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Hamdeni, I.;  Louhaichi, M.;  Slim, S.;  Boulila, A.;  Bettaieb, T. Supplementation in In Vitro Tissue Culture Techniques. Encyclopedia. Available online: (accessed on 16 April 2024).
Hamdeni I,  Louhaichi M,  Slim S,  Boulila A,  Bettaieb T. Supplementation in In Vitro Tissue Culture Techniques. Encyclopedia. Available at: Accessed April 16, 2024.
Hamdeni, Imtinene, Mounir Louhaichi, Slim Slim, Abdennacer Boulila, Taoufik Bettaieb. "Supplementation in In Vitro Tissue Culture Techniques" Encyclopedia, (accessed April 16, 2024).
Hamdeni, I.,  Louhaichi, M.,  Slim, S.,  Boulila, A., & Bettaieb, T. (2022, December 01). Supplementation in In Vitro Tissue Culture Techniques. In Encyclopedia.
Hamdeni, Imtinene, et al. "Supplementation in In Vitro Tissue Culture Techniques." Encyclopedia. Web. 01 December, 2022.
Supplementation in In Vitro Tissue Culture Techniques

The growing demand for native planting material in ecological restoration and rehabilitation for agro-silvo-pastoral ecosystems has resulted in a major global industry in their sourcing, multiplication, and sale. Plant tissue culture is used for producing high-quality, disease-free, and true-to-type plants at a fast rate. Micropropagation can help to meet the increasing demand for planting material and afforestation programs.

in vitro plant propagation afforestation organic growth additives

1. Introduction

Climate change and induced human activities have negatively impacted agro-silvo-pastoral ecosystems across the planet [1][2]. The global push to achieve ecosystem restoration targets has resulted in an increased demand for native plant material that current production systems are unable to satisfy [3]. Using native species in restoration efforts is critical for recreating or maintaining healthy, resistant, and resilient ecosystems and communities [4]. Therefore, the development of methods for the large-scale plantations of selected genotypes of medicinal, pastoral, and forest species has become increasingly important in view of the need for the rehabilitation of marginal and degraded rangelands [5][6][7]. Regeneration of plants via in vitro tissue culture is considered to be an efficient approach for clonal plant propagation. Developing protocols for successful plant tissue culture is complicated because there are various interacting factors. Plant raw material, culture conditions, and culture media composition are determining factors in the quality of the final product obtained in any plant cell culture protocol [8]. The choice of nutritional components and growth regulators is one of the most important factors governing the growth and morphogenesis of the plant tissues in culture [9]. There are many components and additives used in plant micropropagation media which vary according to the plant species, cultivar, or explant type and must be experimentally defined for each case [10]. For the optimal growth of tissues in vitro, nutritional requirements should be present in optimum concentrations [11]. Micropropagation media are generally made up of these components: macronutrients, micronutrients, vitamins, amino acids, sugar, gelling agents, and growth regulators [12]. However, these compounds are expensive and pose risks if added in inadequate amounts [13]. Therefore, many studies have been conducted to explore the modification of culture media composition by adding low-cost organic materials as an alternative to expensive materials without compromising the quality of the produced plants [14][15][16]. A variety of organic growth additives such as coconut water, banana pulp, yeast extract, tomato juice, papaya juice, potato homogenate, and pineapple pulp can be very effective in promoting plant growth and development [17][18]. Such organic growth additives provide undefined mixtures of organic nutrients and growth factors [19]. The reasons for applying organic growth additives to the culture medium, besides being a natural source of carbon, are because they contain natural vitamins, phenols, fiber, hormones, proteins, lipids, and minerals [10].

2. Supplementation of Organic Growth Additives to Enhance In Vitro Culture Techniques

2.1. Vegetable, Fruit, and Plant Extracts

The growth and development of tissues vary for different plants according to their nutritional requirements. Tissues from different parts of plants may also have different requirements for satisfactory growth. Commonly, culture media are supplemented with a variety of organic substances or extracts including coconut milk, pineapple pulp, papaya juice, banana homogenate, orange juice, and tomato juice, to test their effect on growth enhancement [20].
Wiszniewska et al. [21] reported the successful micropropagation protocol of three Daphne species (Daphne caucasica, Daphne tangutica, and Daphne jasminea) on MS media supplemented with organic growth additives, including coconut water and pineapple pulp. Daud et al. [22], on in vitro regeneration of Celosia sp. using five kinds of organic growth additives, showed that young coconut juice (YCJ) at 70 mL/L induced the highest shoot regeneration (14.21), banana and tomato juices promoted the highest shoot regeneration of stem segments at 50 mL/L which produced 9.57 and 9.28 shoots per explants, and papaya juice, at the lowest concentration (20 mL/L), showed the highest shoot regeneration (10.5). Different organic growth additives such as coconut water, coconut milk, grind spinach leaves, grind potato tubers, grind carrot, rice flour, green gram, grind pumpkin, banana fruit, and orange juice were assayed by Manawadu et al. [14] to enhance in vitro regeneration of Raphanus sativus. The best response was observed in MS supplemented with 10% orange juice which produced the highest number of shoots (12 shoots/explant). Banana powder (BW), coconut water (CW), and potato dextrose (PD) were added to a basal seed sowing media of Epidendrum nocturnum. The culture medium with 10 mL/L CW showed the greatest germination percentage (71% and 76.75%) compared to 60 and 90 days after seed sowing. Media with 5 g/L BW + 5 mL/L CW showed greater values of plant length (19.80 mm), number of roots (2.1), and fresh weight (0.08 g) [16]. The effect of various organic additives (coconut water, birch sap, maple sap, and banana powder) on in vitro germination, protocorm formation, and seedling growth from Cypripedium macranthos on one-quarter MS medium was investigated during asymbiotic seed culture. The highest germination and protocorm formation percentages were achieved (70.8% and 74.2%) when 100 mL/L of coconut water was added to the basal medium. With 100 mL/L of birch sap or maple sap, the germination and protocorm formation percentages were over 65% and 68% [15]. Knudson C media supplemented with organic growth additives (coconut water, tomato juice, and banana pulp) at different concentrations for Dendrobium lowii in vitro propagation showed a notable protocorm development when treated with 25 g/L of banana pulp compared to the other supplements. It showed the highest growth index value of 593.3 with 100% of protocorms that successfully developed shoots and 93.3% of protocorms producing roots [23].
Supplementing coconut water (50 and 100 mL/L) for Musa cv. Rajabulu in vitro propagation showed the best results on the number of roots (9.33; 9) and root length (11.6; 10.76 cm). With CW concentrations from 50 to 200 mL/L, acclimatization was 100% [24]. Selakorn et al. [18] found that when CW (at 200 mL/L) was supplemented to MS medium, the highest number of shoots/explant (1.71), the highest shoot length (4.25 cm), and the highest number of leaves/explant (4) were recorded on Musa acuminata in vitro multiplication. For mass propagation of the endangered orchid Dendrobium chryseum, supplementing 10% CW to one-half MS medium resulted in the highest number of shoots as well as the longest [25]. The multiplication rate of Dianthus caryophyllus increased over four times when the shoot tip and nodal segment were used as explants on MS medium supplemented with 10% CW [26]. However, when 5% CW was added to one-half MS medium, the highest germination percentage for Gastrochilus matsuran was 93.3% [27]. Adding 4% coconut water with 3% sucrose increased the shoot elongation of Hylocereus polyrhizus to 2.45 cm [28]. MS media enriched with 2% CW enhanced shoot multiplication for Echinacea purpurea to 2.58 buds per explant [29].
Adding tomato juice (TJ) to promote shoot regeneration and multiplication of Physalis angulata produced the maximum shoot number (12.5) in MS supplemented with 5% TJ while the maximum shoot length (10.7 cm) was obtained with 7.5% TJ [30]. Phytamax media supplemented with 100 mL/L pineapple juice for in vitro propagation of Laelia rubescens resulted in the highest seedling height (1.31 cm), the number of leaves per seedling (3.33), and roots (2.33) [31]. However, at 200 mL/L, pineapple juice showed the highest results with 56% asymbiotic germination and 25.8 in seedling formation for in vitro development of xLaeliocattleya on MS medium [32].
Aloe vera gel (AvG) is the most commonly used part of the plant because of its biological effectiveness and chemical composition (carbohydrates, organic acids, proteins, phenolic compounds, vitamins, minerals, and amino acids) [20]. AvG has gained attention because of its interesting antioxidant and antimicrobial properties. Nowadays, it is proposed as an alternative to conventional additives used in plant micropropagation media [33][34][35]. Hamdeni et al. [33] reported that AvG served as an organic nutritional supplement for the enhancement of A. vera in vitro propagation protocol. The work of Haque and Ghosh [34] on A. vera showed that the highest number of formed shoots per explant (length ≥ 2 cm) was 17.8 shoots on MS medium supplemented with 10% AvG. For root induction, adding AvG (20% and 30%) to one-third of MS increased the response to 100% rooting, the number of roots per shoot (9.8; 9.2), and the length of the roots (3.1; 2.8). The study on the in vitro micropropagation of Bacopa chamaedryoides reported that the best rooting response (100%), number (18.3), and length (2.3 cm) of the shoot were achieved on one-half strength MS medium supplemented with 50% AvG [35].

2.2. Amino Acids, Polyamines, and Proteins

Most plants can synthesize the essential requirements of amino acids for cell proliferation and regeneration. Despite this, the exogenous supply of amino acids to culture media plays an important role in stimulating cell growth and the morphogenesis of tissues. Unlike inorganic nitrogen, amino acids are easily assimilated by plant cells and tissues [36]. Saad and Elshahed [37] suggested that plant cells have a higher capacity to take up and transport nitrogen from organic sources rather than inorganic ones. Several studies have reported using amino acids as an organic nitrogen source during in vitro propagation of several species such as Fragaria × Ananassa duch cv. Chandler, Oryza sativa, Rosa centifolia, Carica papaya, and Hibiscus moscheutos to enhance plant tissue growth and increase their regeneration [38][39][40][41][42]. According to Mandal et al. [43], a combination of all amino acids (methionine, glutamic acid, glycine, tryptophane, proline, lycine, arginine, and glutamine) in 20 mg/L concentrations resulted in the best axillary shoot proliferation response (100%) on Aegle marmelos and average shoot number per explant (2.22) when cultured on MS medium supplemented with 2 mg/L BAP. Baskaran et al. [44] reported that the type and amount of amino acids used in the medium have a significant effect on growth and multiple shoot development. Khatri et al. [45] evaluated three nitrogen sources, adenine sulfate (Ads), casein hydrolysate (CH), and putrescine (PU) for their ability to enhance in vitro shoot multiplication of Chlorophytum borivilianum. It confirmed that including 20 mg/L of Ads to MS medium supplemented with 2 mg/L BAP + 1 mg/L NAA resulted in the best shoot induction response (96.67%). For rooting, one-half MS supplemented with 9 mg/L PU + 2 mg/L IBA was best with 83.33% root induction. Samiei et al. [46] found that adding 600 mg/L of casein hydrolysate to the Vander Salm medium resulted in the maximum shoot number (4.1 shoots/explant) while glutamic acid at 12 mg/L enhanced shoot regeneration and leaf number of Rosa canina. Glutamine at 30 mg/L and asparagine at 20 mg/L concentrations improved shoot multiplication of Orthosiphon aristatus on MS medium supplemented with 1 mg/L BAP and 0.5 mg/L Kn [47]. David et al. [48] reported the rapid development from the protocorms of Vanda helvola (99.5%) treated in Knudson C medium containing 0.1% peptone, which successfully produced 3.10 leaves with an average length of 10.97 mm per responsive explant after 90 days of culture. Casein hydrolysate at 0.05% was most effective on Stevia rebaudiana in vitro propagation, which resulted in 90% regeneration frequency, a maximum of 15 shoots, and a shoot length of 6 cm per explant [49].

2.3. Essential Oils

In vitro microbial contamination is one of the most serious problems when culturing tissue. Using plant extracts such as essential oils for explant disinfection and establishing an aseptic culture medium to replace autoclaving was found to be an alternative procedure for plant tissue culture [50]. Oxidative browning from the accumulation and oxidation of polyphenols in the media is another impediment to in vitro propagation. For the successful establishment of in vitro tissue cultures, it is necessary to limit the oxidation of phenolics, the source of enzymatic browning, and inhibit microbial growth [51]. In the study of Hamdeni et al. [51], Rosmarinus officinalis and Thymus vulgaris essential oils were assayed for their effectiveness in controlling enzymatic browning and contamination of cultures from Aloe vera. While T. vulgaris essential oil inhibited explant growth, R. officinalis induced the highest explant survival percentage (100%) with no signs of browning after four weeks of culture with concentrations of 0.05%, 0.075%, and 0.1%. The lowest infection percentage (10%) was observed for media containing 0.075% and 0.1% of R. officinalis. The highest average number of leaves per explant was 3.71 with 0.1% R. officinalis and the greatest leaf length was 3.18 cm with 0.05%. According to Taghizadeh et al. [52], sterile medium conditions were obtained by using eugenol, carvacrol, or thymol at 0.01% and 0.5% which inhibited the growth of fungi and bacteria contaminations respectively with no autoclaving of the medium and vessels. The inhibitory effect of essential oils from Mentha piperita, Thymus vulgaris, and Cinnamomum camphora against common fungal contamination affecting the tissue culture of Phoenix dactylifera was reported by Jasim et al. [53]. These essential oils at 2% each resulted in 100% inhibition of mycelium growth of fungi species (Alternaria spp., Fusarium spp., and Aspergillus spp.) compared to the control treatment (10% of fungal contamination) with no disinfecting agent. The disinfecting properties of thymol and carvacrol (at 200 mg/L each and for 60 to 120 min exposure time) led to the appropriate control of fungi and bacterial infection of Cynodon dactylon nodal explants [54].


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