Acta agriculturae Slovenica, 118/3, 1–9, Ljubljana 2022 doi:10.14720/aas.2022.118.3.1208 Original research article / izvirni znanstveni članek Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant Mawadda MEHERAT 1, Mohammad SHATNAWI 1, Rida SHIBLI 2, Tamara QUDAH 3, 4, Saida ABU MALLOH 3, Tamadour AL-QUDAH 5 Received July 03, 2019; accepted July 05, 2022. Delo je prispelo 20. julija 2019, sprejeto 5. julija 2022 1 Faculty of Agricultural technology, Al Balqa Applied University, Salt, Jordan 2 Al Ahliyya Amman University (and The University of Jordan), Faculty of Agricultural technology, Amman, Jordan 3 Hamdi Mango Centre for Scientific Research, University of Jordan, Amman 4 Corresponding author,e-mail: t.alqudah@ju.edu.jo 5 Department of Nutrition and Food Technology, Faculty of Agriculture, Mutah University, Karak, Jordan Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant Abstract: Tetragonolobus palaestinus Bioss (Aljalaton) is one of the Jordanian medicinal plants that can be used to treat stomach pain and some infections. This study was done in order to establish optimal in vitro propagation method for T. palaestinus. Factors of in vitro shooting, rooting, and ac- climatization of the in vitro Tetragonolobus palaestinus seed- lings were studied using different growth regulators. For in vitro shooting, different cytokinins including benzylamino purine (BAP), kinetin, TDZ, and zeatin were used in increas- ing concentrations (0.0, 0.3, 0.6, 0.9, 1.2, 1.5, and 2.0 mg l-1). Using benzylamino purine (BAP produced a maximum of 2.0 shoots/explants on Murashige and Skoog (MS) medium supplemented with 0.3 mg l-1. Moreover, the effect of different concentrations of IBA (indole-3-butyric acid), IAA (indole- 3-acetic acid), andnaphthalene acetic acid (NAA) was evalu- ated for in vitro rooting. The highest number of roots (4.06 roots/explant) was obtained on MS medium supplemented with 0.3 mg l-1 IBA. All of the plants (100 %) were grown nor- mally after the acclimatization process. Based on these results simple protocol of T. palaestinus in vitro culture was optimized for the first time which can be utilized to do more studies on cell culture and production of active secondary metabolites. Key words: acclimatization;, in vitro; shoot multiplica- tion;, rooting Klonsko razmnoževanje vrste Tetragonolobus palaestinus Bioss: jordanske zdravilne rastline Izvleček: Vrsta Tetragonolobus palaestinus Bioss (Alja- laton) je jordanska zdravilna rastlina, ki se lahko uporablja za blaženje bolečin v želodcu in zdravljenje nekaterih okužb. Namen raziskave je bil vzpostaviti optimalen način in vitro razmnoževanja te rastline. Preučevani so bili dejavniki in vi- tro gojenja (vkoreninjenja, tvorbe poganjkov) in aklimatizacije sadik te rastline z uporabo različni rastnih regulatorjev. Za in vitro tvorbo poganjkov so bili uporabljeni različni citokinini in sicer benzilamino purin (BAP), kinetin (TDZ) in zeatin v naraščajoči koncentraciji (0,0; 0,3; 0,6; 0,9; 1,2; 1,5 in 2,0 mg.l- 1). Uporaba benzilamino purina (0,3 mg l-1) je dala pri gojenju na Murashige in Skoog (MS) gojišču največ poganjkov, dva na izseček. Učinek različnih koncentracij rastnih regulator- jev (IBA-indol-3-maslene kisline, IAA -indol-3-ocetne kisline in naftalen ocetne kisline NAA) je bil ovrednoten pri in vitro vkoreninjenju. Največje število korenin (4,06 korenin/izseček) je bilo dobljeno na MS gojišču, z dodatkom 0,3 mg l-1 IBA. Vse rastline (100 %) so po obdobju aklimatizacije rastle normalno. Na osnovi teh rezultatov je bil prvič optimiziran enostaven pro- tokol za in vitro gojenje te vrste, ki bi se lahko uporabil v na- daljnih raziskavah na celičnih kulturah in produkciji aktivnih sekundarnih metabolitov. Ključne besede: aklimatizacija; in vitro; namnoževanje poganjkov; vkoreninjanje 2 Acta agriculturae Slovenica, 118/3 – 2022 M. MEHERAT et al. 1 INTRODUCTION Tetragonolobus palaestinus is a wild plant from the Fabaceae family. Its natural habitat can be found in the northern parts of Jordan on the rocks and meadow ar- eas (Afifi & Abu-Irmaileh, 2000). T. palaestinus is a her- baceous plant, it starts to grow after seasonal rainfall in the winter, and has a red flower and a small fruit (pod) which can be served as fresh or boiled to eat, it is well-known among the people as Aljlatoun (Al-Ka- raki, 2000). Most Jordanian wild plants are becoming endangered due to the expansion of urban and rural settlements, uncontrolled deforestation, illegal collec- tion, industrial pollution, and low level of environmen- tal awareness (Al-Bakri et al., 2011). Therefore, to solve such problem, alternative methods for massive plant propagation like plant tissue culture techniques and other biotechnological approaches are used for pro- ducing medicinal plants, isolating medicinal secondary products, conserving and rapid propagating valuable, rare, and endangered plant species (Arafeh et al., 2006; Al-Mahmood et al., 2012; Qrunfleh et al., 2013; Shatna- wi , 2013). Propagation methods of T. palaestinus using seeds are not preferred due to the low germination per- centage (Al-Karaki, 2000). In vitro culture of T. palaestinus can solve propaga- tion problems as it guarantees mass production of plant material without compromising the natural resources and it also improves and conserves this plant (Alenizi et al., 2020; Ebrahim et al., 2007; Shatnawi 2006, Shib- li et al., 2018). The use of in vitro culture technique is the best solution to overcome T. palaestinus propaga- tion problems and can also enhance mass production without threatening the natural resources (Shibli et al., 2003; Makhadmeh & Shatnawi, 2008; Shatnawi et al., 2011, Al Qudah et al., 2011). Also, it is an important tool in both basic and applied studies and for commercial applications (Arafeh et al., 2006; Ahmad et al., 2010). Using micropropagation; the plant developed from this technique is true to type or genetically uniform with the mother plant (Shibli et al., 2003). Production of a large number of genetically uniform disease-free plants is known to be a reliable technique system. The con- ventional method of propagation is done by vegeta- tive methods through root suckers or terminal cutting which is classified as very slow (George et al., 2008). In vitro propagation plays a major role in the rapid production of disease-free planting material of newly improved varieties year rounded basis (Ebrahim et al., 2007; Shatnawi 2006, Shatnawi et al., 2011). Shoot tip culture is a relatively simple in vitro technique for the rapid propagation of selected pathogen-free plant ma- terials. Therefore, many simple protocols have been de- veloped for the rapid multiplication of newly released commercially important genotypes through apical meristem cultures. Successful commercial micropro- pagation protocol depends on successful rooting and acclimatization of in vitro derived plantlets (Ebrahim et al., 2007; Shatnawi 2006, Shatnawi et al., 2011). Till now; there are no available data on the in vitro propagation of T. palaestinus. So, this study was initiated to develop an applicable and simple protocol for in vitro establish- ment, multiplication, rooting, and acclimatization of T. palaestinus. 2 MATERIAL AND METHODS 2.1 ESTABLISHMENT OF IN VITRO CULTURE Plant seeds of wild T. palaestinus were collected in mid of April in north Jordan – “Al-Sareeh, Irbid” (32.3306° N latitude and 35.8951° E Longitude). First- ly, surface sterilization of seeds was done by wash- ing seeds with tap water for 5 min. After that, seeds were immersed in (4  %) sodium hypochlorite for 15 min. The following sterilization steps were performed under sterile conditions in a laminar air flow cham- ber; the seeds were washed 3 times in sterile distilled water, then soaked in 70  % ethanol solution for 30 s and washed several times with sterile distilled water. Seeds were cultured on the surface of hormone free Murashige and Skoog (MS) medium (1962) inside Petri dishes (five seeds/ Petri dish). Murashige and Skoog (MS) medium was supplemented with vitamins and 30 g l-1of sucrose. After the final volume of the MS media was adjusted to 1 l and the pH to 5.75, 8 g of agar was added to the media mixture with constant stirring and heating until the agar was completely dissolved. After that, 100 ml of medium was poured into Erlenmeyer flasks. Then flasks were plugged and autoclaved at 121 ºC for 15 min. After that cultures of seeds were kept in a growth room in the dark and moderate temperature 24 ± 2 ºC for four weeks until full germination. The germinated seedlings were transferred to light condi- tions in the growth room under the light regime (16/8 h (light/dark) and a light intensity of 50 μmol m-2s-1. Afterward, cultures were transferred to the new medi- um and further grown. Then, cultures were transferred to MS medium provided with growth regulators, i.e. 0.3 mg l-1 benzyl amino purine (BAP) and 0.05 mg l-1 naphthalene acetic acid (NAA) with 30 g l-1 sucrose, to increase the growth of the cultures. 3 Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant Acta agriculturae Slovenica, 118/3 – 2022 2.2 SHOOT PROLIFERATION Microshoots of 10 mm in length, were treated with different concentrations of cytokinins for shoot prolif- eration experiments. MS media were supplemented with 0.0, 0.3, 0.6, 0.9, 1.2, 1.5 or 2.0 mg l-1 of BAP, Ki- netin, Thidiazuron (TDZ) or Zeatin. Five replications with three microshoots were used for each treatment. Data were collected after five weeks for the microshoots growth parameters as shown in Table 1. 2.3 ROOT FORMATION OF IN VITRO CULTURES Microshoots, 10 mm in length, were treated with different concentrations of auxins. For root formation MS media were supplemented with 0.0, 0.3, 0.6, 1.2, 1.5 or 2.0 mg l-1 of indole-3-butyric acid (IBA), indole ace- tic acid (IAA) or naphthalene acetic acid (NAA). Ten replications were used with one microshoot / replicate. Data were collected for the number of axillary shoots/ explant, shoot length, root length, and rooting (%) after five weeks. 2.4 ACCLIMATIZATION The fully in vitro rooted microshoots were hard- ened gradually from in vitro tubes. Firstly, the tubes plugs were removed for three days and the cultures were left in the growth room. After that microshoots were gently transferred from test tubes and washed un- til all agar residues were removed and grown in plastic pots that have a suitable mixture of (1 peat : 3 perlite). Cultures were covered with perforated plastic bags for 3 days with continuous wetting with sterile distilled wa- ter. After that, plastic bags were removed and the pots were left for extra 2 weeks under growth room condi- tions with continuous wetting. At the end of the accli- matization experiment, the survival percentage of the acclimatized plants was registered. 2.5 EXPERIMENTAL DESIGN The completely randomized design (CRD) was used with all the experiments. Data were analyzed in SPSS Software with Tukey HSD Multiple Range test at p ≤ 0.05. Means and standards error of means were cal- culated. 3 RESULTS AND DISCUSSION In this study, significant differences in the shoots growth parameters were obtained using BAP, Kinetin, TDZ, or zeatin at different concentrations. At 0.3 mg l-1 of BAP, maximum shoots numbers (2.0 shoot per ex- plants) were obtained (Table 1 and Fig. 1). Additionally, the length of shoots and the number of leaves on aver- age increase up to two- fold (30.0 mm and 12.2 leaves, respectively) in comparison with controls (18.0 mm, 7.0 leaves, respectively). At 0.9 mg l-1 BAP, a maximum fresh mass of 112.0 mg was obtained and it was 1.7-fold higher compared to controls (66.0 mg). Shoot length increased up to 30 mm and 26 mm at the lowest and the highest concentrations of BAP (0.3, 2.0 mg l-1; re- spectively), comparing with the control (18.0 mm). But; at 1.2 and 1.5 mg l-1 BAP the length of the shoots was significantly smaller compared to the length of shoots in medium with 0.3 mg l-1 BAP. Furthermore; BAP at (0.3 and 0.6 mg l-1) concentrations resulted in the highest number of leaves 12 (leaves). One of the best cytokinins that can be used to induce in vitro shoot formation is 6-benzylaminopurine (Singh et al., 2019). Thảo et al. (2013) reported the highest shoot induction in common bean (Phaseolus vulgaris L.) when BAP was used with NAA in media. Similarly, in Securidaca longipedunculata (Fresen) the combinations between BAP and IBA at (1.5 mg and 0.1 mg l-1; respectively) produced a better short number and length per explant than other growth regulator combinations (Lijalem and Feyissa, 2020). Besides that, BAP has been reported in many previous studies for shoot multiplications. For example; Trichosanthes dioica Roxb. was established from nodal explants on MS medium containing 1.0 mg l-1 BAP (Tiwari et al., 2010). BAP also; gave the best re- sults for Prosopis cineraria (L.) Druce in vitro establish- ment (Kumar and Singh, 2010). BAP gave the best out- come for shoot induction in the in vitro grain legume, Phaseolus vulgaris (Malik and Saxena, 1992 ) Similarly, kinetin at 0.3 mg l-1 had increased the shoot length up to 32.0 mm which was 1.7-fold longer than control (18.0 mm) (Table 1, Fig 1). Moreover, maxi- mum dry and fresh mass (140 and 114 mg; respectively) of in vitro Tetragonolobus palaestinus explants were ob- tained at 0.3 g l-1 of kinetin. Increasing concentrations of kinetin inhibited the growth of the shoots in length and their appearance was swelling and short. Kinetin induced expansion of growth by swelling rather than elongation, this was confirmed previously by Naeem (2004). Ahmadi et al. (2011) reported that using kinetin at 2.0 mg l-1 increased the in vitro shoot induction in 4 M. MEHERAT et al. Acta agriculturae Slovenica, 118/3 – 2022 Matthiola incana (L.) W.T.Aiton. In Moringa stenopetala (Baker f.) Cufod.; maximum number of shoots per ex- plant (3.43 ± 1.41) and 7.97 ± 4.18 leaves per explant were obtained on MS medium containing 0.5 mg l-1 ki- netin with 0.01 mg l-1 NAA. (Adugna et al., 2020). The addition of 0.3 mg l-1 TDZ to MS medium re- sulted in longer shoots (32.0 mm) compared to con- trols (18.0 mm), and the highest number of leaves per explant (14.8 leaves per explant) was obtained on MS medium supplemented with 1.2 mg l-1 TDZ. The Growth regulator TDZ had been used in previous stud- ies in order to promote in vitro propagation of differ- ent plants species of the Fabaceae family; such as, in vitro Psophocarpus tetragonolobus (L.) D.C. (Singh et al., 2014); and common bean (Veltcheva et al., 2005). The results from the present work demonstrated that TDZ at low concentration was effective compared to other cytokinins (Table 1). However, it was found to be effective at low concentration. Low concentrations of TDZ (0.01 mg l−1) were the most appropriate for shoot regeneration in Abelmoschus moschatus Medik (Sharma & Shahzad, 2008). The effect of TDZ on growth param- eters is not entirely clear, and more studies are needed to understand its role in plant tissue cultures. (Ugand- har et al., 2012). TDZ in combination with NAA pro- duced relatively shorter shoots when used with Secu- ridaca longipedunculata (Fresen) (Lijalem and Feyissa, 2020). Furthermore; TDZ had been reported to have an adverse effects with Vitex trifolia L. (Ahmed and Anis, 2012). When zeatin was used, a maximum number of shoots (1.4 shoots per explant) was obtained on MS medi- um supplemented with 0.3, 0.9, and 1.2 mg l-1 Zeatin (Ta- ble 1). While the maximum shoot length (28.0 mm) was produced on MS medium supplemented with 0.9 mg l-1 Zeatin. On the other hand, Vikram et al. ( 2012) reported that Zeatin at 1.2 mg l-1 produced a maximum number of multiple shoot formation in Lycopersicum esculentum L..  In addition, a highly efficient organogenesis protocol for in vitro regeneration of eggplant was developed using zeatin (García-Fortea et al., 2020). This may be due to that, zeatin suppress apical dominance which leads to in- crease numbers of multiple shoots and reduce the length of the shoot. 3.1 IN VITRO ROOTING The in vitro rooting of T. palaestnius was signifi- cantly induced at a concentration of 0.3 mg l-1 of IBA with (4.06 roots/microshoot). The rooting percentage was 40 % with 3.33 mm/root long at 0.3 mg l-1 of IBA. Meanwhile; control and other concentrations of IBA showed lower in vitro rooting; as we can show in (Table 2 & Fig 2). Low concentrations of IBA (1.0 mg l-1) also resulted in the highest in vitro rooting in Cicer micro- phyllum Benth. (Singh et al., 2019) and in common bean (Phaseolus vulgaris L.) (Thảo et al., 2013). For rooting with IAA growth regulator; the maxi- mum number of roots per microshoots was (2.14 roots/ explant) obtained at 1.2 mg l-1 IAA with a maximum root length of 2.94 mm. The maximum root percentage (30 %) was also recorded on media supplemented with 1.2 mg l1 IAA. Using 0.3 mg l-1 NAA resulted in 0.5 developed root length of 1.94 mm (Table 2). No callus occurred at mi- croshoots bases. The current study showed that auxin is essential for the induction of root formation of in vitro T. palaestnius cultures. This is because auxin exerts a primary role in root formation by its involvement in successive and inter- dependent phases (Mineo, 1990). The rooting of legumi- nous species is dependent on the auxin type (Dewir et al., Figure 1: In vitro shoot formation of Tetragonolobus palaestinus after five weeks. A) MS with 0.3 mg l-1 (BAP). B) MS with 0.6 mg l-1 (BAP). C) MS medium with 0.3 mg l-1 Kinetin. Bars represent 1.0 cm Acta agriculturae Slovenica, 118/3 – 2022 5 Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant 2016). Using IBA or NAA at a low concentrations such as 0.3 mg l-1 increased cell division and root primordial formation and proved to enhanced rooting percentage, number of roots per rooted explants and, root length as compared to IAA or IBA. Some studies found that NAA was the best rooting auxin in Vigna mungo (L.) Hepper (Mony et al., 2010). Furthermore; different legumes of the Fabaceae family have been in vitro rooted using dif- ferent auxins types. For example; the in vitro rooting in Clitoria ternatea L. which is known as the butterfly pea plant was obtained by the addition of 1.50 mg l-1 NAA with the highest number of adventitious roots (12.86 ± 2.14) (Lee et al., 2021). While IBA had been used in the in vitro rooting of Lotononis bainesii Baker (Fabaceae) at the concentration of 0.049 μM IBA (Vidoz et al., 2012). Also, 83 % of in vitro rooting in Thermopsis turcica Kit Tan, Vural & Küçük. (Fabaceae) was attained on pulsed- IBA treated shoots (Cenkci et al., 2008). The growth reg- ulator IAA, was used at 0.005–0.01 mg l-1 to induce in vitro rooting in Psoralea corylifolia L.(Fabaceae) which is Dry mass/five explants (mg) Fresh mass/five explants (mg) Number of leaves/ explant Shoot length (mm) Number of axillary shoots/explant Concentrations mg L-1 34.0 ± 2.4 c66.0 ± 2.0 b7.0 ± 0.32 c18.0 ± 1.22 c1.0 ± 0.0 bControl 0.0 BAP 34.0 ± 2.4 c66.0 ± 2.0 b12.2 ± 0.74 a30.0 ± 3.1 a2.0 ± 0.32 a0.3 56.0 ± 5.0 b90.0 ± 2.0 ab12.0 ± 0.44 a28.0 ± 2.0 ab1.6 ± 0.20 ab0.6 106.9 ± 15.0 a112.0 ± 22.0 a11.2 ± 0.37 ab26.0 ± 2.4 ab1.6 ± 0.40 ab0.9 52.0 ± 5.0 b92.0 ± 4.0 ab10.8 ± 0.37 b22.0 ± 2.0 b1.0 ± 0.24 b1.2 20.0 ± 5.0 d52.0 ± 0.0 c11.4 ± 0.51 ab22.0 ± 2.0 b1.6 ± 0.00 ab1.5 62.0 ± 5.0 b88.0 ± 17.6 ab11.8 ± 0.37 ab26.0 ± 2.4 ab1.8 ± 0.20 ab2.0 Kinetin 114.0 ± 25.0 a140.0 ± 24.4 a12.4 ± 1.12 a32.0 ± 3.7 a1.2 ± 0.20 a0.3 64.0 ± 9.21 ab96.0 ± 14.7 ab11.8 ± 0.48 a22.0 ± 0.0 b1.2 ± 0.20 a0.6 66.0 ± 5.12 ab92.0 ± 3.7 ab11.8 ± 0.48 a24.0 ± 2.4 ab1.2 ± 0.2 a0.9 20.0 ± 3.1 c44.0 ± 4.0 d8.0 ± 0.20 c15.0 ± 0.0 d1.0 ± 0.0 b1.2 66.0 ± 4.0 ab88.0 ± 2.0 b10.2 ± 0.37 b20.0 ± 0.0 b1.4 ± 0.24 a1.5 14.0 ± 2.0 c30.0 ± 0.0 d4.0 ± 0.24 d15.0 ± 0.0 d1.0 ± 0.0 b2.0 TDZ 58.0 ± 7.3 c84.0 ± 4.4 b10.8 ± 0.37 b32.0 ± 2.0 a1.6 ± 0.24 a0.3 64.0 ± 5.1 b90.0 ± 4.4 ab12.8 ± 0.58 ab20.0 ± 0.0 ab1.8 ± 0.37 a0.6 85.0 ± 5.8 ab84.0 ± 5.1 b10.8 ± 0.37 b22.0 ± 2.0 b1.2 ± 0.20 b0.9 112.0 ± 24.1 a136.0 ± 26.1 a14.8 ± 0.24 a30.0 ± 4.4 a1.8 ± 0.37 a1.2 60.0 ± 5.4 c92.0 ± 2.0 ab10.6 ± 0.40 b24.0 ± 2.4 ab1.6 ± 0.24 a1.5 66.0 ± 6.7 b90.0 ± 6.7 ab11.8 ± 0.41 b22.0 ± 2.0 b1.6 ± 0.24 a2.0 Zeatin 106.9 ± 24.0 a66.0 ± 1.8 b15.4 ± 0.81a26.0 ± 2.45 a1.4 ± 0.25 a0.3 56.0 ± 4.8 b92.0 ± 2.0 ab10.4 ± 0.24 ab20.0 ± 0.0 ab1.2 ± 0.20 ab0.6 74.0 ± 5.1 ab112.0 ± 22.0 a15.4 ± 1.02 a28.0 ± 3.74 a1.4 ±0.40 a0.9 52.0 ± 1.9 b92.0 ± 5.0 ab10.0 ± 0.95 ab24.0 ± 2.44 ab1.4 ± 0.24 a1.2 20.0 ± 6.0 d52.0 ± 2.0 c7.6 ± 0.51 c11.0 ± 1.00 c1.0 ± 0.00 b1.5 62.0 ± 3.0 b88.0 ± 4.0 ab11.60 ± 0.88 ab20.0 ± 00 ab1.2 ± 0.20 ab2.0 Table 1: The effect of different concentrations of the cytokinins on in vitro grown Tetragonolobus palaestinus after five weeks of in- cubations. *Values represent means ± standard error. *Means within the column for each growth regulator having different letters are significantly different according to Tukey HSD at p ≤ 0.05 Acta agriculturae Slovenica, 118/3 – 20226 M. MEHERAT et al. a rare and endangered herbaceous medicinal plant (Rout et al., 2010). We can see from this, that in vitro rooting depend on plant species and the type of auxin used. 3.2 ACCLIMATIZATION The acclimatization process of Tetragonolobus pa- laestinus microshoots was proved its ability to the pro- duction of healthy acclimatized micro shoots. The mix- ture of (Peatmoss: Perlite) was suitable for roots growth and new leaves were formed after 2 weeks (Fig 3). All of the rooted plantlets were survived after acclimatiza- tion process after acclimatization process. No variations were observed visually among the acclimatized plantlets as shown in Fig (3). A lower survival rate of 65 % was obtained in C. microphyllum using different potting cul- ture mixture ( garden soil, vermiculite, and vermicom- post (1:1:1) (Singh et al., 2019) Plantlet needs an acclimatization period and this could be due to the effect of tissue differentiation, growth, and development (Quisen, 2013). The acclimatized plantlet may be affected by the change in environmental conditions during acclimatization, which may be due to the short acclimatization period evaluated in this study (Shatnawi., 2013). Different plants of the Fabaceae fam- ily have been successfully in vitro rooted and acclima- Rooting % Root length (mm) Number of roots / explant Shoot length (mm) Number of axillary shoots/explant Concentrations mg.l-1 20% 2.00 ± 0.70 ab 1.50 ± 0.48 b18.07 ± 7.46 ab 1.01 ± 0.23* b Control 0.0 IBA 40% 3.33 ± 0.90 a 4.06 ± 0.67 a 19.0 ± 9.5 a 1.16 ± 0.32 a 0.3 20% 1.44 ± 1.33b 3.17 ± 0.52 b 17.06 ± 7.06 ab 1.09 ± 0.18 a 0.6 30% 2.94 ± 1.86 a 2.67 ± 0.78 ab 12.28 ± 4.21 b 1.04 ± 0.15 a 1.2 30% 1.94 ± 2.83 b 2.06 ± 0.78 ab 13.33 ± 7.13 b 1.04 ± 0.16 a1.5 20% 3.17 ± 0.86 a 2.94 ± 0.81 ab 12.04 ± 5.29 b 1.06 ± 0.17 a 2.0 IAA 20% 0.89 ± 0.40 b0.56 ± 0.24 b13.78 ± 4.41 ab 1.00 ± 0.22 b 0.3 27% 2.61 ± 0.84 a 0.50 ± 0.20 b 12.61 ± 3.45 b 1.22 ± 0.18 a 0.6 30% 2.94 ± 1.50 a 2.14 ± 0.17 a 19.33 ± 4.02 a 1.06 ± 0.16 ab 1.2 26% 1.83 ± 1.54 ab 0.57 ± 0.60 b 12.28 ± 4.09 b 1.09 ± 0.12 ab 1.5 20% 2.39 ± 1.11 a 1.39 ± 0.41 ab 12.61 ± 3.38 b 1.02 ± 0.13 b 2.0 NAA 30% 0.89 ± 0.40 c 0.56 ± 0.24 a 17.00 ± 4.41 ab 1.00 ± 0.22 b 0.3 27% 1.61 ± 0.84 ab0.50 ± 0.20 a 18.01 ± 3.45 a 1.06 ± 0.18 ab 0.6 20% 1.94 ± 1.50 a 0.44 ± 0.17 b 16.03 ± 4.02 b 1.01 ± 0.16 b 1.2 26% 1.83 ± 1.54 a 0.17 ± 0.60 b 17.08 ± 4.09 ab 1.09 ± 0.12 ab 1.5 30% 1.39 ± 1.11 b 0.39 ± 0.41 ab 17.01 ± 3.38 ab 1.02 ± 0.13 b 2.0 Table 2: The effect of different auxin concentration on in vitro root formation of Tetragonolobus palaestinus after five weeks growth period. *Values represent means ± standard error. *Means within the column for each growth regulator having different letters are significantly different according to Tukey HSD at p ≤ 0.05 Figure 2: The effect of 0.3 mg l-1 indole-3-butyric acid (IBA) on in vitro growth of Tetragonolobus palaestinus after five weeks growth. The bar represents 1.0 cm Acta agriculturae Slovenica, 118/3 – 2022 7 Clonal propagation of Tetragonolobus palaestinus Bioss: A Jordanian medical plant tized. For example, the regenerated plantlets of licorice (Glycyrrhiza glabra L.; Fabaceae) were acclimatized, with a survival rate of 77.7  %, when transferred to ex vitro conditions and showed no morphological abnormalities (Shaheen, 2020). While, the in vitro seedlings of Caesal- pinia ferrea Mart. were acclimated without the presence of roots in different types of the substrate with 73.4 % surviving plantlets after 30 days of growth (Silva et al., 2018). On the other hand, in A. leiocarpa (L.A.S.Johnson ex G.J.Leach) K.R.Thiele & Ladiges plantlets the substrate composition did not affect the survival or growth of in vitro rooted plantlets during acclimatization (Haygert- Lencina it., 2017). We can notice from this, that accli- matization is a critical process and it depends on plant species and the successful adjustment of the environment of the acclimatized plants. 4 CONCLUSION The current results indicat that in vitro propaga- tion method of Tetragonolobus palastinus was successful, with full survival percentage for the first records of this plant species in vitro. The optimum in vitro propagation method of Tetragonolobus palastinus was obtained at 0.3 mg l-1 of Benzylamino purine (BAP) with 2.0 shoots/ex- plants and 0.3 mg l-1 IBA with (4.06 roots/explants) and this method is recommended for in vitro clonal propaga- tion in T. palaestinus. 5 REFERENCES Adugna, A. Y., Feyissa, T., & Tasew, F. S. (2020). Optimization of growth regulators on in vitro propagation of Moringa stenopetala from shoot explants. BMC Biotechnology, 20(1), 1-10. https://doi.org/10.1186/s12896-020-00651-w Adwan, G., Abu-shanab, B., & Adwan, K. (2009). 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