Validating the Efficacy of an Established Micropropagation Protocol for Commercial Propagation of Neolamarckia cadamba

Wong, Sai-Yan; Ho, Wei-Seng; Ling, Kwong-Hung

ABSTRACT

Background and Objective: Micropropagation is an efficient technique for mass-producing superior clones used in establishing planted forests. However, there is a lack of comprehensive reports on the effectiveness and reliability of the established micropropagation protocol for Neolamarckia cadamba. The aim of this study was to demonstrate the effectiveness and reliability of the established micropropagation protocol for mass propagating true-to-type N. cadamba clones. Materials and Methods: Two selected candidates plus trees of N. cadamba were cultured in B5 media supplemented with 0.8 mg L^–1 BAP for shoot multiplication and in ½ B5 media supplemented with 0.1 mg L^–1 PBZ for root regeneration. The growth performance, the presence of phytopathogens and morphological differences were investigated. The collected data were subjected to a two-tailed t-test (p<0.05). Results: The results showed no significant variation (p<0.05) in the number of shoots regenerated from each explant compared to the reference clone N5 (B39 = 4.6, B42 = 4.3 and N5 = 4.8). Moreover, the rooting patterns of the investigated clones (B39 = 14.5 and B42 = 9.4) significantly outperformed clone N5 (6.9), with over 90% successful root regeneration. Phytopathogen analysis using ERIC-PCR assay confirmed that the in vitro regenerants were free of any phytopathogens. Additionally, histological examination revealed no significant differences between the stock plants and in vitro regenerants. Conclusion: This study successfully ascertained the effectiveness and reliability of the established micropropagation protocol for mass propagating true-to-type N. cadamba clones.

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INTRODUCTION

Neolamarckia cadamba, also known as Kelampayan locally in Sarawak, is an essential source of plywood in Malaysia. It is a fast-growing plantation tree species for planted forest development and is proven able to reach a height of 45 m in 8 years^1-3. The maturation period and rotation cycle are relatively short, only five to ten years, compared to some other timber species. Due to its ability to grow in exploited and denuded land, especially in logged-over areas, N. cadamba plantations are known to be able to reduce the logging pressure of natural forests, which results in increasing demand in the wood-based industry^4,5. It also poses a self-pruning ability, leaving a branchless bole of more than 25 m. To meet the increasing demand for the species, in vitro micropropagation of N. cadamba was introduced. Several protocols for micropropagation of N. cadamba were established using different regeneration pathways and types of explants. These included the direct somatic embryogenesis from internodes⁶, direct shoot induction and plant regeneration using apical bud and nodal explants obtained from mature trees⁷, direct adventitious shoot multiplication and plant regeneration through the aseptic cotyledons of N. cadamba in vitro seedlings⁸ and optimized direct organogenesis from nodal segments of in vitro seedlings⁹. However, some studies showed that even though the micro-propagated plants are from the same species, they might respond differently to the same protocol because they have different genotypes. Thus, ascertaining the protocol is essential.

One of the significant advantages of micropropagation is that this technique can produce disease-free plants or is also known as axenic cultures^10,11. Hamill et al.¹² reported that vegetative propagations are troubled by systemic diseases, which often carry the pathogen in the new plants. They mentioned that micropropagation serves as an effective technique to eliminate pathogens from mother plants, producing germ-free cultures. Only a few studies have addressed verifying micro-propagated cultures, specifically identifying phytopathogenic bacteria. Currently, molecular techniques applied in identifying phytopathogenic bacteria are Enzyme-Linked Immunosorbent Assay (ELISA) and Polymerase Chain Reaction (PCR)¹³. ELISA is dependent on the antigen-antibody reaction and it is reported that there is a high possibility of false positive or negative results due to insufficient blocking of immobilized antigens and antibody instability¹⁴. The PCR techniques, meanwhile, are primarily gene-specific. This causes the identification process to be less efficient from the aspects of labour and costs¹⁵. Thus, a technique that can identify multiple bacteria species is recommended.

Numerous studies have shown the efficacy of enterobacterial repetitive intergenic consensus (ERIC) PCR technique for identifying bacteria^16-18. The ERIC sequences are 127 bp imperfect palindromes that Hulton et al.¹⁹ first identified in the genomes of vibrios and enteric bacteria in 1991. According to their findings, the sequences are only found in transcribed regions of the genome, either in untranslated regions upstream or downstream of open reading frames or in intergenic regions of polycistronic operons. Although the chromosomal locations of ERIC sequences vary between species, their nucleotide sequence conservation is high, making the primer universal. The ERIC-PCR method has been used to identify numerous bacterial species^20-22.

The specific objective of this study was to ascertain the efficiency and reliability of the micropropagation protocol established previously⁹ for N. cadamba. The morphological and growth performance of micro-propagated cultures derived from two selected N. cadamba candidates plus trees (i.e., B39 and B42) was comprehensively analyzed. In addition, the ERIC-PCR technique was employed to reaffirm the absence of contaminants in the micro-propagated N. cadamba cultures.

MATERIALS AND METHODS

This study was carried out from July, 2019 to June, 2022. The experiments were carried out at the Laboratory of Forest Genomics and Informatics (fGiLab), University of Malaysia Sarawak (UNIMAS), Malaysia.

In vitro germination and propagation of Neolamarckia cadamba: Dried preserved fruits were procured from the chosen N. cadamba plus trees in the Kelampayan planted forest in Kanowit (N02E00.780'E and 112E03.877'), Sarawak, Malaysia. About 100 seeds were first isolated and pre-treated with a water bath at 35°C for 24 hrs to break seed dormancy. Pre-treated seeds were sterilized in 20% commercial Clorox for 15 min and 70% ethanol for 30 sec. Sterilized seeds were cultured on basal B5 media in Petri dishes. The micropropagation protocol established previously⁹ was applied in this study. Multiplication media (B5+0.8 mg L^–1 benzylaminopurine (BAP)+2% (w/v) sucrose) was used to obtain and multiply nodal segments and subculture was conducted every three weeks until the eighth subculture (S8) was obtained. The regenerated shoots were then excised and cultured on rooting media (½ B5+0.1 mg L^–1 paclobutrazol (PBZ)+2% (w/v) sucrose) for 4 weeks. The number of shoots, the percentage of nodal segments producing shoots, the number of roots and the rooting percentage were recorded and subjected to statistical analysis. Finally, the rooted plantlets were hardened in an autoclaved soil mixture (loamy soil: Compost = 3:1). The number of surviving plantlets (ex vitro) was recorded after three months of acclimatization.

Histological study of leaf and stem structure: Leaves and stem samples from one-month-old in vitro regenerants were studied. The samples were bleached with 70% Clorox before staining with Safranin and Alcian Blue²³. Samples were observed and recorded using a compound light microscope (LEICA ICC50 HD). Parameters of plant histology referring to Boughalleb et al.²⁴ with minor modifications were estimated using ImageJ (Ver. 1.53s)²⁵.

Verification of axenic cultures using ERIC-PCR assay: The DNA extraction in this study adopted the modified Murray and Thompson²⁶ protocol. The DNA from two regenerants of each alternate generation (S2, S4, S6, S8) and acclimatized plantlets (ex vitro) were extracted and quantified in a 1.6% agarose gel using λHindIII (0.1 μg μL^–1) (BioLabs, New Zealand) as a marker. The gel image was analyzed using Gel Analyzer 19.1. The DNA samples were subjected to ERIC-PCR to identify the presence of phytopathogenic bacteria. The ERIC primers (ERIC1R-5' ATG TAA GCT CCT GGG GAT TCA C 3' and ERIC2-5' AAG TAA GTG ACT GGG GTG AGC G 3') were used to amplify the samples. The PCR profile used was 1 cycle of 95°C for 7 min, 30 cycles of 94°C for 1 min, 52°C for 1 min and 65°C for 2 min, followed by an extension cycle of 65°C for 15 min and a final shock at 4°C²⁷. The PCR was performed in a final volume of 20 μL containing 120 ng template, 100 pmol primer, 0.4 mM dNTPs, 1.0 U Taq DNA polymerase, 2.0 mM MgCI₂ and 1x Trans EasyTaq^{^®} buffer. Amplification products were separated on a 1.6% agarose gel under 60 V, 90 A for 1.5 hrs.

Statistical analysis: All the collected data were subjected to a two-tailed t-test (p<0.05) using GraphPad Prism 9 version 9.3.1.

RESULTS

In vitro germination and propagation of Neolamarckia cadamba: Data generated (B39 and B42) were compared with the regeneration data of reference clone N5 (9) to evaluate the growth performance of different clones of the same species under the same protocol (Table 1 and Fig. 1a-i). In general, the shoot regeneration rate was high in both culture clones (97%), which is 3% lower than clone N5⁹. Meanwhile, the average number of shoots produced per explant showed no significant difference between all clones, where the highest shoot regenerated was achieved by clone B39 (4.6), followed by N5 (4.5) and B42 (4.3). In contrast, the shoot length of both culture clones of B39 and B42 showed high similarity but were distinctly different from N5, showing 0.97 cm (B42) and 0.89 cm (B39).

On the other hand, different from N5, in which 100% root regeneration was achieved, clones B39 and B42 showed 97 and 90% root regeneration, respectively. Clones B39 and B42 in this study showed a significantly higher number of roots regenerated than clone N5. Under the four-week culture period, the highest number of roots was filed by clone B39 (14.5), followed by clone B42 (9.4) and clone N5 (6.9). In contrast, clone N5 demonstrated the longest root length (B39 = 2.20, B42 = 2.39 and N5 = 3.30 cm).

Table 1:

Direct shoot organogenesis and root regeneration of N. cadamba

Parameter	B39	B42	N5⁹
Regeneration
Number of shoots	4.60±2.09^a	4.27±1.86^a	4.48±2.56^a
Induction (%)	97	97	100
Growth
Shoot length	0.89±0.47^a	0.97±0.46^a	1.43 ± 0.66^b
Callus formation (%)	86.67	83.33	82.5
Rooting
Number of roots	14.50±7.58^a	9.37±5.326^a	6.88 ± 4.15^ab
Induction (%)	97	90	100
Root length	2.2±0.48^a	2.39±0.62^a	3.30 ± 0.99^ab
Callus formation (%)	10	0	0
Acclimatisation
Survival (%)	67	53	96
^abDifferent letter across the row indicates significant differences (p<0.05) when subjected to two-tailed t-tests

Image for - Validating the Efficacy of an Established Micropropagation Protocol for Commercial Propagation of Neolamarckia cadamba

Fig. 1(a-i):

In vitro propagation of N. cadamba (a), (b) Nodal explants propagated in B5 medium supplemented with 0.8 mg L^–1 BAP (c), (d) The axillary shoots spouting from nodal explants, (e), (f) Elongation of regenerated shoots in B5 medium fortified with 0.1 mg L^–1 BAP, (g) Root regeneration in ½ B5 medium fortified with 0.1 mg L^–1 PBZ, (h) Acclimatization of rooted plantlets and (I) Plantlets were transferred to potting medium containing soil (3)
^{Peat (1) and successfully acclimatized, Bar = 1.0 cm}

As for acclimatization, the survival rate of clones B39 and B42 was significantly lower than that of clone N5. Only 67% of B39 regenerates survived, while B42 showed only a 53% survival rate.

Histological study of micro-propagated Neolamarckia cadamba: Detailed measurements of the stem and leaf anatomical parameters were studied (Table 2 and Fig. 2 (I and II). Generally, no significant anatomical differences were found between stock plants and regenerate but were found significantly different between different clones. Clone B39 had a larger stem and pith compared to clone B42. The mean stem cross section of clone B39 was 2.776 mm (stock plant) and 2.914 mm (regenerates), while clone B42 was 2.423 mm for stock plant and 2.483 mm for regenerants with a mean difference of 0.392 mm between the two clones. The measurements of pith diameter also showed that clone B39 had a mean difference of 0.334 mm larger than clone B42. Clone B39 had a thicker pith than clone B42 by the mean value of 0.322 and 0.244 mm, respectively, leading to a mean difference of 0.079 mm.

Verification of clean culture using ERIC-PCR assay: The ERIC-PCR was performed to identify the presence of phytopathogens in vitro and ex-vitro cultures. Amplification of genomic DNA from 26 samples from clones B39 (Fig. 3a) and B42 (Fig. 3b) produced 104 bands. Amplicons of ~200, ~300, ~450 and ~600 bp were monomorphic across all tested samples.

Fig. 2(a-d):

Leaf and stem histology of one-month-old in vitro propagated N. cadamba clones (I) B39 and (II) B42, (a) Leaf cross-section under 40X magnification, (b) Leaf cross-section under 100X magnification, (c) Stem cross-section under 40X magnification and (d) Leaf cross-section under 100X magnification
^{abe: Abaxial epidermis, ade: Adaxial epidermis, ct: Cortex, Ep: epidermis, M: Mesophyll cells, Ph: phloem, Pt: pith and X: Xylem epidermis}

Fig. 3(a-b):

DNA profiles generated using ERIC-PCR primers, (a) Samples from clone B39 and (b) Samples from clone B42, L = 100 bp plus DNA ladder, 1-8 = In vitro samples and 9-13 = Ex vitro samples

Table 2:

Histological study of N. cadamba between stock plants and regenerants

	B39		B42
Characteristics	Stock Plants	Regenerants	Stock Plants	Regenerants
Stem
Stem cross section	2.776±0.074^a	2.914±0.228^a	2.423±0.226^b	2.483±0.130^b
Epidermis thickness	0.115±0.013^a	0.097±0.024^a	0.087±0.026^a	0.110±0.039^a
Cortex thickness	0.399±0.029^a	0.442±0.133^a	0.445±0.086^a	0.382±0.135^a
Pith diameter	1.286±0.061^a	1.354±0.094^a	0.940±0.122^b	1.032±0.191^b
Xylem thickness	0.110±0.007^a	0.098±0.022^a	0.106±0.059^a	0.119±0.016^a
Phloem thickness	0.096±0.007^a	0.076±0.022^a	0.093±0.030^a	0.087±0.011^a
Leaf
Leaf thickness	0.190±0.028^a	0.190±0.037^a	0.164±0.038^a	0.169±0.034^a
Adaxial epidermis	0.040±0.011^a	0.038±0.007^a	0.032±0.005^a	0.035±0.013^a
Abaxial epidermis	0.021±0.008^a	0.025±0.009^a	0.022±0.004^a	0.024±0.013^a
Mesophyll thickness	0.098±0.025^a	0.112±0.026^a	0.084±0.023^a	0.094±0.025^a
Mid-vein thickness	0.973±0.042^a	0.938±0.072^a	0.925±0.034^a	0.952±0.095^a
Width of bundle sheath	0.498±0.062^a	0.497±0.063^a	0.438±0.013^a	0.466±0.085^a
Pith diameter	0.334±0.055^a	0.309±0.053^a	0.244±0.023^b	0.243±0.020^b
Xylem thickness	0.059±0.005^a	0.053±0.009^a	0.050±0.005^a	0.052±0.008^a
Phloem thickness	0.049±0.009^a	0.045±0.007^a	0.040±0.005^a	0.050±0.012^a
^abDifferent letter across the row indicates significant differences (p<0.05) when subjected to a two-tailed t-test

DISCUSSION

The N. cadamba clone B39 produced the highest number of shoots, with an average of 4.6 shoots, surpassing clones B42 (4.3) and N5 (4.5), as shown in Table 1. Conversely, clone N5 showed the lowest root production, yielding only 6.9 roots in contrast to clones B39 (14.5) and B42 (9.4). These findings provide evidence of the effectiveness and reliability of the micropropagation protocol developed for N. cadamba using B5 medium⁹ instead of the conventional MS medium, thus further validating the micropropagation protocol employed in this study. As reported by Mok and Ho⁹, the highest number of regenerated shoots in the B5 medium (5.4) surpassed the highest value achieved in the MS medium (3.0) by 2.4. This is due to the differences between the composition of the two media. Russowski et al.²⁸ mentioned that the major difference between MS and B5 media is the lower ammonium-to-nitrate salt ratio in the B5 medium. It is possible that this difference in the proportion of nitrogen sources, combined with full-strength concentration, may have influenced the shoot regeneration of N. cadamba.

According to Khaskheli et al.²⁹ BAP effectively lowers apical meristem dominance while also inducing the formation of axillary or adventitious shoots. This study was in agreement that BAP is an efficient cytokinin promoting axillary shoot growth. Numerous plant species, including Musa acuminata, Bacopa monnieri (L.) and Vanilla planifolia, have shown that BAP is more effective than other cytokinins at promoting shoot multiplication^30-32. The application of BAP on shoot induction and multiplication on N. cadamba has also been reported. Kavitha et al.⁷ successfully regenerated 3.18 and 2.34 shoots from apical and nodal explants of a mature tree, respectively, using MS medium supplemented with 1.0 mg L^–1 BAP, while, Deng et al.³³ reported that optimum shoot multiplication was achieved in MS medium with 1.0 mg L^–1 BAP and 0.05 mg L^–1 of indole-3-butyric acid (IBA), producing 3.4 shoots per explant. Huang et al.⁸ showed that 1.0 mg L^–1 BAP and 0.05 mg L^–1 of IBA promoted better shoot multiplication, resulting in 5.92 shoots regenerated per explant.

This study also agreed that a low concentration of BAP is efficient enough in the shoot multiplication of N. cadamba⁹. According to their research, when the explants were cultured at lower concentrations of BAP, healthier and normal shoots developed despite the fact that the shoot induction percentage and shoot multiplication rate was higher when the explants were grown at higher concentrations of BAP. Necrosis in the shoot tips of regenerated shoots was also observed at higher BAP concentrations after two to three weeks of culture. They also highlighted the risk that the elevated cytokinin concentration might promote somaclonal variation in the plantlets following multiple subcultures. Therefore, reducing the concentration of cytokinins in consecutive subcultures is required to minimize the frequency of somaclonal changes.

In protocols that suggested the use of BAP as selected cytokinin in micropropagation of N. cadamba, the results in this study showed little difference in the number of shoots regenerated in lower BAP concentration when compared to those regenerated in higher BAP concentration. Kavitha et al.⁷ found that 2.5 mg L^–1 BAP was the optimal concentration for producing 5.7 shoots per explant, while Deng et al.³³ suggested a concentration of 1.0 mg L^–1 BAP with a combination of another two cytokinins, 0.1 mg L^–1 IBA and 2.0 mg L^–1 GA₃ produced only 3.4 shoots per explant. On the other hand, Huang et al.⁸ suggested using 5 mg L^–1 BAP and 0.05 mg L^–1 naphthalene acetic acid (NAA) produced only 5.9 shoots per explant despite the exceptionally high concentration of BAP applied. Given the risk of inducing somaclonal variations with multiple cytokinin combinations and high concentrations, this study agreed that a lower BAP concentration (0.8 mg L^–1) is sufficient to induce multiple shoot regeneration.

The present study agreed that the rooting medium ½ B5 supplemented 0.1 mg L^–1 paclobutrazol (PBZ) is sufficient in inducing root regeneration in N. cadamba⁹. As PBZ concentration does not induce growth during rooting, there is no callus formation on the regenerated shoots. The PBZ is a triazole-derived plant growth retardant that reduces plant growth without changing developmental patterns or being phytotoxic³⁴. It inhibits gibberellin biosynthesis, reduces internodal growth to give stouter stems and increases root growth^35,36. Multiple studies suggested that PBZ efficiently promotes stress tolerance in vitro cultures. Therefore, it is preferred to be applied at the last stage of micropropagation before acclimatization to withstand the stress generated due to environmental changes^37,38. Hence, it is agreeable that PBZ is applied in this study instead of other auxins in the rooting stage. The number of roots regenerated in this study was significantly higher compared to other protocols using auxins as rooting media. For instance, only 60% root regeneration was recorded using 1.0 mg L^–1 IBA⁷. Deng et al.³³ recorded only 7.8 roots regenerated per shoot using 0.1 mg L^–1 NAA. Huang et al.⁸ applied ½ MS medium supplemented with 0.05 mg L^–1 NAA and 0.05 mg L^–1 IBA for root regeneration and regenerated 5.2±1.5 roots per plantlet. Thus, by achieving over 90% root regeneration with 14.5±7.6 roots (B39) and 9.37±5.3 roots (B42) per regenerated explant, this study agrees that 0.1 mg L^–1 PBZ (paclobutrazol) is more efficient in root regeneration of N. cadamba.

All the studied parameters except for shoot regeneration showed significant differences among the three clones evaluated. This is due to the response of different genotypes toward similar tissue culture conditions. As in vitro propagation aims to produce a large amount of ‘elite’ clones posing the same preferred characteristics, it has already been defined that in vitro culture is highly dependent on the genotype of donor material^39-41. The genotype of the explants strongly determines morphogenic responses such as organogenesis, shoot proliferation and rooting. This indicates that different gene combinations between individuals of the same species tend to express one characteristic more than another^42,43. Hence, it explains the results of this study in which the morphological performance of clone B39 and clone B42 are different from clone N5⁹. This further supports the statement that the suitability of the applied protocol is genotype dependent.

The suggestion that the micropropagation protocol is genotype-dependent and not suitable species-wide was also proposed and validated by Gomes et al.⁴⁰ when they conducted a study to identify the relationship between genotype and morphological performances of Arbutus unedo L. They noted that callus development and shoot multiplication were highly genotype-dependent processes. They examined ten distinct A. unedo clones and the results revealed that genotype significantly impacts multiplication rate (p<0.05) in terms of both shoot length and the number of regenerated shoots. Some genotypes significantly outperformed others, displaying differences in all examined variables. As a result, they noted that the genotype of explants greatly influences a micropropagation protocol’s effectiveness. This finding is similar to what is reported in this study, where the shoot length of both clones B39 and B42 was significantly lower than the reference clone N5⁹.

Mok and Ho⁹ reported different propagation responses to the study by Kavitha et al.⁷ under the same culture condition. When cultured in MS medium supplemented with 0.5 mg L^–1 BAP, Kavitha et al.⁷ successfully regenerated 2.2±0.21 shoots, while Mok and Ho⁹ reported 3.0±1.54 shoots per explant. Similarly, Kavitha et al.⁷ reported 2.3±0.32 shoots regenerated for 1.0 mg L^–1 BAP supplemented MS medium, but Mok and Ho⁹ reported 2.6±1.31 shoots regenerated. This shows a significant difference in the resulting number of shoots when subjected to t-tests but also showed a different trend of the explants reacting to PGRs. Explants in Kavitha et al.⁷ showed an increasing trend of shoot regeneration from 0.5 to 1.0 mg L^–1 BAP and reached the highest proliferation rate at 2.5 mg L^–1 BAP while Mok and Ho⁹ reported a decreasing trend under the same culture conditions. Thus, it is unsurprising that the clones studied in this research showed significantly different shoot lengths, root lengths and the number of regenerated roots compared with Mok and Ho⁹, even though the same micropropagation protocol was applied.

Several studies on varied species have shown that genotype can significantly affect the efficiency of micropropagation protocols and most of them agreed that genotypes have to be tested before applying the established micropropagation protocol^43,44. However, the multiplication rate is the primary factor to be considered when assessing the efficiency of a micropropagation protocol. The multiplication rate of explants is calculated and represented by the number of shoots regenerated per explant. The present results indicate no significant difference in the multiplication rate of the studied clones (B39 and B42) compared to clone N5⁹. Therefore, this finding further ascertains the effectiveness of the micropropagation protocol of N. cadamba.

The growth parameters of the regenerants, such as shoot length, root length and the number of shoots and root regeneration, might not be sufficient to clarify the effects of micropropagation and genotype on the morphology of regenerants. Hence, a histological study was conducted to investigate the anatomical structure of growing cells and clarify the differences observed morphologically. Overall, the results showed no significant differences between stock plants and regenerates for all measured parameters, indicating that the protocol is reliable for producing morphologically identical regenerates.

However, differences were observed between the two clones (i.e., B39 and B42) with different genotypes, specifically in the leaves and stem. Notably, a significant difference was found in the stem cross-section and the pith diameter of both the stem and leaves. Pith, also known as the medulla, is a tissue in the stems of vascular plants⁴⁵. The pith is composed of soft, spongy parenchyma cells that store essential nutrients like water and sugars, which can be transported between the pith and the vascular bundles. In eudicotyledons, a pith is located in the center of the stem, encircled by a ring of xylem followed by a ring of phloem⁴⁶. Clone B39 showed a larger pith diameter than clone B49, causing it to pose a larger stem cross-section when compared to clone B42. However, the two clones have no significant difference in the cortex layer. Thus, the differences identified in the pith showed no importance because, in woody plants such as N. cadamba, the pith will be reduced to a small opening as the pith is replaced with a xylem. The stem cross-section study is essential to those trees that are used for timber production and where the quality of the timber is desired⁴⁷. Thus, clone B39 is preferable for mass production.

The morphological variations between various genotypes of the same species have been extensively studied^48,50. According to Lamhamedi et al.⁵⁰ natural variability occurs within species and since each individual was raised from a single seed, genotype differences were anticipated. Several clones were discovered to differ significantly in height, root collar diameter, branch density, shoot dry mass, root dry mass and needle length in their study of identifying the variations between various genotypes of Picea glauca. They concluded that there were differences in physiology and growth among clones within a family.

A similar statement stating that genotype plays an essential role in determining the morphology of plants and their clones was also reported by Sharma et al.⁵¹. They studied the morphological characteristics of planting stock as indicators of field performance and found that although planted in the same place, different clones performed differently in growth, thus affecting their survival rate in the field. Likewise, Rolletschek et al.⁴⁹ also demonstrated the growth differences due to different genotypes in Phragmites australis. In their study, two adjacent grown Phragmites clones were studied for their physiology and morphology differences during different seasons. They found that the two clones showed significance in many parameters, such as shoot morphology and underground biomass. These variations remained significant even after field transplantation and thus, it is hypothesized that both clones follow distinct eco-physiological strategies causing their morphogenic differences.

The ERIC primers are universal markers that can amplify many sequences across different organisms. The ERIC sequences were highly conserved in non-coding regions of enterobacteria such as Escherichia coli and Salmonella typhimurium¹⁹. The ERIC-PCR is a valuable tool to identify the presence of bacteria strains based on the targeting amplicons of the species, providing references to other studies to identify the presence of certain species. For example, using ERIC-PCR, this study produced monomorphic amplicons (200, 300, 450 and 600 bp). By referring to Mondal and Mani²⁷, they validated the presence of Xanthomonas campestris pv. punicae, 900 bp amplicon was reported as the targeted virulence gene of the species. With the absence of the targeted amplicon, in which the largest amplicon found in only one of the ex vitro samples is only 600 bp, it can be presumed that the cultures in this study are free from X. campestris pv. punicae. Besides that, Mondal and Mani²⁷ also showed amplicons of 75, 150, 400, 700 and 1200 bp, which do not meet any of the amplicons in this study, suggesting that the amplified products have no relationship to the species.

Gillings and Holley⁵¹ suggested that ERIC primers are not specified to amplify ERIC elements only. They claimed that all tested organisms, including bacteria, bacteriophages, fungi, plants and vertebrates, produced complex DNA fingerprints in their study. Thus, they suggested that ERIC-PCR might be useful for distinguishing between organisms or evaluating species diversity, but it does not mean all amplicons produced by this method resembles the presence of ERIC sequences. Hence, it provides evidence that the amplicons produced in this study originated from the genome of N. cadamba itself, suggesting the cultures are axenic and pathogen-free.

CONCLUSION

This study confirmed that low concentrations of cytokinin (BAP) are effective for inducing in vitro shoot multiplication in N. cadamba, resulting in an average of four regenerated shoots per explant. Replacing auxin with PBZ successfully reduced callus formation prior to acclimatization. The ERIC-PCR assay validated the production of axenic cultures and thus, it can be concluded that the evaluated micropropagation protocol is both efficient and reliable for the mass production of N. cadamba.

SIGNIFICANCE STATEMENT

This study aimed to demonstrate the effectiveness and reliability of the established micropropagation protocol for mass propagating true-to-type N. cadamba clones. We comprehensively investigated the growth performance, presence of phytopathogens, histological examination and morphological differences between the stock plants and in vitro regenerants derived from two selected candidates plus trees of N. cadamba. The findings of this study provide additional evidence to support the effectiveness and reliability of the established micropropagation protocol for large-scale production of true-to-type N. cadamba seedlings.

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