Effect of Indole-3-Butyric Acid on Clonal Propagation of Swietenia macrophylla through Branch Cutting

The study discloses the scopes of clonal propagation Swietenia macrophylla through branch cutting treated with IBA. A total of four hundred cuttings were used with four replications to assess the rooting ability. The study exposed significant (p < 0.5) difference of rooted cuttings among the treatments. The highest (62.51%) rooting percent was observed in cutting with 0.4% IBA treatment. In addition, root number and its length per cutting were increased with increasing IBA concentration (p < 0.5). The experiment showed significant (p < 0.5) difference of sprouting among the treatments.The highest (67.27%) percent of sprouting was observed in cuttings with 0.4% solution, which showed a similar fashion of percent of rooting. The study also showed significant (p < 0.5) difference of shoot number per cutting, the length of the longest shoot, and number of leaves per cutting among the treatments. The overall survival of rooted cuttings after transfer to polybag significantly (p < 0.5) differed among the cuttings treated with different IBA solution.The highest survival percentage (69.67%) was observed in the cuttings rooted with 0.4% IBA treatment and the lowest (55.6%) survival was found in cuttings treated with control. The use of 0.4% IBA treatment is suggested for rooting of juvenile leafy branch cutting of S. macrophylla.


Introduction
Swietenia macrophylla King, big-leaved mahogany, a timber species of the greatest economic value of Maliaceae family, is indigenous to the mainland of Central and South America, from Mexico to Peru, Bolivia, and Brazil, which makes it the most extensively dispersed mahogany species [1][2][3].It is an exotic tree species in Bangladesh and becoming close to nature in some part of domestication.It was first introduced in Indian subcontinent in 1872 from Brazil [3,4].S. macrophylla, one of the significant commercially important neotropical timber species [5], grows well from sea level to 1500 m altitude where annual rainfall is between 1400 and 2500 mm with a mean annual temperature of 20-28 ∘ C. The plantation of S. macrophylla performed better on deep, nutrient rich, well drained soil with a pH range of 6.5 to 7.5 but it cannot tolerate water logged conditions.It can be found on alluvial, volcanic, lateritic soils, heavy clays, and soil derived from limestone, granite, and other types of rocks and even on shallow rendzinas within its natural range of habitat [3].S. macrophylla is considered as luxurious timber species for high class furniture and cabinet manufacturing.The bark has been used as a febrifuge and also for dying and tanning leather [3].The seed of this species is used for extracting oil which is edible and performs antimalarial, antibabesial, and antidiarrhoeal activities [6].The seed of this species is used as a popular ingredient of cardiac and blood sugar reducing medicines [7].Raw seeds of S. macrophylla are used for diabetes and hypertension treatment in many countries.Traditionally, seeds are chewed or crushed and swallowed in India and in Malaysia for those purposes [8].This is also practiced in Bangladesh (personal observation).Antioxidant, antidiabetic, anti-inflammatory, antimutagenic and antitumor and antifungal, and antihyperglycaemic activities have been reported in many research articles of this species [6,7,9,10].
Clonal propagation proposes the prospects, changes, and opportunities to generate a dependable and sufficient supply of better-quality planting stock locally, timely, and quickly.Various tropical tree species have been fruitfully propagated by branch cuttings [11].This approach is painstaking the model system for a quick development [12,13] through maintaining certain desirable traits of a threatened species at a low cost using nonmist propagator [14], although many species differ in their rooting requirements [15] and rooting success differs with different concentrations of individual auxins [14,16].In addition, age of cutting and donor plant also influences over the rooting ability of cuttings.Usually the more juvenile the cuttings, the greater the success in rooting [17][18][19][20][21][22].Moreover, rooting success, rooting speed, root length, and root number reduce with increasing the magnitude of maturity of donor plant [21,22].But no serious attempt was taken to propagate Swietenia macrophylla.So, it is crucial to determine the appropriate treatment for optimum rooting of this species for vegetative propagation.This species is very important from different points of view (ecological, economical, aesthetic, etc.) for Bangladesh.Indigenous species are being replaced by exotic fast-growing species in the rehabilitation of degraded tropical forests.A remarkable number of species are required to restore degraded ecosystems properly, particularly in the tropics.However, the irregular flowering and fruiting habits and the recalcitrant nature of the seeds of many species make seed collection problematic.To overcome the unpredictable supply of seeds, many studies have been conducted to investigate the ability of cuttings to propagate and successful rooting has recently been reported for a considerable number of Southeast Asian rainforest species [23].Bangladesh initiates vegetative propagation for tree improvement since 1977.Several methods including side veneer and whiptongue grafting, forkert, Patch and T-budding, and adult and juvenile and nonleafy cuttings and air layering have been tried on 15 forest species but the success varied among the species [24].Vegetative reproduction could open up a new horizon for the multiplication of species for large-scale plantations [25].The species which propagated by vegetative propagation can show disease resistant property because they are the same genotype as the mother plant.For short rotation and industrial species vegetative propagation is an alternative option [26] for conservation.With an aim to assist preserving this species from economic and biodiversity points of view, this experiment was designed to investigate the possibility of vegetative propagation of Swietenia macrophylla by investigating the effects of different IBA concentrations on the rooting ability of juvenile branch cuttings from mother tree.Several studies of S. macrophylla have been conducted (Table 1) but studies related to macro vegetative propagation especially rooting of branch cutting are very scant.Therefore, an attempt was taken to carry on this experiment.The aim of this study was to explore the scope of clonal propagation of Swietenia macrophylla by branch cutting.The main objectives of this study were (i) to find out the effect of IBA treatment on rooting and sprouting ability in juvenile stem cutting and (ii) to examine the survival percentage of rooted cuttings treated with different IBA concentration.Stem cutting [34] In vitro propagation Embryo culture [35] In vitro propagation Tissue culture (seed) [36] In vitro propagation Tissue culture (shoot) [37] Somatic embryogenesis Embryo [38] 2. Materials and Methods

Study Site.
The experiment was conducted in the forest nursery of Forestry and Wood Technology Discipline, Khulna University, Bangladesh.The study area is a low (4 m above the sea level) fertile floodplain dominated by alluvial soils [27].The geographic position of the study area is 22.802 ∘ N latitude and 89.533 ∘ E longitude.The landscape of the study area is located in the southwestern part of Bangladesh near the Sundarbans, the largest unit mangrove forest in the world.The climate of this area is documented as subtropical in nature, in particular winter (November-February), summer (March-mid-June), and monsoon (late June-September), the distinguishing seasons, approximately similar to the other parts of the country.In winter the temperature variation is very low (7 to 12 ∘ C) but in summer it rises from 25 to 32 ∘ C. Sometimes it might be increased up to 40 ∘ C [27].

Construction of Propagator.
The experiment on clonal propagation was carried out in a low cost nonmist propagator.
The propagator was constructed of wooden frames and lined with transparent polythene sheet described by Leakey et al. [14] and modified by Kamluddin et al. [39].The structure was rectangular (length: 1.8 m × width: 1 m × height: 1.5 m) in shape covered with white polythene sheet and closely fitted with a lid, so that the lid was completely airtight.The whole structure was placed on the concrete floor of the nursery.The base of the structure was completely watertight.The polythene base of the propagator was covered with 10 cm thick layer of moist coarse sand mixed with successive layers of fine gravels and small stones.This layer acted as rooting media supported by perforated plastic tray.The propagator was kept under the shade to avoid excessive heat accumulation.Mean temperature within the propagator during rooting was maintained between 25 to 32 ∘ C. To facilitate gas exchange, the propagator was opened for a short time in the morning and in the late afternoon.When the lid of the propagator was opened for inspection, a fine jet of water spraying was used to maintain a low vapor pressure deficit inside the propagator for a enduringly humid environment throughout the propagation period.Air temperature both inside and outside the rooting tent was measured by the thermometers.

Cutting Materials and Design of the Experiment.
Twentyfive mother trees were identified and tagged at home gardens of Jessore, Bangladesh.A total of sixteen healthy branch cuttings per mother trees of closely uniform sized (on average 12-15 cm in length) with one or two leaves trimmed with scissors up to fifty percent of total leaf area were collected from 6-to 10-year-old candidate plus trees for the assessment of rooting behavior of juvenile branch cuttings.Cuttings were then enclosed with polythene bags to protect moisture loss and were then quickly transported to the propagation site.Mother trees, length, and diameter of cuttings were indifferent among the treatments to avoid the variation other than treatments.The cuttings were then immersed shortly in Dithane M45 solution to avoid fungal diseases and kept under shade for thirty minutes in open air.The cuttings were treated with four treatments of indole 3-butyric acid (IBA) solution for a moment.The treatments were T0 (control), T1 (0.2% IBA), T2 (0.4% IBA), and T3 (0.8% IBA).The treated cuttings were placed in the propagator with completely randomized design.Twenty-five cuttings were used with four replications in each treatment.So a total of 400 (25 × 4 × 4) cuttings were placed under four different treatments with four replications.

Observation on Budding and
Rooting.The assessments of rooting success were carried out weekly after the first two weeks of cutting placement in the propagator.A cutting was considered as rooted when it had at least one root exceeding 1 cm long.The rooting of cuttings in the propagator completed within six weeks after putting the cuttings into the rooting media in propagator.The cuttings were subjected to weaning before transferring them into polybag.For weaning, the propagator was kept open at night every alternate day for a week and then every night for another three days.This process was also carried out every alternate day for a week at day time and then every day for another three days to harden the rooted cuttings in prevailing adverse environment outside the propagator.All the rooted cuttings were transferred into polybags filled with a mixture of forest soil and decomposed cow-dung in a ratio of 3 : 1.The observations on sprouting, rooting, number of sprouts/roots/leaves per cuttings, and length of the longest root and shoot were recorded before planting the rooted cutting into polybags.The rooted cuttings were then allowed to grow for ninety days in open sun to count the survival percentages.Watering was done once a day at late afternoon.Weeding was done when necessary.No additional fertilizer and pesticide were used.

Rooting Ability of Cuttings.
The cuttings of S. macrophylla rooted well (Figure 1) in the present study.Rooting percent was varied from 37.24 to 62.51% among the various IBA treatments.The highest rooting percent was observed in 0.4% IBA concentration (T2) and lowest rooting percent was observed in control (T0) (Figure 2).The rooting percent was significantly ( < 0.05) increased with the IBA treatment (Table 2).Again the number of roots per cutting was significantly ( < 0.05) increased with increasing the concentration of IBA.The highest average number of roots per cutting was observed to be 6.68 in 0.8% IBA and the lowest average number of roots per cutting was observed to be 1.26 in control.The average longest root was also significantly ( < 0.05) increased with increasing the concentration of IBA.The average longest root was observed to be 1.56 cm, T0 T1 T2 T3 3.28 cm, 7.66 cm, and 14.12 cm in control, 0.2% IBA, 0.4% IBA, and 0.8% IBA treatments, respectively (Table 2).

Sprouting Ability of Cuttings.
The cuttings of S. macrophylla sprouted well (Figure 1) also in the present study.Sprouting percent was varied from 34.52 to 67.27% among the various IBA treatments.The highest sprouting percent was observed in 0.4% IBA concentration (T2) and lowest sprouting percent was observed in control (T0) (Figure 2).The sprouting percent was significantly ( < 0.05) increased with the IBA treatment (Table 2).Again the number of sprouts per cutting was significantly ( < 0.05) increased with IBA concentration.The highest average number of sprouts per cutting was observed to be 4.81 in 0.8% IBA and the lowest average number of sprouts per cutting was observed to be 1.72 in control.The average longest root was also significantly ( < 0.05) increased with increasing the concentration of IBA.The average longest sprout was observed to be 8.21 cm, 14.16 cm, 15.34 cm, and 15.51 cm in control, 0.2% IBA, 0.4% IBA, and 0.8% IBA treatments, respectively.The average number of leaves per cutting was also significantly ( < 0.05) increased with IBA treatment (Table 2).The average number of leaves per cutting was 6.72, 7.51, 10.43, and 10.74 in control, 0.2% IBA, 0.4% IBA, and 0.8% IBA treatments, respectively (Table 2).

Survival of Rooted Cuttings.
The overall survival of rooted cuttings was very high 90 days after transplanting from propagator to polybag.The rooted cuttings treated with IBA significantly ( < 0.05) increased the overall survival of the rooted cutting (Table 2).The highest survival was found to be 69.67% in 0.4% IBA treatment and the lowest survival of 55.6% was found in control (Figure 2).

Discussion
The use of different rooting hormones for the protocol development of stem/branch cuttings of forest tree species is widely documented [14,17,[40][41][42][43][44][45].Husen and Pal [17] investigated the effect of age of donor plants and use of auxins on rooting in single noded leafy stem cuttings of teak (Tectona grandis).They also carried out another study on effect of branch position and auxin treatment on clonal propagation of Tectona grandis [43].The rooting hormone applied for cuttings, especially IBA, has a significant importance in rooting of various tropical forest tree species [13,16,17,[43][44][45][46][47][48][49].Berhe and Negash [48] reported rooting response through branch cutting of Juniperus procera by using IAA, IBA, and NAA.Negash [49] conducted a successful clonal propagation on threatened Juniperus procera by using different concentrations of IBA.Amri et al. [18] investigated the effects of age of donor plant, IBA treatment, and cutting position on rooting ability of stem cuttings on Dalbergia melanoxylon.But the concentration of using IBA for rooting ability varied among species.In our present study, we observed that highest rooting percent with the application of 0.4% IBA also revealed no significant difference between using 0.2% IBA and 0.4% IBA on rooting percent but further increase of IBA concentration (0.8%) lead to decreasing the percent of rooting.The study revealed very much similar result with Abdullah et al. [44].They observed highest percent of rooting of Psidium guajava with the application of 0.2-0.4% IBA but it reduced with increasing of IBA concentration.The similar results were also observed by Aminah et al. [50] on Shorea leprosula rooting percent.They noticed that the application of IBA significantly increased the rate of root emergence in single noded leafy stem cutting.This result is a contrast to Opuni-Frimpong et al. [19].They observed that rooting percent of Khaya anthotheca and Khaya ivorensis increased with increasing IBA concentration.Ofori et al. [47] observed that IBA has no overall effects on rooting percentage on Milicia excelsa through leafy stem cuttings but they found a strong negative correlation ( = 0.90,  < 0.05) between rooting percentage and auxin concentration.Baul et al. [51] found the highest rooting percentage for 0.4% IBA concentration followed by control on Stereospermum suaveolens.Higher rooting percent for our concerned species under low IBA concentration may be due to the higher level of endogenous auxin content in the cuttings.Hartmann et al. [52] documented variation of auxin content at time of severance.The present study revealed significant difference of average number of roots per cutting and the length of longest root with increasing IBA concentration.Abdullah et al. [44] also found similar trend of rooting ability on Psidium guajava.Various studies on Dalbergia melanoxylon [18], Tectona grandis [43], and Juniperus procera [49] also documented significant difference of root number per cutting among IBA treatments.Husen and Pal [43] documented rooting and sprouting percent, mean leaves number, mean shoots number, and shoot and leaf length, and mean number of roots and length per cutting were significantly influenced by the branch position and auxin treatment on Tectona grandis.Negash [49] observed significant difference of root number per cutting among the IBA treatment.He noticed that the number of rooted cuttings and the subsequent root number declined quickly when cutting was treated with 0.4% IBA. also noticed that mean root number increased over threefold in cutting treated with 0.4% IBA.In contrast, several studies on Pausinystalia johimbe [13] and Holarrhena pubescens [45] documented no significant difference of root number per cutting and the length of roots with IBA treatments.Ofori et al. [47] noticed that the mean number of roots per cutting was increased with auxin application, but there was no significant difference among the IBA treatments.It may be due to the fact that the content of endogenous auxin in the stem cuttings of some species decreases when taken from mature mother trees which may lead to reducing rooting ability [17,45].Baul et al. [51] reported that the differences in mean root number per rooted cutting on Stereospermum suaveolens were not statistically significant among IBA treatment ( > 0.1) but the length of the longest root for all treatment was statistically significant ( < 0.05).
The present study documented the highest percent of sprouting with 0.4% IBA treatment but number of sprouts per cutting and the length of longest sprout significantly increased with increasing IBA concentration.The average number of leaves per cutting also followed similar fashion.The consequence of auxin in promoting rooting of cuttings is well recognized [11,16,18,[43][44][45][46][47][48][49] whereas very few documents are available on the effectiveness of IBA in relation to sprouting.However Husen and Pal [43] carried out an experiment on effect of branch position and auxin treatment on clonal propagation of Tectona grandis and found similar results of highest percent of sprouts and the length of sprouts in 0.4% IBA treatment.They also found similar trends in relation to leaves number per cuttings.
The present study revealed that the survival of rooted cutting after transfer to polybag was significantly higher for the cutting treated with 0.4% IBA which is very much similar with the findings of Baul et al. [45] for Tectona grandis but in contrast, Baul et al. [51] on Stereospermum suaveolens and Minadawati and Rostawati [53] on Agathis loranthifolia documented highest survival percent with low concentration of IBA (0.2%) treatments.It may be due to the fact that the application of higher concentration of IBA treatments seems to be detrimental for survival for some species.Baul et al. [45] postulated that higher concentration of IBA treatment may have some negative impacts against naturally occurring growth hormones in the cuttings.

Conclusion
Swietenia macrophylla is very important from ecological, economical, aesthetic point of view for Bangladesh and clonal propagation through branch cutting might be effective method for its conservation.The present study highlights IBA treatments for maximum rooting, sprouting response, and survival of rooted cutting after transfer to polybag.The highest percent of rooting and sprouting was significantly higher in cuttings treated 0.4% IBA while number of roots per cutting, length of the longest root, number of sprouts per cutting, longest sprouts, and number of leaves per cutting were higher at 0.8% IBA treatment.But there is no significant difference with 0.4% IBA treatment expect in number of sprouts per cuttings.Survival of rooted cutting after transfer to polybag was significantly higher in cuttings with 0.4% IBA treatment.Therefore, considering all the results under different treatments, we recommended that branch cutting with 0.4% IBA treatment may be applicable for this species.However the genetic constituents in the propagules could be determined for future study.

Figure 1 :
Figure 1: Rooting ability of Swietenia macrophylla stem cutting under various IBA treatments.

Figure 2 :
Figure 2: Rooting, sprouting, and survival success of Swietenia macrophylla stem cutting under various IBA treatments.

Table 1 :
Study of seed germination, seedling growth, and propagations related with Swietenia macrophylla.

Table 2 :
Effect of IBA on rooting ability, sprouting ability, and overall survival of rooted cuttings of S. macrophylla.: ± indicates mean ± standard error and the same letter(s) indicate(s) no significant difference at  < 0.05. Note