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In vitro Propagation Protocol for Improving African Mahogany (Khaya senegalensis) Endangered Tree



Mona Ahmed Darwesh, Amal Abdo Naser, Ebrahim El-Abassery Habba, Lobna Salah Taha, Ahmed Mohamed Magdy Gabr and Riham Mohamed Bahgat El-Assaly
 
ABSTRACT
Background and Objective: Khaya senegalensis (K. senegalensis) A. Juss. (Meliaceae), Known as African mahogany, is a multipurpose timber tree with a variety of economic and environmental values. The aim of the current study was to establish a suitable protocol for K. senegalensis micropropagation. Material and Methods: The effect of benzyl adenine (BA) (0.2, 0.4. 0.6 0.8 and 1.00 mg L–1), types of media [Murashige and Skoog (MS), woody plant medium (WPM) and Gamborg medium] and number of subculture on shootlet proliferation were studied. For rooting stage, indole-3-butaric acid (IBA) (1.0, 2.0 and 4.0 mg L–1) and half salt strengths of both MS and WPM, pre-incubation and incubation time were studied. The PCR, RAPD and ISSR, was used to confirm the true to tip between micropropageted plants and the mother plant. One-way and two-way ANOVA is used for statistical analysis. Results: The results showed the highest number of shootlets per explant (5.33) and the highest number of nodes (22.0) was obtained with 0.6 mg L–1. The BA using half strength of MS culture medium for three subcultures caused the highest number of both shootlets formed per explant and nodes, whereas full strength of WPM culture medium caused the longest shoots and repeated subcultures had inhibit effect on shootlet elongation. For rooting stage, the highest number of roots was obtained with pretreatment of the in vitro shootlets with IBA at 4.0 mg L–1 for one week in darkness, then transplanting them to half MS or WPM for 12 weeks, while the longest roots were obtained with half MS culture medium. Plantlets were successful adapted in peatmoss and sand (1:1). The results of molecular analysis of RAPD and ISSR were obviously revealed that there was no variation between the in vitro derived plants and the mother plant. Conclusion: The current study showed the highest shoot proliferation was recorded with 0.6 mg L–1 and with half MS medium. For rooting stage half MS gave the highest root numbers and length. Molecular marker RAPD and ISSR confirmed that this protocol is suitable for propagation of K. senegalensis with confirmation of true to tip.
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Mona Ahmed Darwesh, Amal Abdo Naser, Ebrahim El-Abassery Habba, Lobna Salah Taha, Ahmed Mohamed Magdy Gabr and Riham Mohamed Bahgat El-Assaly, 2017. In vitro Propagation Protocol for Improving African Mahogany (Khaya senegalensis) Endangered Tree. Journal of Biological Sciences, 17: 235-246.

DOI: 10.3923/jbs.2017.235.246

URL: http://scialert.net/abstract/?doi=jbs.2017.235.246
 
Received: July 08, 2017; Accepted: August 28, 2017; Published: September 12, 2017

INTRODUCTION

Khaya (Khaya senegalensis Juss.) family Meliaceae, commonly known African mahogany, is an evergreen tree that usually grows to a height of 15-20 m (up to 35 m on fertile soil)1. Khaya is a multiuse tree with a variety of economic and environmental values1. It is one of the major timber species as it known to be hard and fungus and termite resistant red wood, it is highly valued for carpentry, furniture, ship building and as a decorative veneer. The bark is used as traditional medicine to treat malaria, diarrhea, dysentery, anemia and other diseases2. Recently, the stem bark has been found to contain chemicals (limonoids) that exhibit ant proliferative activity against human cancer cell lines2. In West Africa, the species has become an important urban amenity tree, commonly planted as a roadside or ornamental shade tree. It is also increasingly planted in other countries such as South Africa, Egypt, Australia, Sri Lanka, China, Indonesia, Malaysia and Vietnam for both amenity and timber production3. Natural regeneration of K. senegalensis is poor1 as its seeds lose viability after only two or three weeks under natural conditions4. Very few studies have been done on micropropagation of K. senegalensis as Hung and Trueman5 and Danthu et al.6. The aim of this study was to develop methods for micropropagation of K. senegalensis, including (1) Shoot induction and shoot proliferation to establish a laboratory archive of juvenile clones and (2) Root formation to generate plantlets of the same clones. Reports on micropropagation of other Meliaceae trees have also used MS media containing BA for initiating or proliferating shoots ,with the BA frequently, though not always, supplemented with indole-3-butyric acid (IBA)7-14. Growth of axillary or adventitious shoots is stimulated by the presence of cytokinin in the culture medium such as (BA) at 0.5-1.0 mg L–1 15,16. Regeneration efficiency depends on the medium composition: macro and micronutrients type and concentration of growth regulators. The most commonly used media for tissue culture of dogwood are woody plant medium (WPM)17-20 and medium (MS)21-24. Therefore, the present study was aimed to develop micro propagation protocol for rapid and large scale production of clonal planting material as well as assessment of genetic stability using RAPD and ISSR markers.

MATERIAL AND METHODS

This investigation was carried out at Department of Plant Biotechnology , National Research Centre (NRC) Egypt during years 2014 and 2017 to establish in vitro protocol for shootlet multiplication from seed of K. senegalensis plants.

Plant material: Seeds of K. senegalensis were collected from the Agricultural Research Centre Giza, Egypt (2014). Seeds were refrigerated at 4°C until use.

There are three methods of sterilization followed to improve seed germination:

T1: Seeds were surface sterilized by the process of successive rinsing in 70% ethanol (sigma-aldrich) contain Tween-20 for 1 min and in 15% sodium hypochlorite for 10 min, then seeds were washed three times with sterile distilled water. The seeds were then left to germinate in conical flask (250 mL) containing 50 mL basal liquid 1/2 MS medium21
T2: Seeds were dipped in 70% ethanol and directly burned with the flam till the seed put off the faire. Batches of five seeds were left to germinate in conical flask (250 mL) containing 50 mL basal liquid 1/2 MS medium
T3: Before the sterilization process, the teguments and seed coat were removed manually. These seeds were surface sterilized by the process of successive rinsing in 70% ethanol for 30 sec and then they were washed three times with sterile distilled water. The seeds were then left to germinate in conical flask (250 mL) containing (50 mL) basal liquid 1/2 MS medium

The medium was supplemented with 30% sucrose and adjusted to pH 5.8 with 1 M KOH and subsequently sterilized by autoclaving at 121°C for 20 min. The seeds were then germinated in a growth chamber at 25°C under standard cool white fluorescent tubes, with a light intensity of 35 μmol sec–1 m–2 and a 16 h photoperiod.

Effect of BA concentration on shoolets proliferation: The epicotyls, 5 weeks old germinated seeds, were transferred to full salt strength modified MS described by Lobna25 on Eucalyptus globules MS macro and micro element supplemented with De Fossard vitamins media MS supplemented with De Fossard26 [4.9 mg L–1 Nicotic acid+0.88 mg L–1 Calcium pantothenate+3.6 mg L–1 ribophlavin+ 1.8 mg L–1 Ascorbic acid+1.4 mg L–1 choline chloride+14.5 mg L–1 L-cystain]+0.01 mg L–1 NAA+25 g L–1 sucrose+7g L–1 agar and with different concentrations of BA (0.2, 0.4, 0.6, 0.8 and 1.00 mg).

Cultures were incubated on growth chamber at 25±2°C under standard cool white fluorescent tubes, with a light intensity of 35 μmol sec–1 m–2 and a 16 h photoperiod.

Data were recorded as, number of shootlet, number of nods and shootlet length.

Each treatment contains nine explants and each jar contains one explants (total explants 45= 45 jars).

Effect of type of culture media and strength on shootlet proliferation: The best BA concentration from the previous experimental was used in combination with different types of culture media: MS21, woody plant medium17 (WPM)27 B5 medium at two strengths full and half strengths and for three repeated subcultures.

Cultures were incubated on growth chamber at 25±2°C under standard cool white fluorescent tubes, with a light intensity of 35 μmol sec–1 m–2 and a 16 h photoperiod.

Data were recorded as: number of shootlet, number of nods and shootlet length.

Each treatment contains nine explants and each jar contains one explants (total explants 54 = 54 jars).

Biochemical analysis
Photosynthetic pigments
Extraction and determination of pigments:
Half gram of fresh samples were sectioned into minute pieces in three replicates and freshly macerated and extracted with 80% cold methanol for 24 h at 0°C, then extracted twice 80% methanol for 24 h. The combined extract was filtered and adjusted to a known volume for quantifying the endogenous chlorophyll (a and b) and carotenoids. According to Saric et al.28 the color density was measured using (spectronic) at 660, 640 and 440 nm wave length against the blank methanol.

The contents were calculated20 as follows:

Chlorophyll a = (E.a at 660 nm×9.784)-(E.b at 640 nm×0.99)
Chlorophyll b = (E.a at 640 nm×21.426)-(E.b at 660 nm×4.65)
Carotenoids = [E. a at 440 nm×4.695-(E.a at 660 nm+E.b at 640)×0.268]
E = Scale reading

The amounts of pigments were calculated as mg/100 g F.W.

Total indoles and phenols: The total indoles were determined by using "Erlic's reagent" according to Larsen et al.29 While, total soluble phenols was calorimetrically determined using Folin Ciocaltea reagent30.

Effect of pretreatment with different concentrations of IBA and re-culture on half MS or WPM salt strength: In this experiment, Shootlets were pre-cultured on different concentration of IBA (1.0, 2.0 and 4.0 mg L–1) for 1 week and incubated under complete dark conditions, then shootlets were re-subcultured on free half of MS or WPM for four periods of time (3, 6, 9 and 12 weeks) and incubated under standard cool white fluorescent tubes, with a light intensity of 35 μmol s–1m–2 and a 16 h photoperiod.

Data were recorded as, rooting (%), number of root and root length.

Each treatment contains 12 explants and each jar contains one explants (total explants 48 = 48 jars).

Acclimatization: For the experiments on acclimatization, 30 days old rooted plantlets on MS or WPM medium were removed from Jars, washed free of agar, then transferred to plastic pots containing a 1:1 mixture of sand and Peatmoss and were covered with polythene bags. The survival percentage, stem length (cm) and root length (cm) were recorded after five weeks from acclimatization.

At all stages of acclimatization, the plants were kept in the culture room, under identical culture conditions to those used for the tissue culture studies.

Molecular marker analysis: In the present study, use of two PCR-based techniques, RAPD and ISSR, for the identification of the variation between the mother plant and acclimatized K. senegalensis. The use of two types of markers, which amplify different regions of the genome, allows better chances for identification of genetic variation in the plantlets.

Total genomic DNA extraction: Total genomic DNA was extracted from mother plant and the new branches, which taken at the end of acclimatization K. senegalensis and grinded into a fine powder in liquid nitrogen using a pestle and morter following the steps of CTAB (hexa decyltri methyl ammonium bromide) protocol31.

RAPD-PCR analysis: A set of three random primers was used in the detection of polymorphism among mother plant and acclimatized K. senegalensis. The amplification reaction was carried out in 25 μL reaction volume containing 1×PCR buffer, 1.5 mM MgCl2, 2 mM dNTPs, 1 U Taq DNA polymerase and 25 mg templates DNA and1 μM primer from each of random primers (Operon) Table 1. The PCR (Bio-Rad USA, T100) amplification was performed in a biometera Uno thermal cycles programmed to fulfill 40 cycles after an initial denaturation cycle for 5 min at 94°C. Each cycle consisted of a denaturation step at 94°C for 1 min, an annealing step at 36°C for 1 min and an elongation step at 72°C for 1.5 min. The primer extension segment was extended to 7 min at 72°C in the final cycle. The amplification products were resolved by electrophoresis in a 1.5% agarose gel containing ethidium bromide (0.5 μg mL–1) in 1×TBE buffer at 90 volts. Gel was photographed under UV light with Tracktel GDS-2 gel documentation system. The size of the amplification products was estimated from 100 bp DNA ladder.

ISSR-PCR analysis: The ISSR scorable primers were designed and screened for PCR amplification. The PCR was performed in 25 μL reaction volume containing 1×PCR buffer, 1.75 mM MgCl2, 5 mM of each dNTPs, 40 μM oligonucleotide primer from each of the ISSR primers (Operon) Table 2, 25 ng genomic DNA and 1 U of Taq DNA polymerase. A high stringency touch down and hot start thermo cycling profile was used as follows: An initial denaturation step for 5 min at 94°C followed by ten touch down cycles (94°C/30 sec, 65-55°C/45 sec, 72°C/1 min). This was followed by 35 the PCR products were separated by electrophoresis in a 2.0% agarose gel containing ethidium bromide (0.5 μg mL–1) in 1×TBE buffer at 90 volts. Gel was photographed under UV light with Tracktel GDS-2 gel documentation system. The size of the amplification products was estimated from 100 bp DNA ladder.

Table 1:Random primers, names and sequences, used for RAPD analysis

Table 2:ISSR primers, names and sequences, used for ISSR analysis

Statistical analysis: All experiments were set up according to a completely randomized design. Data for numbers of shootlet, number of nodes and length of shootlet, were analyzed by two-way ANOVA, data for chemical characters were analyzed by one-way ANOVA. Variance in treatment means and standard errors were determined, followed by the least significant difference (LSD) test at p<0.05 to compare means. All Data were analyzed by factorial one and two-way ANOVA was performed on data using COSTATV-6332.

RESULTS AND DISCUSSION

Effect of BA concentration on shoolets proliferation: The positive effect of BA concentrations (0.2, 0.4, 0.6, 0.8 and 1.0 mg L–1) for three repeated subcultures on shootlets proliferation of K. senegalensis had been clearly indicated in Table 3 and Fig. 1. The results revealed that culturing of explants on MS medium supplemented with 0.8 mg L–1 BA caused the highest number of shootlets/explant, followed by 0.6 mg L–1 of BA which also led to the highest number of nodes. The interaction effect of BA concentrations and subcultures number on shootlets proliferation was recorded in Table 1. The data cleared that BA concentrations (0.6 and 0.8 mg L–1) and repeated subculture for three times led to the highest number of shootlets/explant, whereas the highest number of nodes (22.0) was obtained with 0.6 mg L–1. It can also notice that with increasing BA concentration above 0.6 mg L–1 (0.8 and 1.0 mg L–1), the length of shootlets was decreased to the lowest values. Results were in agreement with those found by Danthu et al.6 on K. senegalensis when tested shoot induction with BA at 0, 2.2, 8.9 and 22.2 μM and found the highest induction in the media containing 2.2 or 8.9 μM of BA.

Table 3:Effect of BA concentrations on shootlets proliferation of K. senegalensis during three repeated subcultures
*Same latter means non-significant difference; different letter mean significant difference at 0.05 probabilities. aHighly significant then significant decrease with the following letters

Fig. 1(a-g):
Micropropagation of Khaya senegalensis from seed (a) Seed with coat, (b) Seed without coat, (c) Shoot induced on modified free Ms medium, (d) Shoot proliferating on modified MS medium supplemented with 0.8 mg L–1 BA, (e) Shoot proliferating on modified MS medium supplemented with 0.6 mg L–1 BA, (f) Shoot proliferating on modified1/2 MS supplemented with 0.6 mg L–1 BA and (g) Shoot proflierating on modified 1/2 B5 supplemented with 0.6 mg L–1 BA
  *Modified: Supplemented with De Fossard vitamins

On the same species, Cao Dinhand Stephen5 observed that shoot proliferation over four passages was higher in media containing BA 4.4 μM. Moreover, Lobna et al.33 mentioned the promotion effect of BA at 1.0 mg L–1 on the in vitro shootlets formation of Paulownia kawakamii this might attributed to the important physiological role of cytokinins to stimulate cell division and elongation as well as activate RNA, protein synthesis and enzyme activity, as was reviewed by Kulaeva34.

Table 4:Effect of Media type and strength on shootlets proliferation of K. senegalensis during three repeated subcultures
*Same latter means non-significant difference; different letter mean significant difference at 0.05 probabilities. aHighly significant then significant decrease with the following letters

Effect of culture media type and strength on shootlet proliferation: Data presented in Table 4 and Fig. 1 showed the significant effect (p<0.05) of various types of culture media (MS, WPM and B5) at two strengths (full and half) for three repeated subcultures on shootlets proliferation of K. senegalensis. Culturing the explant on half strength of MS medium gave the highest number of shootlets and nodes proliferated per explant, meanwhile full strength of WPM or B5 media caused the lowest ones (5.56, 4.22; 12.67 and 9.44, respectively). These values were in maximum rates with the third repeated subculture (12.11 and 31.67, respectively). The same trend was observed in the interaction effect between culture media treatments and the number of repeated subcultures. Using half strength of MS culture medium for three subcultures caused the highest number of both shootlets formed per explant (21.00) and nodes (66.33) as compared to other media types and strengths for any repeated subcultures. Whereas full strength of WPM culture medium caused the longest shoots and repeated subcultures had inhibit effect on shootlet elongation. Similarly, different types of basal media like White medium, Nitsch and Nitsch medium, MS medium and Gamborg medium have been reported for micropropagation35,36 but MS21 was the most favored culture medium and widely used because it contains all the nutrients essential for in vitro plant growth. In comparison to other media formulation, MS consider a high level of nitrogen, potassium and some of the micronutrients, particularly boron and manganese37. Moreover, strength of media is one of one of important factor to optimize the regeneration protocol28. Results are confirmed with those obtained by Yaussef 38 on Melaleuca armillaris, who indicated that culturing of explants on MS medium increased the shootlets proliferation rates 11.6- fold higher than culturing on WPM and B5 medium. Gray and Benton39 on Vitis rotundifolia observed that shoot proliferation rate on half strength of MS medium was as good as half strength.

Effect of different concentrations of IBA pretreatment under darkness and culture media types on rhizogenesis behavior: In this experiment, Shootlets were pre-cultured on different concentration of IBA (1.0, 2.0 and 4.0 mg L–1) for one week and incubated under dark conditions, then shootlets were resubcultured on free half of both MS and WPM for four periods (3, 6,9 and 12 weeks) and incubated under light conditions. The results in Table 5 and Fig. 2 shows that the highest percentage of rooting (100%) and was obtained with pre-culturing the shootlet explants on MS medium supplemented with high concentration (4.0 mg L–1) of IBA then subcultured on half strength of WPM medium with no significant differences between this value and those when the shootlets were precultured on MS medium supplemented with 2 or 4 mg L–1 of IBA, then transplanted to half strength of MS or WPM (83.33 and 75%, respectively). Increasing periods above 3 weeks (6, 9 and 12 weeks) showed the best results for rooting percentage. The highest number of roots (5.22 and 4.33) was obtained with pretreatment of IBA 4.0 mg L–1, then reculturing the shootlets on half strength of both MS and WPM medium, while the longest roots (9.04 cm) was obtained on the same treatment, but with MS at half strength. These values of roots number and length were highest when the data were recorded after 12 weeks. It seems that the promotion effect on both rooting percentage and roots number was attributed to the pretreatment with high concentration of IBA (4.0 mg L–1) and lowering the strength of media (MS or WPM).

Fig. 2(a-f):
Rooting and acclimatization of Khaya senegalensis, (a) In vitro rooted shootlet on 1/2 MS medium after treatment with 4 mg L–1 IBA, (b) In vitro rooted shootlet on 1/2 WPM medium after treatment with 4 mg L–1 IBA, (c) In rooted shootlet on 1/2 MS medium after treatment with 2 mg L–1 IBA, (d) 2 weeks "before polythene cover removed" acclimatization of a plantlet, (e) Five weeks "after polythene cover removed" acclimatization of a plantlet and (f) Two months acclimatization of a plantlet

This may attributed to high salt of culture medium content is not necessarily for root formation. Confirmed results was recorded by Kumar and Reddy40, who mentioned that the use of dilute media formulations has promoted better formation of roots, since high concentration of salts may inhibit root growth, even in presence of auxins in the culture medium. Regarding the effect of IBA concentration on rhizogenesis behavior41 on Quercus ruber observed that rooting was achieved on in vitro regenerated shoots in an IBA concentrated solution. Contrary, Ostrolucka and Bezo42 cultured the explants of Quercus species (Q. ruber, Q. cerris and Q. rubra) on woody plant medium with IBA at various concentrations. They observed that the highest rhizogenesis rates were obtained in case of using low concentration of IBA.

Table 5: Effect of different concentrations of IBA pretreatment under darkness and culture media types on rhizogenesis behavior
*Same latter means non-significant difference; different letter mean significant difference at 0.05 probabilities. aHighly significant then significant decrease with the following letters

Chemical constituents
Photosynthetic pigments:
It is clear evident that using full strength of WPM significantly augmented photosynthetic pigments such as chlorophylls a, b and carotenoids content to highest values (105.12, 43.30 and 79.35 mg/100 g F.W., respectively) followed by full strength of MS. While, using full or half strength of B5 medium caused the lowest values of these pigments content in the in vitro shootlets as shown in Table 6. The enhancement of pigments accumulation in shootlets tissues with WPM (full strength) medium may be due to the ingredients of culture medium influence on these pigments43. Same finding was previously reviewed by Youssef38 on Melaleuca armillaris comparing with MS and B5 medium.

Total indoles and phenols: Results in Table 6 show that by using various culture media types at half and full strengths exhibited significant increase (p<0.05) on the levels of total phenoles and indoles in shootlets tissues as shown in results of table 6. The highest levels of these constituents (394.64 and 1085.88 mg/100 g F.W., respectively) were obtained as a result of using B5 medium at half strength, meanwhile the lowest ones with half strength of MS, full or half strength of WPM for total phenols and full strength of MS for total indoles. In this share, Lobna44 on Melaleuca armillaris studied various culture media types (MS, B5, QL and WPM) at quarter, half and full strengths on the biosynthesis of both total indoles and phenols in shootlet in vitro. The total indoles was found in greatest amount when WPM medium was used, the most effective concentration was quarter strength. The total soluble phenols was detected in maximum value when B5 or WPM were used with no significant effect of three different concentrations of culture media which were tested.

Molecular analysis: True-to-type clonal fidelity is one of the most important prerequisites in the micropropagation of any species. A major problem encountered with the in vitro culture is the presence of somaclonal variation amongst sub-clones of one parental line, arising as a direct consequence of in vitro culture of plant cells, tissues or organs. A better analysis of genetic stability of plantlets can be made by using a combination of two types of markers which amplify different regions of the genome45. Hence, in the present study, two PCR based techniques, RAPD and ISSR were adopted for evaluation the variation between the mother plant and acclimatized plants which irrigated with diatom.

Fig. 3(a-f):
DNA amplification pattern obtained for 1: Mother plant, 2 and 3: Acclimatized micropropagated plants generated by RAPD primers, (a) OP-A07, (b) OP-B07, (c) OP-B11 and ISSR primers, (d) HB-8, (e) HB-11 and (f) HB-12 and M: 100-1500 pb DNA Ladder

Table 6:Effect of different media type and strengths on chemical constituents of K. senegalensis(after three repeated subcultures)
*Same latter means non-significant difference; different letter mean significant difference at 0.05 probabilities. aHighly significant then significant decrease with the following letters

RAPD-PCR analysis: Three random primers (OP-A07, OP-B07 and OP-B11) were screened in RAPD analysis for their ability to produce sufficient amplification products. In the present study, OP-A07 present the highest number of fragments produced by the three primers (36 with 12 fragments/primer) (Fig. 3), with no variations between mother plant and micropropagated plants. While, primer OP-B11 present the lowest number of fragments (12 with 4 fragments/primer). That is confirmed our protocol to produce true to tip plants between mother plant and micropropagated plants. In this respect, RAPD markers have been able to assess the genetic stability of micropropagated plants of Deutzia scabra and the effect of slow growth preservation of the same plant almonds45,46 turmeric47 and yams48. As RAPD markers amplify different regions of the genome, their simultaneous analyses give a better interpretation of the genetic stability of the in vitro regenerates49.

ISSR-PCR analysis: Four ISSR primers were used in this study, the three ISSR primers produced good reproducible and scorable patterns and the amplification profiles were screened for the presence of polymorphisms among and within the mother plants and micropropagated plants (Fig. 3). The three primers showed no polymorphic bands obtained between the three primers. These results are confirmed the true to tip between mother plant and the micropropagated plants. The ISSR amplify different parts of the genome, the data generated from this analysis was obtaining more balanced values for genetic variation among genotypes targeted and representation of their relationships. This allowed us to explore the DNA polymorphism in the collection of genotypes analyzed and generate many polymorphic markers ensuring a good coverage of the genome49,50.

CONCLUSION

It can be concluded that seeds without seed coat is the suitable starting plant material for micropropagation. Half MS salt strength supplemented with 0.6 mg L–1 BA is the best for shootlets multiplication. Pre-culturing the shootlet explants on MS medium supplemented with high concentration (4.0 mg L–1) of IBA then subcultured on half MS gave the highest root numbers and length. Acclimatized plants showed no variation differences with the mother plant by using PCR techniques RAPD or ISSR.

SIGNIFICANCE STATEMENTS

This study developed a substantial and sustainable protocol for micropropagation of K. senegalensis, including shoot induction and shoot proliferation to establish a laboratory archive of juvenile clones and root formation to generate plantlets of the same clones. Thus, this study will help the researcher to the production of African mahogany tree with multi uses of the environment in huge numbers as there are very few studies that have been carried out on it.

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