Impaginato 243 Adv. Hort. Sci., 2023 37(3): 243­253 DOI: 10.36253/ahsc­13895 In vitro propagation and microtuberiza­ tion of potato (Solanum tuberosum L.) Spunta variety in Lebanon M. Dalleh 1, J. Borjac 1, G. Younes 2, E. Choueiri 3, A. Chehade 4, A. Elbitar 4 (*) 1 Beirut Arab University, Faculty of Science, Department of Biological Sciences, Debbieh, Lebanon. 2 Beirut Arab University, Faculty of Science, Department of Chemistry, Debbieh, Lebanon. 3 Lebanese Agricultural Research Institute, Tal Amara Station, Department of Plant Protection, Zahleh, Lebanon. 4 Lebanese Agricultural Research Institute, Tal Amara Station, Department of Plant Biotechnology, Plant Tissue Culture Unit, Zahleh, Lebanon. Key words: In vitro culture, microtubers, shootlets proliferation, Solanum tuberosum L., Spunta. Abstract: One of the factors that causes low productivity of potatoes in Lebanon is the limited availability of certified seeds. The aim of this study was to establish a rapid protocol for in vitro propagation and microtuberization of potato (Solanum tuberosum L.) of Spunta variety. Meristems culture associated to thermotherapy (one month/37°C) constituted the first step. The highest per­ centage of reactive meristem (92%) was observed on MS medium devoid of growth regulators while MS medium containing Kin 0.4 mg.l­1, GA3 0.5 mg.l­1 and IBA 0.5 mg.l­1 yielded the highest average number of shootlets (7.8) in the seventh subculture. The lowest number of days obtained for microtuber forma­ tion was 10 and the highest average number of microtuber (1.49) was obtained with shootlets incubated under C2 culture conditions (16­h day/8­h night for initial 7 days at 25±2°C; for remaining period: continuous dark at 17±2°C). Contrary the highest microtubers average length (10.75 mm), average width (7.41 mm) and average weight (646.26 mg) were produced under C1 culture conditions (16­h day/8­h night at 25±2°C). Medium supplemented with 5 mg.l­1 BAP and 6% sucrose presented the highest average number of microtubers of 2.36 and 1.94 respectively. Type and concentration of cytokines and sucrose concentration did not have significant effect on the average length, width and weight of microtubers produced. 1. Introduction Potato (Solanum tuberosum L.) is the most important non­cereal food crop in the world (Bamberg et al., 2016; Nikitin et al., 2018). It is cultivat­ ed in about 150 countries (Basera et al., 2018). Potato cultivation is con­ sidered as strategic as it occupies the world fourth place in production (*) Corresponding author: abitar@lari.gov.lb Citation: DALLEH M., BORJAC J., YOUNES G., CHOUEIRI E., CHEHADE A., ELBITAR A., 2023 ­ In vitro propaga‐ tion and microtuberization of potato (Solanum tuberosum L.) spunta variety in Lebanon. ­ Adv. Hort. Sci., 37(3): 243­253. Copyright: © 2023 Dalleh M., Borjac J., Younes G., Choueiri E., Chehade A., Elbitar A. This is an open access, peer reviewed article published by Firenze University Press (http://www.fupress.net/index.php/ahs/) and distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Competing Interests: The authors declare no competing interests. Received for publication 25 October 2022 Accepted for publication 30 August 2023 AHS Advances in Horticultural Science https://doi.org/10.36253/ahsc-13895 http://www.fupress.net/index.php/ahs/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/ Adv. Hort. Sci., 2023 37(3): 243­253 244 after Maize, Wheat, and Rice (FAO, 2019). In potato production, the quality of seed potatoes planted is an important determinant of the final yield and quality (Roy, 2014). When farmers use their farm­saved seed potatoes for several cropping cycles without renewing the seed lot from a reliable source, seed­borne diseases accumulate and cause severe yield and quality losses (Fuglie, 2007). Potato propa­ gation takes place primarily asexually, through tubers and microtubers (Zhang et al., 2006). Potato microtubers are minute tubers produced through in vitro culture technique yielding disease­ free and high­quality seeds that can be preserved for a long time (Badoni and Chauhan, 2009). Extensive physiological research has revealed that tuberization is controlled by several factors, such as hormonal combination, ratio of photoperiod, nutrient composi­ tions among others (Naresh et al., 2011). Many researchers used different growth regulators for in vitro induction of microtubers in potato (Hossain, 2005). Cytokinins, such as benzylaminopurine (BAP), and kinetin (Kin) are among these growth regulators (Al­Safadi et al., 2000; Sarkar et al., 2006; Aksenove et al., 2009). In addition, sucrose, the cheap and safe disaccharide, is considered as a superior agent and a critical stimulus for inducing microtubers (Hussain et al., 2006; Nistor et al., 2010). In Lebanon, potato production is important for food security as well as a source of revenue in rural areas. It is a strategic crop for Lebanese agriculture, covering about 19,000 ha in the Bekaa plain (MOA, 2012) and with production of approximately 300,000 tons per year constituting the greatest field crop ton­ nage in Lebanon. It is cultivated mainly in the Bekaa valley (central­eastern Lebanon, 900­1000 m above the sea level, 70% of total area) and in Akkar plain (northern Lebanon, 25­30% of total area) (Choueiri et al., 2017). Lebanon does not produce certified potatoes’ seeds, they are imported mainly from the European Union (EU) Member States. In the early years of the 21st century, Lebanon imported between 15,000 and 20,000 tons of potato seeds each year and relied heavily on this import with prices ranging between $750 and $1,000 per ton (Abou­Jawdah et al., 2001). The absence of a seed certification program, the introduction and exchange of potato seeds of unknown sanitary status and the lack of phytosani­ tary measures resulted in increased incidence and severity of potatoes’ diseases (Abou­Jawdah et al., 2001). To prevent the further spread of these dis­ eases and to avoid the introduction of new pathogens, it is important to reinforce the seed certi­ fication scheme that mandates using and trading of only certified potato seeds. Such a scheme will help the Lebanese farmers to improve their production, get involved in a certified seed production program and reduce the incidence of diseases, and thus eco­ nomical losses. The implementation of a potato seed production program in Lebanon is possible; especially that Lebanon is characterized by a wide range of microclimates favorable to produce them (Abou­ Jawdah et al., 2001). In a report published by the International Potato Center (IPC) in 1975, areas in Lebanon including the Laklouk, Daher el Beidar, and Northern Bekaa were listed among the qualified areas for potato seeds production. Hence, the present study was initiated with the objective to determine optimum concentration of sucrose, the effect of two cytokinins (BAP and Kin) on the microtuberization capacity of the potato cultivar Spunta under different incubation conditions. 2. Materials and Methods Plant material The experiment was conducted at the Lebanese Agricultural Research Institute (LARI), Department of Plant Biotechnology. The mother plants used in this study were of Spunta variety. Thermotherapy com­ bined with meristem culture has been successfully established for efficient eradication of the potatoes’ viruses. About 10 tubers were subjected to a temper­ ature of 37°C for 50 days, under a photoperiod of 16 hours/day. At the end of the thermal treatment, the sprouts from the treated tubers were separated in portions of 3 cm and surface sterilized using 70% ethanol for 1 min followed by 5% (v/v) sodium hypochlorite containing two drops of Tween­20 for 10 minutes. Finally, the explants sources were washed 4 times with sterile distilled water (10 min each wash) before being used for the collection of meristems. In vitro propagation Meristems have been cut with the first pair of leaf primordial and cultivated in 9 cm Petri dishes on 3 MS basal medium (Murashige and Skoog, 1962). P1 medium lacked any additional growth regulators. P2 medium contained Kinetin (Kin), Gibberellic acid (GA3) and indole­3 butyric acid (IBA) at concentra­ Dalleh et al. ‐ Potato micropropagation 245 tions of 0.2 mg.l­1, 0.5 mg.l­1 and 0.5 mg.l­1 respective­ ly. P3 medium was supplemented with Kin at 0.4 mg.l­1, GA3 at 0.5 mg.l­1 and IBA at 0.5 mgl­1. In addi­ tion, to all media, a vitamin mixture consisting of nicotinic acid (5 mg.l­1), ascorbic acid (20 mg.l­1), pyri­ doxin (5 mg.l­1), thiamin (10 mg.l­1) and myo­inositol (100 mg.l­1) was added. The pH of all media was adjusted to 5.7 prior to the addition of agar (0.7%). The media were autoclaved at 121°C for 20 min. Each medium was prepared in ten replicas with 5 meris­ tems per replica for testing. The in vitro cultivated 150 meristems were then shifted to a culture growth room at 25±2°C with 16 h photoperiod under white light intensity of 3000 lux. Thirty days later, survival rate of meristems was recorded. The regenerated shootlets from the meristems were fragmented to obtain uninodal cuttings and transferred onto fresh medium. Subcultures of 30 days interval were re­con­ ducted seven times using the same 3 initiation media i.e., P1, P2 and P3, under the same culture conditions described above with a set of 60 shootlets per medi­ um (10 repetitions of 6 shootlets). The newly regen­ erated shootlets per explant were recorded by the end of each subculture to calculate the multiplication rate as follows: Multiplication rate= Number of new shootlets/Number of initial shootlets Sanitary control To check the sanitary status of the shootlets that arose from the thermotherapy­treated meristems, the Tissue­Blot Immunoassay (TBIA), a reliable, rou­ tine and cost­efficient serological test that allow pro­ cessing of large numbers of individual plant samples serological test and that is commonly used to detect plant viruses in field and vegetable crops (Makkouk and Kumari, 1996) was performed to detect six main potato viruses PVA, PVX, PVY, PVM, PVS and PLRV. In addition, all samples were tested by the double­anti­ body sandwich enzyme­linked immunosorbant assay (DAS ­ ELISA, Loewe, Germany) using specific anti­ bodies (Clark and Adams, 1977). All shootlets result­ ing from the meristems culture were subjected to the sanitary control. Each shootlet was numbered and divided into 2 parts, the first part was sent to the plant protection laboratory at LARI to be analyzed, while the second part of each plant remained in the culture medium pending the sanitary control results. Pre‐tuberization Shootlets from the fifth subculture were aseptical­ ly transferred into culture­tubes containing 10 ml of pre­tuberization medium (P1). Cultures were grown for 20 days in culture growth room at 25±2°C and 16 h photoperiod under white light intensity of 3000 lux. Tuberization Eight MS Liquid media with two sucrose concen­ trations 6% and 8% respectively, supplemented with 2 cytokinins, Kin (0, 2 and 4 mg.l­1) or BAP (0 and 5 mg.l­1) or their combinations were used as an induc­ ing medium for microtuber production. Ten ml of each liquid medium were added to the tubes con­ taining the pre­tuberization MS solid medium after 20 days of culture. For each culture medium, two cul­ ture conditions were studied. In the first culture con­ dition, the incubation was in culture growth room for 16­h day, 8­h night at 25±2°C and 3000 lux for 60 days. In the second condition, an initial incubation at 16­h day, 8­h night at 25±2°C and 3000 lux, for 7 days followed by continuous dark at 19±2°C to 60 days. Conditions are summarized in Table 1. For each cul­ ture medium and condition, 32 replicas were pre­ pared. Microtubers produced were harvested at day 60. Data were recorded on days to microtubers for­ mation, average number, average length, average width and average weight of microtubers. Statistical analysis Means ± standard deviations were recorded for each step of the propagation protocol and analyzed by using standard analysis of variance (ANOVA). Duncan’s multiple range test was used to show dif­ ferences among the treatments’ means. All statistical analyses were performed using SAS for Windows (SAS Institute Inc., 1995). 3. Results In vitro propagation The survival rate of meristems was recorded after 30 days. The highest percentage of reactive meristem was formed on P1 medium that is devoid of growth regulators with an average of 920±4.8%, whereas a significant decrease of surviving reactive explants of 74±5.8% and 58±5.0% (p<0.05) was obtained on P2 and P3 media respectively as shown in figure 1 and 2 (A­C). No significant changes in percent of reactive meristems was observed between P2 and P3 media. The effect of growth regulators added in the cul­ ture medium is summarized in Table 2. Medium P3 Adv. Hort. Sci., 2023 37(3): 243­253 246 presented the highest multiplication rate of 7.8±1.2 in the seventh subculture. Whereas in medium P2, the best multiplication rate obtained was 6.9±0.7 in the sixth subculture. Illustrations related to shootlets proliferation are presented in figure 2 (D and E). It is important to note that a l l s h o o t l e t s obtained in the meristems’ cultures coupled with thermotherapy were f ree of the tested PVA, PVX, PVY, PVM, PVS and PLRV viruses. In vitro microtuber formation In vitro tuberization was obtained after propaga­ Medium Medium composition Culture condition C1 M1 20 ml of MS solid medium with 6% sucrose + 0 Hormones 16­h day, 8­h night at 25±2°C M2 20 ml of solid MS medium with 8% sucrose + 0 Hormones 16­h day, 8­h night at 25±2°C M3 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M1 medium + 5 mg.l­1 BAP 16­h day, 8­h night at 25±2°C M4 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M2 medium + 5 mg.l­1BAP 16­h day, 8­h night at 25±2°C M5 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M1 medium + 4 mg.l­1 Kin 16­h day, 8­h night at 25±2°C M6 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M2 medium + 4 mg.l­1Kin 16­h day, 8­h night at 25±2°C M7 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M1 medium + 5 mg.l­1BAP + 2 mg.l­1Kin 16­h day, 8­h night at 25±2°C M8 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M2 medium + 5 mg.l­1BAP + 2 mg.l­1Kin 16­h day, 8­h night at 25±2°C Medium Medium composition Culture Condition C2 M1 20 ml of MS solid medium with 6% sucrose + 0 Hormones For initial 7 days, 16­h day, 8­h night at 25±2°C, for remaining period continuous dark at 19±2°C M2 20 ml of solid MS medium with 8% sucrose + 0 Hormones For initial 7 days, 16­h day, 8­h night at 25±2°C, for remaining period continuous dark at 19±2°C M3 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M1 medium + 5 mg.l­1 BAP For initial 7 days, 16­h day, 8­h night at 25±2°C, for remaining period continuous dark at 19±2°C M4 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M2 medium + 5 mg.l­1 BAP For initial 7 days, 16­h day, 8­h night at 25±2°C, for remaining period continuous dark at 19±2°C M5 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M1 medium + 4 mg.l­1 Kin For initial 7 days, 16­h day, 8­h night at 25±2°C, for remaining period continuous dark at 19±2°C M6 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M2 medium + 4 mg.l­1 Kin For initial 7 days, 16­h day, 8­h night at 25±2°C, for remaining period continuous dark at 19±2°C M7 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M1 medium + 5 mg.l­1BAP + 2 mg.l­1Kin For initial 7 days, 16­h day, 8­h night at 25±2°C, for remaining period continuous dark at 19±2°C M8 MS solid medium, after 20 days of culture, addition of 20 ml of liquid M2 medium + 5 mg.l­1BAP + 2 mg.l­1Kin For initial 7 days, 16­h day, 8­h night at 25±2°C, for remaining period continuous dark at 19±2°C Table 1 ­ Culture media and culture conditions tested for in vitro microtubers production of potato Fig. 1 ­ Effect of culture medium on the percentage of reactive meristem. Means with the same letter are not signifi­ cantly different according to Duncan’s. Fig. 2 ­ Represent the stages of the in vitro propagation and microtubers production of a representative under the three pre­tuberization conditions. A= Sprout of the treat­ ed tuber. B= Meristem initiation. C= Shootlets elonga­ tion. D= Uniodal cutting explants. E= Shootlet prolifera­ tion. F= Shootlet on pretuberization medium. G and H = Shootlets on tuberization medium. I= Microtubers pro­ duced under culture conditions C1. J= Microtubers pro­ duced under culture conditions C2. Dalleh et al. ‐ Potato micropropagation 247 tion of the shoots (Fig. 2 E­H). The effect of BAP, Kin, or their combination in the presence of sucrose (6% or 8%) under both culture conditions were studied for microtubers’ formation and development. Table 3 summarizes the results of microtubers’ formation under all treatments. Both the cytokinin type and its concentration sig­ nificantly (p<0.0001) affected the average number (AN) of microtubers produced. Media containing BAP alone or in combination with Kin was superior to other media. The highest AN of microtubers per shootlet, i.e. 2.36 and 2.22, were produced when shootlets were propagated on M3 and M7 media with 5 mg.l­1BAP or 5 mg.l­1BAP + 2 mg.l­1Kin respec­ tively under C2 conditions. On the other hand, no sta­ tistically significant effect was observed when varying the type nor the concentration of the cytokines on the average length (AL) and average width (AW) or the average weight AWe of microtubers (p=0.12, p=0.44 and p=0.39 respectively) as shown in Table 3. There is a linear relation between size and weight of microtuber implying that each factor that influ­ enced the microtuber weight directly influenced microtuber size. Effect of culture condition on microtuber formation Results showed that the effect of culture condition was highly significant with p value <0.0001 for AN, AL, P1= medium without any additional growth regulators P2= medium contained Kinetin (0.2 mg·l­1), Gibberellic acid (0.5 mg·l­1) and indole­3 butyric acid (0.5 mg·l­1). P3= medium was supplemented with Kinetin at 0.4 mg·l­1, Gibberellic acid at 0.5 mg·l­1 and indole­3 butyric acid at 0.5 mg·l­1 Table 2 ­ Effect of culture media on the average number of shootlets per explant as recorded along the 7 subcultures Table 3 ­ Effect of cytokinin type and concentration on the average number (AN), average length (AL), average width (AW) and average weight (Awe) of microtubers produced Means followed by different letters in each column are significantly different for P≤0.05. Media Average number of shootlets per explant Subculture 1 Subculture 2 Subculture 3 Subculture 4 Subculture 5 Subculture 6 Subculture 7 P1 0.70±0.3 1.17±0.2 3.87±0.4 4.07±0.5 5.08±0.5 5.4±0.2 4.00±0.6 P2 1.0±0.0 0.9±0.5 2.63±0.7 6.2±0.7 6.07±1.1 6.9±0.7 5.40±0.4 P3 1.2±0.6 1.5±1.0 5.57±0.7 7.33±0.9 6.00±0.2 7.4±0.9 7.80±1.2 Culture media Culture conditions Average number of microtubers Average length of microtubers (mm) Average width of microtubers (mm) Average weight (mg) M1 C1 0.22±0.42 h 8.07 cd 5.05 e 375.3 d M2 C1 0.78±0.70 fg 11.14 ab 7.28 abc 622.2 abc M3 C1 1.53±0.62 cd 12.00 a 7.34 abc 673.1 ab M4 C1 1.44±0.91 cde 10.07 abc 6.90 abcd 593.4 abcd M5 C1 1.00±0.71 efg 11.8 a 7.96 a 734.5 a M6 C1 0.94±0.82 fg 10.78 ab 7.23 abc 621.9 abc M7 C1 1.58±0.95 cd 11.07 ab 7.43 ab 635.4 abc M8 C1 1.25±0.85 def 11.1 ab 7.75 ab 643.3 abc M1 C2 0.87±0.50 fg 9.45 bcd 6.8 abcd 535.4 abcd M2 C2 0.55±0.80 gh 7.24 d 5.00 e 369.1d M3 C2 2.36 a±1.05 8.89 bcd 5.64 de 473.5 bcd M4 C2 1.56±0.98 cd 8.84 bcd 5.91 cde 561.5 abcd M5 C2 1.00±0.65 efg 9.12 bcd 5.86 cde 487 bcd M6 C2 1.58±1.05 cd 7.85 cd 5.34 e 406.3 cd M7 C2 2.22±1.15 ab 7.85 cd 5.08 e 416.3 cd M8 C2 1.80±1.16 bc 9.78 abc 6.26 bcde 600.7 abcd 248 Adv. Hort. Sci., 2023 37(3): 243­253 AW and AWe of microtubers (Table 4). AN was 1.49 for shootlets incubated under C2 condition and 1.09 for shooltlets cultivated under C1 condition. Values of AL, AW and AWe are 10.75 mm, 7.41 mm and 646.26 mg for microtubers produced under C1 condition and 8.63 mm, 5.65 mm and 481.23 mg for microtubers obtained under C2 condition respectively. Under C1 culture condition, most of the cultures produced green microtuber, and this could be due to synthesis of the alkaloid solanine (Hoque, 2010). On the other hand, brown­colored tuber was observed in C2 culture condition (Fig. 2, I and J). Effect of sucrose concentration on microtuber forma‐ tion The effect of sucrose on microtuber production is presented in Table 5. Results revealed a highly signifi­ cant effect of sucrose concentration on the AN of microtubers produced. The media added with 5 mg.l­ 1 BAP or 5 mg.l­1 BAP + 2 mg.l­1 Kin, both supplement­ ed with 6% of sucrose, presented the highest AN of microtubers, 1.95 and 1.9 respectively. On the other hand, sucrose concentration had no effect on AL, AW and the AWe of microtubers (Table 5). Time‐dependent effect of growth regulators, sucrose concentration and culture condition on microtuber formation Microtuber formations was followed in a time dependent manner (Fig. 3). Shootlets incubated under C1 conditions in media supplemented with sucrose (6% or 8%) devoid of any growth regulators, i.e. M1 and M2, needed 60 days for microtubers for­ mation as compared to C2 conditions that needed 40 and 45 days respectively. When incubated in the presence of the diverse cytokinins (M3 till M8) under C1 conditions, microtubers’ formation was evident at day 30. Shootlets incubated in M3 and M7 media, supplemented with 5 mg.l­1 BAP and 5 mg.l­1 BAP + 2 mg.l­1 Kin respectively, showed the fastest microtu­ bers’ formation at day 10. Other cytokinin combina­ tions (M4, M5 and M8) required between 15 to 20 days. This confirm that M3 and M7 combination were the best for microtubers’ formation. Table 4 ­ Effect of culture condition on the average number (AN), average length (AL), average width (AW) and average weight (Awe) of microtubers produced C1 culture conditions= 16­h day, 8­h night (25±2°C), C2 culture conditions= For Initial 7 days, 16­h day, 8­h night (25±2°C), For remaining period: continuous dark (19±2°C) Means followed by different letters in each column are significantly different for P≤0.05. Culture conditions Average number of microtubers Average length of microtubers (mm) Average width of microtubers (mm) Average weight (mg) Colour of microtuber C1 1.09 b 10.75 a 7.41 a 646.26 a Green C2 1.49 a 8.63 b 5.65 b 481.23 b Brown Table 5 ­ Effect of culture condition on the average number (AN), average length (AL), average width (AW) and average weight (Awe) of microtubers produced C1 culture conditions= 16­h day, 8­h night (25±2°C), C2 culture conditions= For Initial 7 days, 16­h day, 8­h night (25±2°C), For remaining period, continuous dark (19±2°C) Means followed by different letters in each column are significantly different for P≤0.05. Culture media Sucrose concentration Average number of microtubers Average length of microtubers (mm) Average width of microtubers (mm) Average weight (mg) M1 6% 0.55 d 8.76 a 5.93 a 455.35 a M2 8% 0.67 d 9.19 a 6.14 a 495.65 a M3 6% 1.95 a 10.45 a 6.49 a 573.3 a M4 8% 1.5 b 9.46 a 6.41 a 577.45 a M5 6% 1 c 10.46 a 6.91 a 610.75 a M6 8% 1.26 bc 9.32 a 6.29 a 514.1 a M7 6% 1.9 a 9.46 a 6.26 a 525.85 a M8 8% 1.53 b 10.44 a 7.01 a 622 a Dalleh et al. ‐ Potato micropropagation 249 4. Discussion and Conclusions Accumulation of seed­borne diseases is a major cause of low yield and quality losses in agriculture. Potato is a strategic crop for many populations. Producing disease­free potato microtubers is vital to improve its production. This study aimed to deter­ mine the best conditions and agents (sucrose and cytokinins) on the microtuberization capacity of the potato cultivar Spunta. As meristem tips are free from viruses, elimination and generation of virus free plants were shown by many researchers through meristem culture to obtain disease free potato plantlets (Jha and Ghosh, 2005; Bhuiyan, 2013). Our findings provide evidence on the importance of having media devoid of growth regulators on the formation of reactive meristem in order to obtain the highest percentage. These results are in contradiction with others where Al­Taleb et al. (2011) indicated that the medium supplemented with 0.5 mg.l­1 of IBA was the best for shoots development and where Hajare et al. (2021) showed that best shoot initiation was obtained on MS medium supplemented with 1.5 mg.l­1 BAP + 3.0 mg.l­1 NAA for Gudiene variety, whereas 1.0 mg.l­1 BAP and 2.0 mg.l­1 NAA produced more shoots in Belete variety. According to a study done by Kanwal et al. (2006) who investigate the in‐ vitro micropropagation of potato cultivar Kuroda, the increase of added BAP concentrations in MS medium induced an increase in the rate of shoot formation. According to Salem and Hassanein (2017) who studied the effect of genotype on multiplication and micro­tuberization of potato in vitro, the highest number of microshoot was formed by cv. Hermes after cultivation in a medium supplemented with 1 mg.l­1 BAP + 0.5 mg.l­1 GA3, whereas the least one was formed by cv. Spunta implying that the number of obtained microshoots is cultivar­dependent. At the level of the multiplication phase, our results showed that the media P3 containing Kin 0.4 mg.l­1, GA3 0.5 mg.l­1 and IBA 0.5 mgl­1 caused the highest number of shootlets/plant. Similar results were obtained by Emaraa et al. (2017) who supplemented their media with Kin (0.2 mg.l­1) and naphtaleneacetic acid (NAA, 0.2 mg.l­1) and obtained maximum number of shootlets/plant. Among the different cytokinins used by researchers when studying micropropagation of plants, Kin stands among the best to promote shoot formation and shoot length (Van­Staden et al., 2008; Hoque, 2010). El Dessoky et al. (2016) also showed that the highest percentage of shoot initia­ tion and multiplication were observed on MS medium containing Kin (0.1 mg.l­1) and GA3 (5 mg.l­1). Concerning microtuber formation, this study revealed that the addition of BAP into the culture medium was more effective in inducing in vitro tuber­ ization in comparison to kinetin. The highest average number (AN) of microtubers was produced when propagated shootlets were subcultured on M3 medi­ um containing 5 mg.l­1 BAP. These results agree with others (Aksenove et al., 2009; Hoque, 2010; Sota et al., 2020) who also showed that BAP has greater potential for microtuberization than kinetin and had an effect on reduction of total sugar and subsequent­ ly have increased starch content (Sarkar et al., 2006). Furthermore, Liljana et al. (2012) observed that tuberization occurred only in the presence of BAP (2 mg.l­1) + IAA (1 mg.l­1). Wang and Hu (1982) and Badoni and Chauan (2010) also reported that the optimum condition for in vitro tuberization of virus­free potatoes were in a medium containing BAP at 10 mg.l­1. Vural et al. (2018) used BAP (2.5 mg.l­1) with NAA (0.5 mg.l­1) in their in vitro micro­ propagation of potato and found that these cytokinins had a positive effect on micro tuber forma­ tion and can be recommended to use them commer­ cially in mass production. On the other hand, these cytokinins showed no significant effect on the average length (AL), average width (AW) and average weight (AWe) of microtu­ bers. Our results contradict those of Aryakia and Hamidoghli (2010) who showed that BAP and Kin induced changes in microtuber size and weight in the in vitro microtuberization of two potato cultivars, Arinda and Diamant. Prat (2004) reported that Kin played a significant role in creating sink during plant growth, and through Fig. 3 ­ Days to microtuber formation under different conditions. Adv. Hort. Sci., 2023 37(3): 243­253 250 regulating the expression of a gene involved in the partition of assimilates towards the stolon as observed in potato. Our study reports that kin exert­ ed no significant effect on growth, diameter and weight of microtuber and this is in agreement with Kefi et al. (2000) and Kanwal et al. (2006). In general, a linear relation between size and weight of microtuber exists (Liu and Xie, 2001). Any factor that influences microtuber weight will directly influence microtuber size. The microtubers parame­ ters AN, AL, AW, and AWe obtained differed based on the culture condition. Growth condition C2 proved to be better for microtuber formation, where­ as condition C1 was better for AL, AW, AWe. Under C1 culture condition, most of the culture produced green microtubers and this could be due to the syn­ thesis of the alkaloid solanine that has fungicidal and pesticidal properties and that is considered as one of the plant’s natural defenses. Hoque has also shown that when potato tubers are exposed to light, they turn green and increase glycoalkaloid produc­ tion (Hoque, 2010). On the other hand, culture condi­ tion C2 yielded brown­colored tubers (Fig. 1, I and J). These results are in agreement with those of Salem and Hassanein (2017) who showed that dark condi­ tions were better for microtuber formation com­ pared to a 16­h photoperiod. Similarly, Sakha et al. (2004) also reported that microtuber formation fre­ quency is higher under dark than light conditions. In addition, García and Bolaños (2017) showed that under the light condition with a photoperiod of 16­ hour light and 8­hour dark, the number of microtu­ bers was lower, but their biomass was higher and greener. On the other hand, under the dark condi­ tion, the number of microtubers was greater, with a lower biomass average and cream color. Garner and Blacke (1989) Al­Hussaini et al. (2015), showed in periods of total darkness, the microtuber weight is reduced. Other study also concluded that the light condition of 8 hours may be of benefit to the in vitro tuberization and increased the size and uniformity of microtubers compared to the in vitro tuberization under dark conditions (Pruski et al., 2002). Thus, an appropriate combination of light and dark conditions with short days can synchronize and accelerate the initiation and development of microtubers as well as increase their numbers. Finally, concerning the effect of sucrose on micro­ tuber formation, lower sucrose concentration (6%) resulted in an increase in the average number of the produced microtubers. Whereas sucrose presence had no effect on the length, width and weight (Table 5). Our results agree with those of Fufa and Diro (2014) with respect to the effect of 6% sucrose on the AN of the microtuber. Aslam et al. (2011) also found that a medium containing 6% sucrose was optimal in terms of minimum time of induction, aver­ age tuber number and weight of microtubers per sin­ gle nodal explant in cultivar Desiree. Imani et al. (2010) also reported that MS medium supplemented with 6% of sucrose yielded the maximum number and the highest AL and Aw of microtubers. In plant tissue culture, most plant requires an exogenous car­ bohydrates source because of the limited photosyn­ thesis that occurred in vitro (Lian et al., 2014). Tuberization is known to be regulated by carbohy­ drates availability such as sucrose which is the trans­ ported form of sugar required for starch synthesis (Abelenda et al., 2019). Sucrose is essential for the in vitro tuberization as an energy source and a signal for microtuber formation (Donnelly et al., 2003; Fufa and Diro, 2013; MotallebiAzar et al., 2013). In this study, to obtain microtubers of a sufficient weight, potato microshoots were cultured for 7 days in 16­h photoperiod followed by 50 days in dark. This type of treatment was referred to as light/dark treat­ ment (Table 1). It increased microtuberization by enhancing tuberonic acid synthesis that was shown to play an important role in in vitro tuber formation (Alisdair and Willmitzer, 2001). In conclusion, the best combination for rapid microtuberization obtained in our study was 6% sucrose, 5 mg.l­1 BAP for Spunta potato cultivar cul­ tured under C2 culture condition was within 10 days. Hossain et al. (2015) who investigated the effect of sucrose, growth regulators and potato varieties (‘Diamant’ and ‘Cardinal’) on rapid microtuberization found that the best combination was 9% sucrose at 5 mg.l­1 of 6­benzyl aminopurine cultivar within 6­8 days while Zakaria et al. (2008) who studied the opti­ mum level of Benzyl Adenine (BA) and Chloro Choline Chloride (CCC) to obtain large­size microtubers of the potato cultivar Diamant showed that early microtu­ ber induction occur by using 500 mg/l of CCC within 15.9 days or by using 10 mg/l of BA within 13.3 days. In the present study, the effect of two growth reg­ ulators, Kinetin and BAP, and the presence of sucrose and different photoperiodic conditions on the perfor­ mance of microtubers in the potato cultivar Spunta was evaluated. Microtubers were produced from in vitro grown plantlets regenerated in MS medium sup­ plemented with kin 0.4 mg.l­1, GA3 0.5 mg.l­1 and IBA https://link.springer.com/article/10.1007/s10535-017-0715-x#auth-J_-Salem https://link.springer.com/article/10.1007/s10535-017-0715-x#auth-A__M_-Hassanein Dalleh et al. ‐ Potato micropropagation 251 0.5 mg.l­1. The highest number of microtuber was obtained in M3 medium during C2 condition supple­ mented with 60 g.l­1 sucrose whereas C1 condition was better for AL, AW, and AWe of microtubers. The results indicated that microtuber induction of potato was highly dependent on sucrose, growth regulator and photoperiod conditions interaction. Production of large microtubers is important for successful uti­ lization of microtubers in seed potato production. 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