sanjaya et al., 2005), D. strictus (Pandey and Singh, 2012), B. balcooa, B. bambos, D. stocksii and G. angustifolia (Rathore et al., 2009). Our study showed a decrease in multiplication rate as bigger propagule sizes (more shoots) were being utilized. This finding was in concordance with those reported by previous study in which propagules with more than 3 shoots displayed a decline in multiplication rate after 4 weeks in D. asper (Arya et al., 1999). The same authors also concluded that three-shoot propagules were the most suitable propagule size for shoot multiplication.

 

Effect of the state of culture medium during shoot multiplication and rooting

During shoot multiplication, clusters of three shoots were transferred into semi-solid and liquid MS media containing different concentrations of BAP hormone. After four weeks, the results displayed that the means of length and number of shoots obtained from the explants cultured in liquid media were overall higher compared to semi-solid media. The overall highest number of shoots was obtained from the liquid culture supplemented with 1.0 and 4.0 mg L-1 BAP with an average of 3.5 shoots per explant (Figure 1). The same culture supplemented with 0.5 mg L-1 BAP in a liquid medium resulted in the highest length of shoots (3.09 ± 0.19 cm) compared to other treatments (Figure 2). Meanwhile, in a semi-solid medium, the number of shoots obtained in 4.0 mg L-1 BAP was the highest with an average of 2.9 ± 0.6 shoots. In the same medium type, the highest shoot length was observed in the treatment with only MS media without supplementation of plant growth regulators at 2.13 ± 0.09 cm. In short, D. asper explants in liquid medium had better growth performance compared to in semi-solid medium (Figure 3). Overall, the mean number and length of shoots did not differ significantly (p < 0.05) for both media types and different BAP concentrations.

 

 

Previous studies have also reported similar findings, highlighting the benefits of utilizing liquid media in bamboo tissue culture. Ogita et al. (2008) suggested that half-strength liquid MS media had a positive effect on the elongation of axillary buds of P. meyeri. The multiplication of D. hamiltonii explants was also more efficient in liquid culture compared to semi-solid media (Sood et al., 2002). Another species, B. bambos favoured liquid MS medium supplemented with 0.1 mg L-1 BAP, 0.1 mg L-1 NAA and additives during shoot multiplication stage (Rathore et al., 2009). Dos Santos et al. (2019) cultured the D. asper species in both semi-solid and liquid media supplemented with Plant Preservative Mixture (PPM) and discovered that culture in liquid medium resulted in lower contamination rates. However, study on the same bamboo species by Singh et al. (2012b) revealed that culturing in liquid medium was not as effective as compared to semi-solid medium for multiplication of shoots.

In tissue culture, the in vitro shoots are often transferred to medium containing certain concentration of auxin to induce rooting (Ribeiro et al., 2020). Without supplementation of this type of hormone, the formation of roots in the explants might become challenging. In this study, the induction of rooting was performed in both semi-solid and liquid media supplemented with different range of IBA concentrations (1.0–5.0 mgL-1) using propagules of three to five shoots. After five weeks, the observation of rooting response and root growth were observed where explant cultures across all IBA concentrations tested in semi-solid media failed to exhibit root development (data not shown). Meanwhile, in liquid media, rooting response was observed in at least 50% of the explants aside from culture supplemented with 1 mg L-1 IBA (Table 2). Mean root length ranged from 8.22 cm to 11.36 cm and showed a trend of increasing with increasing IBA concentration, however the highest length can be observed in culture supplemented with 1 mg L-1 IBA hormone. The same trend was observed in the mean number of roots, with the highest number of roots (5.20) being observed in the culture supplemented with 5 mg L-1 IBA.

 

Table 2: Effect of different IBA concentrations on D. asper growth in liquid media after 4 weeks.

IBA concentration (mg L-1)	Rooting response (%)	Mean root length (cm)	Mean root number
0	50%	8.22±0.57a	1.00±0.63a
1	25%	11.68±1.39b	2.20±2.20a
2	50%	10.53±0.74ab	3.00±2.32a
3	50%	10.3±0.74ab	3.20±2.52a
4	50%	11.01±0.96ab	4.80±3.01a
5	75%	11.36±0.64b	5.20±2.96a

Mean with the same letters does not differ significantly at p < 0.05

 

The result obtained for this study was corroborated with a study conducted by Shirin and Rana (2007), where the best concentration that supported induction of rooting in Bambusa glaucescens was 5 mg L-1 IBA in MS liquid medium. However, there were previous studies that reported that utilizing lower IBA concentration (1 mgL-1) managed to result in the highest number of roots in the same species (Banerjee et al., 2011; Kumar and Banerjee, 2014). Another study utilized half-strength MS medium instead and managed to obtain the highest growth rate along with elongated shoots in the liquid culture supplemented with 2 mg L-1 IBA hormone. Though no root growth in the semi-solid media was observed in the current study, past research had recorded otherwise. Rooting in Bambusa tuldoides and Bambusa vulgaris explants were successful in half-strength MS semi-solid media supplemented with IBA hormones (Desai et al., 2019; Sharoti et al., 2022). The same species with the current study also managed to form roots in semi-solid media with addition of 1.0 mg L-1 IBA, resulted in the highest length and number of roots (Kumar et al., 2018).

Explants often thrive better in liquid media since they are directly in contact with the media, improving the absorption of the media content which further enhances the growth proliferation (Rai et al., 2022). Particularly, the partially submerged shoots in the media provide large surface absorption, allowing PGRs such as BAP and other hormones to be efficiently assimilated into the explants (Rathore et al., 2009). Compared to liquid media, the presence of gelling agent might cause the nutrients and PGRS to be released at a slower rate, causing the absorption of those chemicals to delay which eventually affects the growth rate of the explants (Singh et al., 2013). Though utilizing liquid media offers more benefits as compared to semi-solid, prolong submersion in liquid could lead to hyperhydricity, a physiological disorder causing biochemical changes and disturbance of the structure of the explant’s tissues (Polivanova and Bedarev, 2022). To reduce the occurrence of this event, sterile filter paper bridge was being used to prevent the whole explants from being fully immersed in the liquid medium, along with providing support to the explants in the culture system.

Acclimatization

The rooted plantlets were pre-hardened in a mixture of vermicompost and vermiculite before being transferred to the greenhouse for acclimatization. The use of a combination mixture of soil, cocopeat and compost resulted in 100% survival of the plantlets after 4 weeks.

Conclusions and Recommendations

In conclusion, the micropropagation protocol developed for the multiplication of D. asper bamboo using different sizes of propagules and comparing their growth in liquid and semi-solid MS media has provided valuable insights. The study found that propagules containing three shoots produced the highest multiplication rate, and cultures in liquid media showed a higher shoot proliferation and rooting induction compared to semi-solid media. Additionally, the rooted plantlets were successfully pre-hardened and acclimatized in a greenhouse, demonstrating the potential for large-scale propagation of D. asper bamboo using micropropagation techniques. Further studies could explore the long-term growth and development of the propagated bamboo plants in different environmental conditions, as well as the potential application of these micropropagation techniques on a commercial scale.

Acknowledgements

The authors are grateful to the Ministry of Higher Education (MOHE) Malaysia for the research grant through Fundamental Research Grant Scheme (FRGS) with code FRGS/1/2021/WAB04/UITM/02/12 and Universiti Teknologi MARA (UiTM) Shah Alam, Malaysia.

Novelty Statement

The study demonstrates a significant enhancement in the in vitro growth of Dendrocalamus asper when cultured in liquid media as compared to semi-solid media, providing critical insights into optimizing propagation techniques for this economically and ecologically valuable bamboo species.

Author’s Contribution

Wan Nurfarzana Wan Mohamad Zani: Wrote the manuscript and data analysis.

Norrizah Jaafar Sidik: Assisted in data analysis, funding acquisition and editing the manuscript.

Asmah Awal, Nurul Izzati Osman, Mohd Khairi Nordin, Lyena Watty Zuraine Ahmad: Reviewed the manuscript.

Conflict of interest

The authors declared no conflict of interest.

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