Effects of Woody Plant Media (WPM) and Plant Growth Regulators (PGRs’) on Micropropagation of Morinda lucida Benth., Using Shoot Systems and Seeds.

Ajongbolo Febisola Bukola² ,  Oyetunji Olusola Jacob¹

¹ Department of Botany, University of Ibadan, Ibadan, Nigeria.

² Department of Biotechnology, National Centre for Genetic Resources and Biotechnology (NACGRAB), Moor Plantation, Ibadan, Nigeria.

INTRODUCTION

Morinda lucida Benth is commonly known as Brimstone tree. Its a tropical plant that can be found in the Africa rainforest. The plant belongs to the family of Rubiaceae (Adewole et al., 2021). It is one of the most widely used plants in this region for medicinal purposes (Zapfack and Ngobo, 2002). It is an evergreen shrub of small to medium-sized tree measuring up to 18-25 m tall, with bole and branches often crooked or gnarled; bark smooth to roughly scaly, grey to brown, often with some distinct purple layers. It grows in grassland, exposed hillsides, thickets, forest, often on termite mounds, sometimes in areas which are regularly flooded, from sea level up to 1300m altitude (Ken, 2023).

Propagation of M. luicda is possible by seeds and cuttings, but no details are known (Jansen and Cardon, 2015). Low viability of the seeds limits natural reproduction and commercial propagation of the plant (Zimudzi  and Cardon, 2005). Previous study on breaking of the seeds dormancy revealed that propagation can be attained via seeds with the pre-treatment applications of chemical (30mg/L GA3) coupled with mechanical scarification (nicking) but no report on propagation techniques (Ajongbolo et al., 2018).

Several reports describe the application of Morinda species; in production of anthraquinones, using cell suspension cultures (Hagendoom et al.,1994), (Bassetti et al., 1995). However, no previous experience on the in-vitro propagation of M. lucida has been recorded (Elio  et al., 2011). Morinda lucida plant has numerous richness in medicinal properties and health benefits. The leaves can be used to treat and manage several health problems. Several benefits and usefulness are associated with the Leaves, roots and stem bark of Morinda lucida , such as;  Anti-inflammatory, Antibacterial, and Antiviral proper (Christian and Newman , 2013), Anti-anemia properties (Joppa  et al., 2019), Antipyretic properties (Nwobodo  et al., 2021),  Anti-malarial Properties  (Joseph et al., 2016), and so on. The interest of the Scientific community for the use of M.lucida has increased, hence there is need for scaling up via in-vitro propagation technique to enhance mass production for field establishment (Wilken et al., 2005).

MATERIALS AND METHODS

Study Location

The study was carried out in the Tissue Culture Laboratory of National Centre for Genetic Resources and Biotechnology (NACGRAB), Moor Plantation,  Ibadan, Nigeria (Lat. 7⁰23^’37.69944”N and Log. 3⁰50’22.58916” E). The Herbarium Voucher number of the mother plant was done at the Herbarium Unit of Botany Department, University of Ibadan Nigeria, with Herbarium Voucher number; (UIH-23264).

Explant Collection

Three major shoot systems of M. lucida plant (Shoot tips, Nodes, Leaves) and Seeds were collected from NACGRAB medicinal gardens inside vials, containing water as transport media and brought to the Laboratory. Thorough rinsing of the explants was attained, using running tap water with tween twenty.

Shoot Systems Disinfection

Disinfections of Shoot tips, Leaves and Nodes were achieved by double disinfection techniques, using 70% ethanol for 5minutes as surface sterilization and later used 3% and 1% w/v sodium hypochlorite (NaOCl) for 20 and 10minutes respectively, with final rinsing in sterile water for 3 to 4 times prior excising, under the Laminar flow cabinet.  Each disinfected shoot system were inoculated on the prepared media supplemented with different concentrations of cytokinins (Benzene Amino Purine and Kinetin) and constant concentration of Auxins (Indole-3- Acetic Acid and Naphthalene Acetic Acid) and Gibberellic acid (GA₃). They were later transferred to the growth room for further growth and development. Some were cultured on ordinary Woody Plant Media without any plant growth regulators (PGR’s), as controls.

Seeds Disinfection and Pre-treatment Application                                   

The seeds were subjected to different disinfection protocols, but the best was attained by using 2% w/v disinfectant (NaOCl), with 70% ethanol and was later rinsed thrice, with sterile water. Thereafter, the seeds were soaked in 30mg/L Gibberellic Acid (GA₃) for 5minutes prior inoculation to break the seed dormancy. The disinfected seeds soaked with Gibberellic Acid (GA₃) were divided into two fractions;

The first fraction was scarified prior inoculation (Nicking method).

The second fraction was not scarified prior inoculated (without Nicking)

These were cultured on Woody Plant (WP) media supplemented with Plant growth regulators, while some were cultured on ordinary Woody Plant media as controls.

Media Constituents and Plant Growth Regulators

In-vitro propagation of M. lucida was achieved, using the modified method described by (Elio et al., 2011), on Morinda royoc.  Media preparation for propagation of  Morinda lucida shoot systems  (Nodes ,Shoot tips  and Leaves) and Seeds were achieved with the usage of  Woody Plant Media basal constituents; WPM powder (2.30g/L), inositol (10mg/L), vitamin (5ml /L), sucrose (3%w/v), gelling agent (0.5% /w/v),  plant preservative mixture (2.5mls/L) , p^H (5.7) supplemented with two major cytokinins (Benzyl Amino purine  and Kinetin) respectively, at different standard concentrations, varying  from 1.0mg/l to 5.0mglL,  with Auxins (Indole-3-Acetic Acid and Naphthalene Acetic Acid) at a constant concentration of 0.5mg/l,  2mg/L constant concentration of GA₃  and control, which was ordinary WP media without adding plant growth regulators.  Sterilization was achieved at 121⁰C for 15 minutes at 1.05kg/cm².

Inoculation of Explants and Growth Room Condition

Morinda lucida explants were excised and cultured on prepared media under the Laminar flow cabinet prior proper arrangement inside growth room condition. This was a controllable environment with photoperiods of 16hrs day light and 8hrs darkness. Temperature is 25⁰C±2⁰C, relative humidity of 50-55% and illumination was between 3000 to 4000 lux. The cultures were subjected to this condition for proper growth and development.

Data collection and Experimental Design

M. lucida seeds and shoot systems cultured and excised via in-vitro propagation were monitored on daily basis and the data were recorded fortnightly. Experimental design was a Completely Randomized Design with five replicates.

Statistical Analysis

Data collected were subjected Analysis of Variance (ANOVA), using Statistical Tool for Agriculture and Research (STAR). Means were separated by Duncan Multiple Range Test (DMRT) at 5% significant level.

RESULTS

Effect of Woody Plant Media (WPM) Fortified with Plant Growth Regulators on In-Vitro Propagation of M. lucida Shoot Systems and Seeds.

The Woody Plant media basic constituents augmented with Plant growth regulators (PGRS), revealed effects of different concentrations of cytokinins (BAP and KIN) Gibberellic acid (GA₃) and auxins (NAA and IAA) on growth performances and morphological characterization of in-vitro propagated M. lucida shoot systems and seeds (Table 1). The highest growth performance of plantlets propagated was obtained in seeds propagated on Woody Plant media, fortified with (1mg/LKIN+2mg/LGA₃+0.5mg/LIAA), as shown in (Plate 1b). M. lucida shoot systems and seeds propagated on ordinary Woody Plant media (control) did not respond to regeneration. Instead of growth, virtually all the cultures turned brownish and contaminated. It was observed that shoot tips, nodes, leaves and seeds of M. lucida can’t be propagated in-vitro on ordinary Woody Plant media, compared to media fortified with plant growth regulators.

Analysis of variance for morphological characters of the in-vitro propagated shoot systems and seeds of M. lucida, using Woody Plant media augmented with Plant growth regulators (PGRs’).

The result of analysis of variance shown in Table 1.1 revealed that, there were significant differences among the morphological characters of propagated shoot systems and seeds via in-vitro technique at P≤0.05 (Table 1.1).

Correlation Associated with morphological characters of the in-vitro propagated shoot systems and seeds of M. lucida, using Woody Plant media augmented with Plant growth regulators (PGR’s).

The result of the correlation associated with the morphological characters of propagated shoot systems and seeds propagated via in-vitro technique, using woody Plant media with Plant growth regulators indicated positive responses and effects with significant differences (P≤0.05) as shown in (Table 1.2). There were strong correlation among the morphological characters.

Hardening and Transplanting of in-vitro Propagated M. lucida Plantlets.

Plates 2a shown successful hardening technique of fully grown in-vitro propagated plantlets under ex-vitro condition with no mortality rate, while plate 2b indicated effective transplanting procedure in screen house condition prior field establishment for further utilization. Both hardening and transplanting techniques gave positive effects under ex-vitro condition Plates 2a and 2b.

Table 1.  Effect of Woody Plant Media (WPM) Fortified with Plant Growth Regulators on In-Vitro Propagation of M. lucida Shoot Systems and Seeds.

Values are means and standard errors of five replicates. Values with different letters are significantly different at P≤0.05, using Ducan Multiple Range Test.

BAP= Benzene Amino Purine, KIN= Kinetin, IAA=Indole Acetic Acid, NAA=Napthaline Acetic Acid, WPM= Woody Plant Media

Table 1.1  Analysis Of Variance For In-Vitro Propagation of M. lucida Shoot systems and Seeds, Using Woody Plant Media Fortified With Plant Growth Regulators (PGRs’).

Table 1.2 Correlation Of Characters Associated with  In-Vitro Propagation Of M. lucida, Using Woody Plant Media With Plant Growth Regulators (PGRs’).

Correlation of growth parameters associated with in-vitro propagation of M. lucida, using shoot systems and seeds.

Highly significant at P≤0.01 and significant at P≤0.05.

Plate 1a: Morinda lucida Node and Shoot tips cultures, propagated on Woody Plant Media augmented with Plant growth regulators (PGR’s).

Plate 1b: Plantlets emerged from M. lucida seed cultures propagated on Woody Plant Media, supplemented with KIN, GA₃ and IAA (PGR’s).WPM=Woody Plant Media,  PGR’s=Plant growth regulators, KIN=Kinetin, GA_{3= Gibberellic Acid, IAA=Indole -3-Acetic Acid.}

Pla 2a: Hardening of fully grown in-vitro propagated plantlets of M. lucida, under acclimatization chamber.

Plate 2b: Transplanted seedlings of M. lucida propagated via in-vitro techniques.

DISCUSSION

In-vitro propagation of M. lucida shoot systems and seeds, using Woody Plant media  (WPM) as basic constituents, revealed high significant differences in plant height, number of shoot, number of leaf and number of root in treatment KI1 ( 1mg/L KIN +2mg/L GA₃+ 0.5mg/L IAA). This was similar to the report of Afolabi et al. (2020), which stated that best shoot regeneration was obtained from Noni plant (Morinda citrifolia) at the presence of 1.0 to 2.0mg/L BAP/KIN. 

The leaf cultured on Woody Plant media did not response to noticeable regeneration but rather induced callus at higher concentrations of Plant growth regulators. This report was concurred with the observation of De-Klerk (2002), on callus formation as a mechanical barrier to nutrient and water uptake for in-vitro cultures.

In-vitro propagation of shoot tips, node and leaf on ordinary Woody Plant Media showed no response to regeneration, this might be as a result of inadequate chemical nutrients in the media. This supported the observation affirmed by Shahina and Anwar (2014), in their report which stated that nutrients composition and other factors have influence on the induction and proliferation of multiple shoots.

Highest germination and growth performance was obtained from M. lucida seeds cultured on WPM fortified with lower concentration of Kinetin(1mg/L), GA₃(2mg/L) and IAA(0.5mg/L). This was in agreement with the findings of Fletcher et al., (2000) and Redemacher (2000) which stated that plant growth hormones are known to stimulate seed germination.

Also, the result gotten from the study showed that Woody Plant media, supplemented with kinetin  and Indole-3- Acetic Acid are good plant growth regulators for the germination of M. lucida seed cultures. This was concurred with the work reported by Fletcher et al., (2000), that growth influences in plant may be explained with Indole-3-Acetic Acid (IAA), which had the highest germination percentage.

The M. lucida (seed cultures) excised embryo with cotyledon propagated on Woody Plant Media basic constituents with different concentrations of Plant Growth Regulators (PGRs’) revealed good and better germination with growth performance compared to the one propagated on ordinary Woody Plant media. This report was in accordance with the report of Afolabi et al. (2023), which stated that higher germination of cultured matured Noni embryo was favored by high levels of KIN and BAP combinations.

For seeds cultured on ordinary Woody Plant Media, the study showed that both scarified and not scarified seeds has no suitable basic constituents and appropriate plant growth regulators that can enhance germination and growth. This may be as a result of exposure of seeds to improper chemical constituents in-vitro (https;//www. Plant Cell Technology.Com, (2022). Woody Plant Media basic constituent supplemented with Plant Growth Regulators revealed optimum regeneration, germination and growth performance of seeds and shoot systems via in-vitro technique. This showed that Woody Plant media was very effective for propagation of M. lucida shoot systems and seeds. This was in agreement with the report of Takuya et al., 2008 which stated that Woody Plant Media is more effective for propagation of tree plants.

Hardening and transplanting of fully grown in-vitro propagated plantlets of M. lucida were successful with no mortality rate via ex-vitro condition. This was in accordance with the previous study on hardening of in-vitro propagated wine Grape by Rahul et al. (2019) and Ajongbolo et al. (2024).

CONCLUSION AND RECOMMENDATIONS

In-vitro propagation technique can be utilized as alternative means of conservation and mass production for M. lucida shoot systems and seeds. This technique will also enhance all year propagation of M. lucida within a short period of time for future availability and utilization. Woody Plant media fortified with 1mg/LKIN+2mg/LGA₃+0.5mg/LIAA are recommended with pretreatment applications (scarification and soaking in 30mg/L GA₃) for propagation of M. lucida seeds. Propagation of nodes and shoot tips are also attainable via in-vitro technique, using Woody Plant media augmented with 4 and 5mg/LBAP with constant concentration of GA₃ (2mg/L) and 0.5mg/L IAA.

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