An Efficient Propagation Approach to Forcing Softwood Shoots from Epicormic Buds and Subsequent Rooting of Paulownia elongata S. Y. Hu

The current research describes the multiplication of Paulownia elongata S. Y. Hu, a timber plant, through the forcing of softwood shoots from epicormic buds under glasshouse conditions in spring and fall seasons. Different growth media were used to compare their effect on the forcing potential of epicormic buds. For this, 25–30-cm-long and 1.2–2-cm-diameter stem segments taken from the lower juvenile portion of a mother plant were placed horizontally in flat trays containing media, i.e., sterilized well-moistened sand, peat moss, perlite, and vermiculite individually. Furthermore, 4–6-cm-long forced softwood shoots were detached and treated with various concentrations of IBA (indole-3-butyric acid) and NAA (α-naphthyl acetic acid) either individually or in combinations for subsequent rooting. The response of shoot forcing was better in spring as compared to fall in terms of shoot length (cm), and number of shoots or leaves; however, an earlier bud break was observed during fall after 30 days of the initial experiment. The use of peat moss and vermiculite proved to be equally suitable for early bud break in both seasons, whereas in terms of shoot and leaf number as well as the shoot length (cm), the best outcome was observed in sand. Best rooting was observed at 3 gL−1 IBA + 3 gL−1 NAA in terms of root number per shoot, root length (cm), and days to root initiation while using sand as the growth medium after 50 days of the rooting experiment. The successfully established plantlets were further shifted to soil at Botanical Garden, University of the Punjab, Lahore, Pakistan, exhibiting an 87.5% survival rate. On the basis of the results obtained, it may be concluded that reasonable softwood shoot forcing in P. elongata may further be exploited for its mass scale nursery propagation as well as use in future in vitro studies.


Introduction
Paulownia elongata S. Y. Hu is a hardwood tree species.Tis exceptional genotype produces a lot of biomass, making it economically signifcant and a great source of feedstock for the biofuel sector [1,2].It controls soil and air pollution, facilitates land reclamation, and produces eye-catching fowers as an ornamental plant [3].It is a deciduous tree, indigenous to China, and has since been introduced to Australia, Brazil, Europe, Japan, and the United States [4,5].In China, its growing habit ranges from temperate to nearly tropical zones [6].
Te wood derived from Paulownia is valued for its physical toughness, light to medium fne-textured grain, and nice color as well as the fact that it is light and fexible but does not crack or distort easily [7].Its wood is frequently used to make toys, furniture, plywood, aircrafts, and musical instruments [8].As the demand for wood and wood products is increasing worldwide on a daily basis, it is crucial to meet the demand, especially in a country with a low forest land coupled with high pace of deforestation.In order to address the issues of demand for wood as well as CO 2 emissions [9], it is necessary to look for prized tree species like Paulownia elongata with rapid growth and high CO 2 absorption capacity.
Paulownia has already gained interest as short-rotation and potential bioenergy plant that can be used for both transportation fuel and carbon sequestration [10,11].Terefore, it is very important that this commercially signifcant plant needs to be domesticated outside of its natural habitats, such as in Pakistan where wood and energy demand is ever rising.
It is possible to reproduce Paulownia through seeds, but there are some limitations including seed dormancy and slower growth of seedlings than the plants developed through root or shoot cuttings or tissue culture [12].In comparison with root cuttings, stem cuttings were considered quite challenging to grow [13].Nonetheless, there are associated problems with growing root cuttings as well, such as physical injury to the root cortex and epidermis, deterioration from pathogen attack, and exposure to high temperatures [14].It appears that none of these conventional propagation techniques are suitable for creating signifcant quantities of planting materials for the chosen elite trees [15,16].As a result, other vegetative propagation techniques such as softwood shoot forcing are essential to be worked out for Paulownia's clonal propagation.
A variety of commercially signifcant but recalcitrant woody plant species can be multiplied by forcing softwood shoots from epicormic buds of perennial plants [17].In reality, latent (epicormic) buds are dormant meristematic tissues that were established during the juvenile stage of plant growth, which produce xylem and phloem during the favorable conditions to produce softwood sprouts [18].Furthermore, it produces a relatively cleaner juvenile plant material with easier disinfection compared with explants obtained from feld-grown plants.Te word "cleaner" refers to the fact that in comparison with the plant shoots obtained from the feld, the softwood shoots derived from the epicormic buds grown in a controlled environment of a glasshouse are relatively cleaner and, thus, better suited for further greenhouse or in vitro manipulation although they also need surface sterilization if the intended use of these softwood shoot-derived plant materials is, indeed, in vitro [19,20].Tis approach has been used to vegetatively propagate several woody plants successfully such as Fraxinus pennsylvanica Marsh.and Acer saccharinum L. [17], Tectona grandis L. [21], and Gingko biloba L. [22] for the sustainable availability of relatively healthy and cleaner explants for their in vitro establishment.On the other hand, there is a dearth of such research on Paulownia elongata S. Y. Hu that was chosen for this study as no published study on its softwood shoot forcing from the epicormic buds is currently accessible.
In this regard, forcing softwood shoots from epicormic buds was intended to be investigated as a novel method to produce young, disease-free Paulownia elongata plants as well as explant source materials by making use of various growth media (sand, peat moss, vermiculite, and perlite).Softwood shoots of Paulownia elongata as produced in this study can also be further used in in vitro research.Additionally, diferent IBA (indole-3-butyric acid) and NAA (α-naphthyl acetic acid) concentrations were employed to examine the impact on the rooting capacity of forced softwood shoots using diferent growth media.Successfully rooted plantlets were then acclimatized and established in the feld conditions.

Softwood Shoot Forcing Using Stem Segments.
For softwood shoot forcing from epicormic buds, large stem portions were removed from the lower juvenile parts of 3year-old trees [Paulownia elongata S. Y. Hu, Taxon I.D # 200020795 (Quart J. Taiwan Mus. 12 : 41. 1959)] established at a local nursery at Model Town, Lahore.Tese trees were initially grown from seeds imported from China National Tree Seed Corporation, Jia 34 # Shenggunanli Chaoyang district: Beijing 100029, and the stock established in the growing area located some 15 km south-east of Lahore before their shifting to the retail nursery mentioned above.Te stem portions were then cut into 36 sections, 25-30 cm long and 1.2-2 cm in diameter, three each to be placed horizontally, half embedded in a well-moistened specifc medium preflled in an iron-alloy tray (55 × 32 × 6.6 cm).Twelve fat trays were used, three trays (three replicates) for each of four growth media (sand, peat moss, perlite, and vermiculite) with 3 logs per replicate.Te four tested media in this study were local commercially available coarse sand (bulk density 1.6 g•cm −3 , total porosity 35%, and pH 5.5-6.5),peat moss (Nord Agri Ltd.Elizabetes iela, Riga, LV1001, Latvia, bulk density 0.086 g•cm −3 , total porosity 80%, and pH 5.0-5.6),perlite (Gulf Perlite Dubai, UAE, bulk density 0.1-1.1 g•cm −3 , total porosity 70-85%, and pH 6-8.5), and vermiculite (Gulf Vermiculite Dubai, UAE, bulk density 0.07-0.1 g•cm −3 , total porosity 80-85%, and pH 7-7.5), thus making a total of 12 trays with each medium replicating thrice and placed under glasshouse conditions mentioned below.
Two sets of softwood shoot forcing experiment were performed in the third week of February 2020, with an average temperature of 18 °C and 55% relative humidity (RH) as well as in the last week of September 2020, with an average temperature of 36 °C and 70% RH.A 16-h photoperiod and a light intensity of 200-250 μmol•m −2 •s −1 were maintained.Te same experiment was repeated in mid-February 2021 with 17 °C average temperature and 60% RH and in the end of September, 2021 with an average temperature of 39 °C and 75% RH.Te global positioning system coordinates (GPSs) of the experimental site were 31 °30′04.4″Nand 74 °18′27.7″E.Regular watering to these stem sections was done manually with 650 ml water per tray per day with the help of spray bottles.On alternate days, 0.18% H 2 O 2 solution was sprayed in order to protect stem sections from microbial or fungal growth by following the method of Aftab et al. [17].Data were tabulated for days to bud break, shoot number, number of leaves per shoot, and length (cm) of softwood shoots after 30th day of the initial experiment.

Rooting Potential of Harvested Soft Wood Shoots.
After 30 days of the experiment's initiation, epicormic softwood shoots of 4-6 cm in length were removed from the stem sections with some basal tissue for the rooting response.Tese shoots were taken during April 2020 and March 2021.Basal ends of the excised shoots were placed in distilled water instantly after cutting to avoid drying, and the larger leaves were cut into half transversally to reduce 2 Scientifca transpiration.Te treatment of the basal parts of forced softwood shoots was done by dipping them for 40 seconds in either 1, 2, or 3 gL −1 solution of NAA (Sigma-Aldrich ® )] or IBA (Sigma-Aldrich ® ) alone or in combination.Te treated shoots were placed vertically to an anchorage of 2-2.5 cm in 9-cm-deep and 11.5-cm-wide plastic pots flled up with little more than three-fourth sterilized sand.Tese plastic pots were covered with perforated polythene bags of appropriate size to maintain humidity, which were then placed on racks equipped with cool white forescent 40-watts tube lights (Philips Pakistan Ltd.Karachi) at 25 ± 2 °C under 16-h photoperiod (32 μmol•m −2 •s −1 ).Foliar water spray on these shoots was also carried out every day, while 0.18% H 2 O 2 (v: v) solution was sprayed on alternate days.Data were collected for days to root induction, root number, and root length (cm) after 50 days of rooting experiment initiation.

Acclimatization.
Rooted softwood shoots were then shifted to earthen pots (14 cm wide and 11 cm deep) each flled with course sand, garden soil, and peat moss (2 : 1 : 1; v/v) for 5 weeks to establish them further in the glasshouse.Te moisture content of the earthen pots was maintained by regular watering as well as by covering the pots initially (for 3 weeks) with polythene bags of appropriate size.After 5 weeks, the plants in pots were shifted to exposed nursery conditions (outside the glasshouse) for another 2 weeks.Te successfully established plants were further transplanted in soil at Botanical Garden, University of the Punjab, Lahore.Transplantation survival was noted.

Experimental Design and Data Analysis.
For this study, a completely randomized design was adopted.Softwood shoots were forced from epicormic buds with three replicates of four growth media during spring and fall 2020 followed by its subsequent run in 2021.Similarly, two independent trials were conducted for the rooting potential of softwood shoots in pots using nine auxins treatments each with three replicates (pots) with one shoot per pot in two independent trials.Te data were subjected to the analysis of variance (ANOVA) and t-test (p � 0.05) using SPSS Release 21.0.PCA for interactive correlation between variables was done using OriginPro ® 2024.

Results and Discussion
3.1.Softwood Shoot Forcing Using Stem Segments.Results on softwood shoot forcing were quite comparable in both yearly runs of the experiments, i.e., the one conducted in the spring and fall of the year 2020 and the other during the same time in 2021.In both seasons, softwood shoot forcing was successful; however, an experiment conducted in spring (February) resulted in better growth parameters in terms of shoot length (cm), shoot, and leaf numbers (Figures 1-4).Similar to our fndings with Paulownia elongata, Preece et al. [23] had observed earlier that the optimal time for softwood shoot forcing using stem portions of various plant species, e.g., oak-leaf hydrangea, common lilac, and sugar maple, is from midwinter until the end of spring, depending on the kind of woody plants.However, forcing of softwood shoots is also possible in some plant species until fall.It is probably due to the dormancy of epicormic buds prior to the onset of the winter season in response to environmental stimuli [24].In plants, the control of dormancy is believed to be because of genetic, physiological, environmental, and hormonal factors [25,26].After passing through the chilling temperature, normally dormancy is released as the cell cycle, hormonal activity, and starch degradation into soluble sugars begin [27,28].It is believed that woody plant species of temperate regions have to undergo the dormant stage during the winter season to survive under unfavorable conditions and then start to grow again in spring under suitable temperature and humidity [29].
During the spring time experiments, considering overall performance, sand proved to be the most favorable growth substrate for softwood shoot forcing using stem sections as compared to perlite, peat moss, and vermiculite in the current study (Figure 1).Minimum days to bud break (10.33) were noted in peat moss, while maximum days (12.67) were in sand (Figure 1(a)).Softwood shoot forcing in peat moss was signifcantly diferent (p � 0.05) from sand, but no statistical diference was observed with vermiculite and perlite.A higher number of shoots were observed in sand (9.33) whereas the least (4.00) in peat moss (Figure 1(b)).Te number of softwood shoots in sand was signifcantly diferent from peat moss, perlite, and vermiculite in which perlite was also signifcantly diferent from peat moss and vermiculite.According to Pramanik et al. [30], cocopeat supported the highest growth of plantlets in external conditions as compared to perlite, vermiculite, and sand in Bacopa monnieri L. Panigrahi et al. [31], on the other hand, found the mixture of vermiculite, sand, and organic matter (1 : 1 : 1 v/v) as the best growth substrate for plants instead of using them alone for Chlorophytum borivilianum Santapau & R. R. Fern.Best growth parameters were noted under natural habitat using garden soil, sand, and farmyard manure (2 : 1 : 1; v/v) in Gloriosa superba L. [32].Gantait et al. [33] proved course sand to be the most suitable growth medium for the optimal multiplication of Bambusa balcooa Roxb through culm cuttings in comparison with other three media (soil, vermiculite and 1 : 1 (v/v) soil + sand).In our study, the longest softwood shoots (6.23 cm) were observed in sand while the smallest (2.35 cm) in vermiculite (Figure 1(c)).Data with respect to the shoot length in sand have also shown a statistical diference from all other growth media used.Vermiculite had the least efcient efect on softwood shoot forcing exhibiting rotting or necrosis of the leaves probably due to the defciency of O 2 by excessive water retention [34].Similarly, a maximum leaf number (10.23) was observed in sand whereas the minimum (4.63) in vermiculite (Figure 1(d)).A signifcant diference was observed in sand in terms of number of leaves from vermiculite, perlite, and peat moss.Similar fndings were observed by Noreen and Aftab [22] for Ginkgo biloba.Tey also observed a maximum number of leaves and a maximum shoot length in sand while a minimum in vermiculite.

Scientifca
Te experiment setup in fall has shown a similar response but with a lesser number of softwood shoots and leaves and a decrease in the shoot length (Figures 1(b)-1(d)).However, an early response to bud break was noted during fall as shown in Figure 1(a).Moreover, similar to the springtime experiment, sand was found to be a better medium supporting a better shoot length, and shoot and leaf number during fall as well, except for the bud break response, which was earliest in the case of vermiculite (Figure 4).

Principal Component Analysis (PCA).
Te interaction of the two seasons (spring and fall) and diferent growth media along their correlation with the variables is displayed in a principal component analysis (PCA)-based biplot (Figure 2).Te combined biplot displayed 94.14% of the total variation, with PC1 contributing the maximum variation (78.45%) and PC2 showing 15.69%.Te parameters presented in the same quadrate are nonsignifcantly diferent.Among diferent growth media, sand was noted as the signifcant medium in both seasons presented in the same quadrate and showed a positive correlation with SN, SL, and LN.Peat moss and vermiculite showed negative correlation with observed parameters.Perlite gave positive correlation with DB.Overall, the spring season resulted in better than fall for most of the parameters.Terefore, this technique can be employed to propagate Paulownia elongata as multipurpose tree round the year even during the fall season to fulfll the demand for high-quality wood along with other benefts such as a source of solid biofuel, role in pharmaceutical industry, and ornamental aspect in regions where this plant is exotic.

Rooting Potential of Harvested Softwood Shoots.
Sand was found to support quite reasonable rooting of softwood shoots in 50 days (Figure 5) compared with peat moss, perlite, or vermiculite used either individually or in combinations (data not shown).As Karrenberg et al. [35] reported that the characteristics of the growth substrates being employed were the key determinants of root anchoring, this might have been the case with sand in our study that proved to be a good substrate to support rooting in Paulownia.Sand was also found to be the best anchoring medium in a similar work by Akram and Aftab [21] and Noreen and Aftab [22] on Teak and Ginkgo, respectively.Gaintait et al. [33] also concluded that the sand gave the quickest root initiation presumably due to high aeration characteristics than vermiculite.Te porosity of 35%, ≤2 mm particle size, enough 4 Scientifca moisture holding capacity and a sufcient oxygen difusion rate are the characteristics of sand [36,37].Sand may have been efective because of these characteristics both for forcing softwood shoots and for later root induction in Paulownia.Furthermore, sand provides a sufciently permeable environment for root establishment without sufering injury as reported for Azadirachta indica A. Juss [38].Similarly, studies of Pill and Goldberger [39] indicated that sand was an efcient rooting medium as compared to other media such as perlite.Perlite has got a larger particle size and 70-85% of total porosity, which may not furnish sufcient anchorage to little harvested forced softwood shoots.
Our fndings with regard to rooting were also in line with those of Sardoei [40] who reported signifcant rooting in sand as compared to perlite in Psidium guajava L. However, Stuepp et al. [41] have also recommended vermiculite and carbonized rice hulls (1 : 1) for efcient root induction in mini-cuttings of Paulownia fortunei var.Mikado stumps.
In this study, individual treatments of either NAA or IBA (1 gL −1 each) were least efective in terms of root length, root number, or days to root initiation.Increasing concentrations of NAA and IBA from 1 to 3 gL −1 supported better rooting, but it was with both hormones in combination that supported the most efcient rooting.Earliest root initiation (45.22 days) was recorded by using  Scientifca 5 3 gL −1 NAA, while a delayed response (59.13 days) was exhibited at 1 gL −1 NAA.Higher concentration of NAA (3 gL −1 ) proved to be more efective with respect to the root number (3.66 ± 0.06) and root length (6.07 ± 0.43 cm).
Using IBA, the results were also in line with NAA.A delayed response to root initiation (57.18 ± 0.09 days) was observed at 1 gL −1 IBA, while an early rooting response (37.73 ± 0.08 days) was observed at its 3 gL −1 level.Similarly, the maximum root number (4.36 ± 0.24) and length (7.55 ± 0.03 cm) were also observed at 3 gL −1 IBA as shown in Figure 6.Te results indicated that IBA was more effective for root development than NAA when used at the same concentrations.Similar results regarding the role of IBA on adventitious root induction of Gingko biloba were recorded by Pandey et al. [42].In fact, Zayova et al. [43] also observed that IBA was more efective for root development than NAA and IAA with the best results at 0.5 mgL −1 IBA using ½ MS (Murashige and Skoog) medium [44].Noreen and Aftab [22] reported that a rooting response in the softwood shoots derived from epicormic buds of Ginkgo biloba was best on sand after IBA treatment.Shepherd et al. [45] worked on the rooting of cuttings obtained from epicormic shoots of tea tree (Melaleuca alternifolia (Maiden & Betche) Cheel) under a variety of propagation circumstances using IBA.Muhamad et al. [12] also concluded that IBA was more productive than NAA for in vitro rooting in Paulownia spp.when supplemented with MS.NAA and IBA in combination (3 gL −1 each) resulted in best rooting in terms of maximum number of the longest roots per plantlet (Figure 6).Earliest root initiation (34.45 days), maximum number (7.23 ± 0.40), and longest roots (23.78 ± 0.27 cm) were obtained at 3 gL −1 each of NAA and IBA in combination.We support the fndings of Filipova et al. [46] who reported the combination of 0.5 mgL −1 NAA and 4 mgL −1 IBA for an efcient root development system in Paulownia.Our reported concentrations for the two growth regulators though diferent do indicate that a range of levels Scientifca of NAA and IBA may work for the rooting of this important tree species.Diferent combinations of IBA, IAA, and NAA have also been reported to be superior for the rooting of stem cuttings of Ginkgo [42,47].As the activation of polysaccharide hydrolysis is afected by auxins to release physiologically active sugars and, therefore, energy for root induction-related meristematic activity [48], the combined efect of IBA and NAA as reported in this study might have triggered better polysaccharide hydrolysis, resulting in an efcient rooting in Paulownia.

Acclimatization of Rooted Plantlets.
As sand proved to be the best growth medium for rooting response, hardening of 40 rooted plantlets was initiated using sand enhanced with soil and peat moss (2 : 1 : 1 v/v) for further growth and establishment in the feld (Figure 7).Transplantation survival percentage was 87.5.A key component of a successful acclimatization was maintaining the humidity quotient, which was ensured by water spraying and subsequent covering the potted plantlets with polythene bags [49,50].According to Gantait et al. [33], using sand, farmyard manure, and cocopeat (1 : 1 : 1 v/v/v) increased the rate of survival of fresh roots in Bambusa balcooa by 100%.Cocopeat was found by Pramanik et al. [30] to be the best substrate for Bacopa monnieri, with a 100% survival rate.In their investigation into the acclimatization protocol for Paulownia species, Muhamad et al. [12] discovered that the best growth substrate mixture with 100% survivability was sand and peat moss (1 : 2 v/v).Akramian et al. [51] reported cocopeat and perlite (2 : 1 v/v) as the best horticulture substrate with 100% survivability while studying Philodendron cv.Birkin.

Conclusion
Te successful forcing of softwood shoots of Paulownia elongata and their subsequent rooting is a step towards future exploitation of this as-yet-unexploited technique for growing this important tree species outside of its natural habitat.We recommend using sand as the growth medium and the combination of IBA and NAA (3 gL −1 each) for their rooting.As sand is readily available and considerably less expensive than other growth media, it may also fulfll the requirement of cost-efectiveness, a major consideration in the mass scale propagation of ornamental or economically important plants.Spring in both years is proved to be the best time to force softwood shoots probably due to suitable mild temperature and relative humidity.Te fndings of the current investigation highlight the fact that the propagation of Paulownia elongata through softwood shoot forcing and its subsequent root induction holds good promise for the mass propagation of this plant species.In addition, this method can be employed to obtain a relatively cleaner plant material and suitable explants for the use in various aspects of in vitro research.
Te following suggestions for future research may be considered: (i) Relatively cleaner and juvenile explant source for in vitro studies.
(ii) It can be employed for the propagation of Paulownia under nursery conditions and then used for further studies.(iii) As this elite plant is known for precious wood, largescale production can be achieved through this effcient technique.(iv) Softwood shoot forcing experiment can be compared under controlled culture conditions and a natural environment.(v) Diameter of the stem segments can be compared for shoot vigor.

Figure 1 :
Figure 1: Efect of sand, perlite, peat moss and vermiculite on various growth parameters of softwood shoots forced from epicormic buds in Paulownia elongata S. Y. Hu. (a)-Days to bud break, (b)-shoot number, (c), shoot length (cm) and (d)-leaf number values shown are means (± SE).Variation in alphabets represents statistical diference among growth media (p � 0.05).

Figure 2 :
Figure 2: Principal component analysis (PCA) biplots presenting the interactive correlation of diferent growth media along with two seasons (spring and fall) on measured growth parameters (days to bud break: DB; shoot number: SN; shoot length (cm): SL; leaf number: LN).

Figure 3 :
Figure 3: Forcing of softwood shoots from epicormic buds using diferent growth media in Paulownia elongata during spring season after 30 days of initial experiment.(a) Forced softwood shoots in sand.(b) Large stem segments showing the emergence of softwood shoots in perlite.(c) Browning of stem segments and less number of shoots in vermiculite.(d) Minimum shoot number in peat moss.Bar � 2 cm.

Figure 4 :Figure 5 :Figure 6 :Figure 7 :
Figure 4: Forcing of softwood shoots from epicormic buds using diferent growth media in Paulownia elongata during the fall season after 30 days of the initial experiment.Overall, growth media: (a) sand, (b) perlite, (c) vermiculite, and (d) peat moss, gave lesser response during fall time experiment as compared to spring in terms of shoot number, shoot length, and number of leaves.Bar � 4 cm.