Shoot tip necrosis of in vitro plant cultures: a reappraisal of possible causes and solutions

Vol.:(0123456789) 1 3 Planta (2020) 252:47 https://doi.org/10.1007/s00425-020-03449-4 REVIEW Shoot tip necrosis ofinvitro plant cultures: areappraisal ofpossible causes andsolutions JaimeA.TeixeiradaSilva1,2· EsmaeilNezami‑Alanagh3,4· MaríaE.Barreal3 · MafatlalM.Kher5· AdhityoWicaksono6 · AndreaGulyás2· NorbertHidvégi2· KatalinMagyar‑Tábori2· NóraMendler‑Drienyovszki2· LászlóMárton2· MarianaLandín7 · PedroPabloGallego3 · JohnA.Driver8· JuditDobránszki2 Received: 19 March 2020 / Accepted: 27 August 2020 / Published online: 3 September 2020 © The Author(s) 2020 Abstract Main conclusion Shoot tip necrosis is a physiological condition that negatively impacts the growth and development of invitro plant shoot cultures across a wide range of species. Abstract Shoot tip necrosis is a physiological condition and disorder that can arise in plantlets or shoots invitro that results in death of the shoot tip. This condition, which can spread basipetally and affect the emergence of axillary shoots from buds lower down the stem, is due to the cessation of apical dominance. STN can occur at both shoot multiplication and rooting stages. One of the most common factors that cause STN is nutrient deficiency or imbalance. Moreover, the presence or absence of plant growth regulators (auxins or cytokinins) at specific developmental stages may impact STN. The cytokinin to auxin ratio within an invitro plant can be modified by varying the concentration of cytokinins used in the culture medium. The supply of nutrients to invitro shoots or plantlets might also affect their hormonal balance, thus modifying the occurrence of STN. High relative humidity within culture vessels and hyperhydricity are associated with STN. An adequate supply of calcium as the divalent cation (Ca2+) can hinder STN by inhibiting the accumulation of phenolic compounds and thus programmed cell death. Moreover, the level of Ca2+ affects auxin transport and ethylene production, and higher ethylene production, which can occur as a result of high relative humidity in or poor ventilation of the invitro culture vessel, induces STN. High relative humidity can decrease the mobility of Ca2+ within a plant, resulting in Ca2+ deficiency and STN. STN of invitro shoots or plantlets can be halted or reversed by altering the basal medium, mainly the concentration of Ca2+, adjusting the levels of auxins or cytokinins, or modifying culture conditions. This review examines the literature related to STN, seeks to discover the associated factors and relations between them, proposes practical solutions, and attempts to better understand the mechanism(s) underlying this condition invitro. Keywords Boron· Calcium· Chloride· In vitro shoots· Mineral nutrient deficiency· Physiological disorder· Plant growth regulators Introduction Shoot tip necrosis (STN) is a term that was originally coined by Sha etal. (1985). STN is sometimes also referred to as shoot tip abortion (Millington 1963), tip burn (McCown and Sellmer 1987), apical necrosis (AmoMarco and Lledo 1996; Koubouris and Vasilakakis 2006; Machado etal. 2014), apex necrosis (Rugini etal. 1986), top necrosis (De Klerk and ter Brugge 2011), shoot tip damage/injury (Ahmed and Palta 2017b), or shoot dieback (Barghchi and Alderson 1996). STN occurs when the shoot tip of a plant, both ex vitro and invitro, shows Communicated by Anastasios Melis. Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0042 5-020-03449 -4) contains supplementary material, which is available to authorized users. * Jaime A. Teixeira da Silva jaimete[email protected] * Adhityo Wicaksono [email protected].id; [email protected] Extended author information available on the last page of the article Planta (2020) 252:47 1 3 47 Page 2 of 35 signs of browning and death during multiplication, elongation and/or rooting stages, despite growing in apparently ideal conditions (Vieitez etal. 1989; Bairu etal. 2009b). Invitro, STN can ultimately result in the inhibited growth of the entire plantlet or it can be localized at affected shoots. The affected area spreads basipetally down from the shoot tip to lower parts of shoots. However, shoot formation from basal axillary shoot buds is not necessarily inhibited, as was observed for pistachio (Pistachia vera L.) (Barghchi and Alderson 1996). STN is also not always fatal to the plant, and apical dominance can be assumed by the next closest axillary bud, at least in sweet chestnut (Castanea sativa Mill.) and oak (Quercus robur L.) (Vieitez etal. 1989). If growing axillary branches develop STN, then a “witches’ broom” pattern develops (Fig.1; McCown and Sellmer 1987). On some occasions, the shoot tip can outgrow STN, leaving behind a scarred part of the stem with deformed leaves (McCown and Sellmer 1987). Sudha etal. (1998) observed axillary branching after STN in jivanthi (Holostemma annulare (Robx.) K. Schum., i.e., Holostemma ada-kodien Schult.) invitro cultures. STN is problematic not only for stock cultures of invitro plantlets, but also for commercial production (Sha etal. 1985). The precise mechanism underlying STN still remains unclear, although some possible reasons have been proposed, including mineral deficiency or the presence of high concentrations of plant growth regulators (PGRs) in the medium. One of the most cited reasons is calcium (Ca) deficiency. Ca deficiency is also a reason for the tip-burn disorder in the leaves and stems of field-grown fruits such as strawberry (Fragaria × ananassa Duchesne) and vegetables (Mason and Guttridge 1974, 1975; Saure 1998) and its symptoms closely resemble those of STN. This review aims to examine the literature that exists on this physiological disorder, including an earlier review by Bairu etal. (2009b), while exploring new literature published over the past decade. One objective is to attempt to better identify some of the possible reasons for the occurrence of STN and to suggest practical solutions to alleviate this physiological disorder invitro. Shoot tips are a popular explant in plant tissue culture. On occasion, shoot tip explants necrose (e.g., Krishna etal. 2008). In this review, the necrosis of shoot tip explants, i.e., explant necrosis, is not considered to be STN, which relates exclusively to the shoot tip of a tissue-cultured invitro plantlet. Shoot tip necrosis: occurrence andalleviation A wide range of plants display STN in invitro cultures (Table1; Suppl. Table1). Among all published studies, the occurrence of STN is particularly prominent in trees and woody shrubs (58.9% and 21.9%, respectively, of studies in Suppl. Table1; Suppl. Figure1). Studies on pistachio represent the largest proportion (10.8%) of studies on STN invitro, followed by pear (Pyrus spp.) (8.1%) (Suppl. Figure2). The incidence of STN in micropropagation, especially at the rooting stage, is shown in Fig.2. STN, at least according to the reported literature, has occurred most frequently in the Rosaceae (20.5%), followed by the Anacardiaceae (12.3%) (Suppl. Figure3). We caution readers that relative values might simply indicate the popularity of a studied species and not necessarily the actual incidence of STN in plan species or families studied to date. For example, only a single report on STN exists for an orchid, hybrid Cymbidium (Guha and Usha Rao 2012), so the incidence for the Orchidaceae is in fact 100% of studies, but the relative incidence (relative to all other species studied in Table1) is only 1.4%. De Block (1990) found that STN was linked to Ca deficiency and associated with the use of Woody Plant Medium Fig. 1 Schematic diagram of shoot tip necrosis (STN). An imbalance in minerals, nutrients, plant growth regulators, or other invitro conditions, lead to STN. This results in the blackening and death of the terminal shoot tip, the branching of axillary buds, and in some cases, STN in axillary shoots, leading to the formation of a “witches’ broom” pattern Planta (2020) 252:47 1 3 Page 3 of 35 47 Table 1 Shoot tip necrosis: observations and possible solutions* Scientific name and cultivar Stage, medium and observed problems Reason(s) provided for incidence of STN Solution provided to halt, reduce, or prevent STN, and other observations References Azadirachta indica A. Juss Optimum SMM: MS + 1.11µM BA + 1.43µM IAA + 81.43µM AdS. STN = UQ Basal medium microand macronutrient concentration Addition of 0.42mM Ca(NO3)2, 0.70mM Na2SO4, and 0.57mM K2SO4 Arora etal. (2010) Begonia homonyma Steud Optimum SMM: MS + 15µM BA + 5µM NAA. STN (all on MS) = 18% (15µM mTR + 5µM NAA for 12weeks → 2µM mTR + 0.5µM NAA for 6 weeks), 34% (15µM TDZ + 5µM NAA for 12 weeks → 2µM TDZ + 0.5µM NAA for 6 weeks), 44% (15µM BA + 5µM NAA for 12 weeks → 2µM BA + 0.5µM NAA for 6 week) Use of PGRs. High BA conc. and/or use of BA as the CK Use of mTR. Use half-strength MS rather than MS, reduce BA conc. to 0.5µM and add 2 or 5µM GA3: STN = 10–36% in various media defined in column 2 Kumari etal. (2017) Butea monosperma (Lam.) Taub Optimum SGM: half-strength WPM + 5mg/l BA (± 10mg/l fructose). 90% STN in terminal 2–3mm No substantiated reason provided. Only theoretical observation with no supporting data Addition of fructose eliminated STN in 95% of STN-positive cultures. Some phenolics were exuded from cut ends Kulkarni and D’Souza (2000) Castanea dentata (Marsh.) Borkh. cv. B’ville, Iowa #2, BDW Optimum RIM: half-strength MS + AC. SEM: WPM salts + NN vitamins. SMM = 500mg/l PVP 40 + 500mg/l MES + 0.89µM BA. STN = 25–67%, depending on genotype and treatment. STN reduced to 19–21% across three genotypes in replication trial Wounding, developmental stage, genotype Low concentration of BA (0.22µM) at an advanced stage of root initiation reduced STN. When on SEM, wounding had no effect on STN (~ 30–38%, 13–30%, 20–25% for B’ville, Iowa #2, and BDW, respectively). STN increased to 67% and 88% in Iowa #2 and BDW, respectively, when cuttings were plated on SMM (no change for B’ville, at 38%) Xing etal. (1997) Castanea sativa Mill. clones 431, T-13, 812; Quercus robur L Optimum RIM: half-strength MS + 3mg/l IBA (7 day) or dip in 1g/l IBA (20–60s) for chestnut; half-strength Gresshoff and Doy (1972) basal + 0.5g/l IBA (8min) for oak. STN UQ (only axillary shoot development) In SEM, when BA was removed, or in RIM, STN developed Addition of 0.01mg/l BA to RIM, but this reduced rooting in chestnut and oak, but axillary shoots developed marginally more (+ 1%) in chestnut clone T-13. When the cut surface of shoot tips was added to BA-impregnated agar, rooting was reduced in both trees, but axillary shoot development increased, the amount depending on the day of decapitation Vieitez etal. (1989) Castanea sativa Mill. cv. Garrone rosso, Clone 46 Optimum RIM: MS + 0.044µM BA + 5µM IBA (8 day) then same medium without IBA. STN = 23% after 8 day, 77% after 26 day for Clone 46; UQ for Garrone rosso Ca deficiency; lack of BA Clone 46 formed > twofold more STN than Garrone rosso (68% vs 25%). A block of agar containing 3mM CaCl2 and/or 5µM BA that was placed around shoot tips eliminated or delayed STN Piagnani etal. (1996) Planta (2020) 252:47 1 3 47 Page 4 of 35 Table 1 (continued) Scientific name and cultivar Stage, medium and observed problems Reason(s) provided for incidence of STN Solution provided to halt, reduce, or prevent STN, and other observations References Cercis canadensis var. mexicana SMM: WPM + MS vitamins + 11.1µM BA. STN UQ Excessively high concentrations of 2iP (25–74µM), TDZ (presumably 5 or 11–23µM) and kinetin (concentration range NR) General suggestions on how to improve shoot growth, but no specific details, or data, about how to improve STN Mackay etal. (1995) Corylus avellana L Optimum SMM: half-strength Cheng (1975) basal + 25µM BA (15 days) → same medium but + 0.5 or 2.5µM BA (25 days); optimum RIM: half-strength liquid Cheng (1975) basal + 50µM IBA (5 days) → same medium (solid) (15 days). STN = UQ High IBA concentration Reducing IBA from 50µM at the rooting stage to 10 or 25µM, or by reducing exposure period to IBA to 8 days Pérez etal. (1985) Cydonia oblonga Mill. rootstock clone C Optimum SMM: MS + 5µM BA. STN = 15% in SM of control cultures Ca deficiency Raising Ca2+ (in the form of Ca(NO3)2) from 3 to 18mM reduced STN, but this also reduced shoot proliferation. Link between Ca deficiency and hyperhydricity unclear Singha etal. (1990) Cymbidium hybrid Via del Playa Yvonne Optimum SMM: MS – MgSO4 + Na2SO4. STN = 5% (control), 35%, 60%, 80% (10, 15, 20µM SNP, respectively) Addition of SNP, a nitric oxide donor Nitric oxide, a positive and negative regulator of stress, could not prevent STN Guha and Usha Rao (2012) Dalbergia latifolia Roxb Optimum SMM: ¾ (macro) MS or WPM + 5mg/l BA + 0.5mg/l NAA. STN = UQ Tended to find STN associated with leaf abscission, but not linked to poor aeration or high humidity Doubling Ca2+ concentration in MS or WPM media did not reduce STN. Solution only provided to reduce leaf abscission by adjusting the NH4/NO3 ratio, but not STN Lakshmi Sita and Raghava Swamy (1993) Dipterocarpus alatus Roxb., D. intricatus Dyer Optimum seedling establishment: MS or WPM + 0.1µM BA. STN = UQ Nitrogen level Removal of NH4NO3 from WPM. High humidity likely not the cause because of high aeration of vessels Linington (1991) Dwarf rose (Rosa gymnocarpa Nutt.) cv. Starina Optimum RIM: auxin-free MS. Lowest incidence of STN = 6% (on RIM). When 1mg/l IAA was added, STN increased from 6–22% to 16–62% (range caused by the treatment) Inclusion of auxin, specifically IAA In the absence of auxin, 2.5–10mg/l AgNO3 reduced the incidence of STN from 22% to 2–12%. In IAAcontaining RIM, 1.5 × Ca2+ levels decreased STN from 58 to 28%. In IAA-containing RIM, 1.5 × Ca2+ levels + 2 × Mg2+ levels decreased STN from 58 to 24% Podwyszyńska and Goszczyńska (1998) Ensete ventricosum Welw. cv. Oniya Optimum SMM: MS + 11µM BA + 6µM IAA The term STN was not used However, STN was induced since shoot tips were split vertically down the center for micropropagation. 40% of greenhouse-derived shoot tips died due to blackening (aka STN; 0% in invitro shoot tips) Diro and van Staden (2005)a Planta (2020) 252:47 1 3 Page 5 of 35 47 Table 1 (continued) Scientific name and cultivar Stage, medium and observed problems Reason(s) provided for incidence of STN Solution provided to halt, reduce, or prevent STN, and other observations References Gaultheria hispidula (L.) Muhl. ex Bigelow, Rhododendron cv. Chinsayii, Rhododendron dauricum L Optimum SMM: Anderson (1984) basal + 15.9–16.3mg/l 2iP (across three plants). STN = UQ Presence of BA in medium at any concentration (0.1–10mg/l). Use of 2iP did not induce STN No suggestions Norton and Norton (1985) Haloxylon persicum (Bunge ex Boiss & Buhse) Optimum SMM: MS + 0.5µM TDZ. STN = 100% (0 or 2µM TDZ), 86% (0.5µM TDZ), 90% (1µM TDZ); all with 10µM Kin: 74% (2mM CaCl2), 65% (4mM CaCl2), 21% (2mM CaCl2 + 0.1mM H3BO3), 16% (4mM CaCl2 + 0.1mM H3BO3), 23% (2mM CaCl2 + 0.2mM H3BO3), 19% (4mM CaCl2 + 0.2mM H3BO3) Low Ca2+ and BO− 3 Addition of 4mM CaCl2 + 0.1mM H3BO3 + 10µM Kin. Use of several sugars (sucrose and maltose (60– 180mM), or fructose and glucose (110–330mM)) with 10µM Kin did not reduce STN (range = 89–100% for all treatments), except for 120mM sucrose (STN reduced to 84%) Kurup etal. (2018) Harpagophytum procumbens [(Burch) de Candolle ex Meissner] Optimum SMM: half-strength MS + 6mM Ca2+. STN = 27% (PGR-free MS), 25–35% (MS + 5µM BA, mT or mTR), 33–62% (MS + 5 or 10µM BA, mT or mTR + 2.5µM IAA), 80% (halfstrength MS), 20–133% (half-strength MS + 6–9mM Ca2+ alone or in various combinations with 0.2–0.5mM BO− 3 ) High CK (BA) level. Addition of auxin (IAA) Addition of 6–9mM Ca2+ with or without 0.2–0.5mM BO− 3 , or only 0.5mM BO− 3 , and in IAA-containing medium, 5 or 10µM mT or mTR reduced STN Bairu etal. (2009a) Harpagophytum procumbens [(Burch) de Candolle ex Meissner] Optimum SMM: MS + 8.8µM BA. STN = 88, 90, 86% (full-strength MS, NN and WPM); 29, 27, 27% (halfstrength MS, NN and WPM); 14, 26, 28% (quarter-strength MS, NN and WPM); 18, 21, 25, 26% (1, 2, 3, 4% sucrose); 29, 37, 64, 76% (sucrose, glucose, fructose, maltose at 0.086M); 19, 28% (2-week subculture; 4-week continuous culture) High mineral content of MS, NN, or WPM. High sucrose concentration (> 3%). Use of non-sucrose sources or carbohydrates. Lack of subculture Reducing basal media to half strength. Use of low sucrose concentration. Use of 2-week subcultures Jain etal. (2009) Harpagophytum procumbens [(Burch) de Candolle ex Meissner] Same as Bairu etal. (2009a) Active CKs may be converted to other inactive or irreversible forms of CKs, e.g., 9-glucosides Selection of CK, and the choice of CK:auxin ration can influence endogenous level of CKs, and thus the outcome of STN Bairu etal. (2011) Harpagophytum procumbens [(Burch) de Candolle ex Meissner] Optimum SMM: MS + 1.5mg/l BA + 6.2mg/l H3BO3. STN = 53% (SMM + 10mg/l H3BO3), 13% (SMM + 10mg/l H3BO3 + 5mM Si in the form of sodium silicate solution) No reason provided Addition of Si as SiO2Lišková etal. (2016) Planta (2020) 252:47 1 3 47 Page 6 of 35 Table 1 (continued) Scientific name and cultivar Stage, medium and observed problems Reason(s) provided for incidence of STN Solution provided to halt, reduce, or prevent STN, and other observations References Hibiscus rosa-sinensis L. cv. Cassiopeia Wind Yellow, Caribbean Pink Optimum SMM: MS + 2.2µM BA. STN = UQ Low Ca2+ level (independent of BA concentration) STN only assessed visually, but photographic evidence provided Christensen etal. (2008) Juglans nigra L Optimum SMM: half-strength DKW. STN = 11% or 17% in stage 1 (3–4 week culture) when Zea = 5 or 12.5µM, respectively, (44% and 33% in stage 2, which is 5–8 week of culture). 53% STN on MS when 12.5µM Zea was used, and measured in stage 2 Use of BA at 25µM or Zea at 12.5 or 25µM. Basal medium (decreasing level of STN): half-strength DKW > DKW > MS > WPM (stage 1) or MS > half-strength DKW > DKW > WPM (stage 2) Increasing TDZ from 0.5 to 1µM or reducing BA from 25 to 12.5µM BA improved percentage of spontaneous shoots, i.e., reduced STN Bosela and Michler (2008) Lavandula angustifolia Mill. cv Provence Blue Optimum SMM: MS + 1µM BA (40 day subculture). STN = 10% (1320mg/l CaCl2), 21% (440mg/l CaCl2) for a single subculture; 30% (1320mg/l CaCl2), 51% (440mg/l CaCl2) for a second subculture Low Ca2+ level. Subcultures Including CaCl2 at 1320mg/l. Only subculture once Machado etal. (2014) Lens culinaris Medikus cv. Titore Optimum SMM: MS + 0.4–0.8mg/l. STN = 85%, 70%, 56% and 49% in MS + 0.2, 0.4, 0.6 and 0.8mg/l BA, respectively (91%, 87%, 75% and 73% in B5; 0–3% in MS + 440mg/l CaCl2; 18%, 16%, 5% and 5% in B5 + 750mg/l CaCl2) Low BA conc. or reduced levels of Ca2+ Increasing BA conc. or adding 750mg/l CaCl2 to basal medium Ye etal. (2002) Lonicera caerulea f. caerulea; L. caerulea f. edulis SMM: 9/10 × MS + 8.9µM BA, 2.4µM pyridoxine HCl. STN = 17% on halfstrength MS; 6% on 75% MS; 9% on MS Insufficient microand macronutrients in MS; high day/night temperatures In caerulea form, 0% STN at 24°C/20°C (6% at 26°C/20°C, 17% at 28°C/21°C). In edulis form, 1% STN at 24°C/20°C (23% at 26°C/20°C, 49% at 28°C/21°C) Karhu (1997) Macadamia tetraphylla L.A.S.Johnson Optimum SMM: MS + 2mg/l BA. RIM: SM + 3mg/l IBA. STN in RIM: 40% at 3mM Ca2+ (20%, 70%, 85% at 6, 12 and 24mM). Mulwa and Bhalla (2000) reported 76% STN in RIM Inadequate aeration, high humidity Application of < 6mM Ca2+ in RIM Mulwa and Bhalla (2000); Bhalla and Mulwa (2003) Malus × domestica (Borkh.); Camellia sinensis (L.) Kuntze; Populus tremula L. × P. alba L.; Gerbera jamesonii Bolus ex Hooker f SMM/RIM: MS + 2.2µM BA + 5.3µM NAA. STN = 49, 53, 3 and 5% in shoots of apple, tea, poplar and gerbera, respectively Lack of exogenous CK (BA) in medium; lack of endogenous hormones in plants CK required in medium but details not provided Kataeva etal. (1991) Planta (2020) 252:47 1 3 Page 7 of 35 47 Table 1 (continued) Scientific name and cultivar Stage, medium and observed problems Reason(s) provided for incidence of STN Solution provided to halt, reduce, or prevent STN, and other observations References Musa spp. cv. Grande Naine (GN; AAA), Dwarf Cavendish (DC; AAA), Nendran (AAB), Quintal Nendran (QN; AAB) Optimum SMM: MS + 6.66µM BA. STN = 27% and 29% in GN and DC after seven subcultures on SMM (18% and 19% at the rooting stage); 38% and 40% in Nendran and QN after five subcultures on SMM (26% and 27% at the rooting stage) Low Ca2+ level Reducing the culture period, modifying salt strength in basal medium, addition of various PGRs (Kin, NAA, and IBA), adjusting levels of sucrose, fructose, and AgNO3 did not improve STN levels. Addition of 50–100mg/l CaCl2 for at least two subcultures after the fourth and sixth subculture (for bananas and plantains, respectively) allowed 91–97% of shoots (across all four cultivars) to be recovered (unclear if recovered shoots were free of STN) Martin etal. (2007) Paeonia suffruticosa Andr SMM: WPM + 0.3% AC. STN UQ Low Ca2+ level Adding 6mM CaCl2 to WPM Wang and van Staden (2001) Pimelea spicata R.Br Optimum SMM: half-strength MS + 0.5 or 1.0mg/l BA. STN = 38% (MS), 73% (MS + ventilation), 18% (half-strength MS), 56% (half-strength MS + ventilation), 32% (half-strength MS + 440mg/l CaCl2) Addition of Ca2+. Application of ventilation to culture flasks Using half-strength MS; not ventilating flasks; not adding supplementary Ca2+ Offord and Tyler (2009) Pistacia integrima × P. atlantica rootstock UCB1 Optimum SMM: MS + 0.5mg/l BA. STN = 42% (1 × CaCl2, 1 × H3BO3), 29% (1 × CaCl2, 2 × H3BO3), 38% (1 × CaCl2, 3 × H3BO3), 21% (1.5 × CaCl2), 19% (1.5 × CaCl2, 2 × H3BO3), 32% (1.5 × CaCl2, 3 × H3BO3), 19% (2 × CaCl2), 17% (2 × CaCl2, 2 × H3BO3), 30% (2 × CaCl2, 3 × H3BO3) (all × levels relative to MS) Low Ca2+and BO− 3 Increasing CaCl2 level to 3 × MS level, and doubling MS level of H3BO3 reduced STN to 17%. High level of KNO3 (2280mg/l) with 1320 or 1650mg/l NH4NO3 eliminated STN from 10% at all other concentrations Nezami etal. (2015) Pistacia integrima × P. atlantica rootstock UCB1 Optimum SMM: MS + 0.5mg/l BA, Gamborg vitamins. STN = 41% (control, no CNTs), 37% (50µg/l CNTs), 30% (100µg/l CNTs), 23% (150µg/l CNTs), 13% (200µg/l CNTs) CNTs promote or improve physiological processes Use of 200µg/l CNTs Kermani etal. (2017) Planta (2020) 252:47 1 3 47 Page 8 of 35 Table 1 (continued) Scientific name and cultivar Stage, medium and observed problems Reason(s) provided for incidence of STN Solution provided to halt, reduce, or prevent STN, and other observations References Pistacia vera L. cv. Mateur SMM: MS + 3mg/l BA tested for STN after 5 week. STN = 100% in 28-day cultures Ca Ca2+ and BO− 3 deficiency First STN symptoms in 12 days, affecting the whole aerial portion by 16 days. 100, 500 or 1000µM of B, as H3BO3, reduced STN but 200µM increased STN. 500 and 1000µM B stunted shoots. Ca2+ as 0.3 and 3mM CaCl2, and 15 and 30mM CG increased shoot number and length, but only 15 and 30mM could reduce STN, but eliminate it. Shoots immersed in liquid medium + 15mM Ca2+ prevented STN Abousalim and Mantell (1994) Pistacia vera L. cv. NR SMM: unrooted shoots on MS + 4mg/l BA after 4 week. STN = partly quantified High humidity in culture jars slowing nutrient flow Addition of 12mM CaCl2 reduced STN the most from 2.7/cultured explant to 1.1/cultured explant, but 3–24mM was an effective range. Ca acetate could also reduce STN but also caused shoot stunting. H3BO3 at 100–800µM reduced STN from 2.6 (100µM) to 0.4 (800µM), but above 200µM, shoot multiplication was reduced while shoot stunting occurred at 100 and 200µM. No Caor B-free controls were used. Increasing ventilation of adding a liquid medium overlay did not reduce STN Barghchi and Alderson (1996) Pistacia vera L. cv. NR Optimum SMM: DKW + 5µM BA + 0.5µM IBA + 0.01g/l AA. STN = 25% (DKW), 45% (MS), 60% (WPM) Use of CG, shoot density in flasks, flask ventilation, flask volume, bottom cooling Improvements in STN when using bottom cooling (50% STN), reducing shoot number per flask from 7 to 5 (52% STN), or use of ventilated jars with larger volume (58% STN), relative to the control (75% STN) or the addition of 3mM CG (80% STN) García etal. (2011) Pistacia vera L. cv. Ohadi, Kalleghochi SMM: unrooted shoots on MS + 4mg/l BA + 0.25mg/l NAA tested for STN after 4–6 weeks; callus production and media browning also observed. STN = UQ NAA inhibited CK production; callus that formed at base of shoots used nutrients; rooted shoots may absorb nutrients; insufficient Ca2+ uptake No suggestions. STN initially detected in Barghchi and Alderson (1983) Barghchi and Alderson (1985) Planta (2020) 252:47 1 3 Page 9 of 35 47 Table 1 (continued) Scientific name and cultivar Stage, medium and observed problems Reason(s) provided for incidence of STN Solution provided to halt, reduce, or prevent STN, and other observations References Platanus acerifolia (Ait.) Willd Optimum SMM: MS + 1.33µM BA + 0.27µM IAA. STN = 57% (gelled medium), 69% (liquid medium), but wide variation (~ 20–69%) depending on the genotype Use of liquid medium Use of solid medium (gelled with 7g/l agar) Alegre etal. (2015) Populus alba L. × P. tremula L.; P. trichocarpa Torr. & A.Gray ex. Hook. × P. deltoids W.Bartram ex Marshall Optimum SMM: WPM + 0.5mg/l MES + 0.02mg/l TDZ. STN in transformation experiments = UQ NO− 3 / NH+ 4 ratio, especially < 0.8mM NH+ 4 in medium; medium pH < 4.9; Ca deficiency Medium without TDZ could not form shoots; after 7 days, NH+ 4 conc. decreased from 5.0mM to 1.6mM; use of 650mg/l CG + 0.5mg/l MES + 2.5µg/l BA allowed shoot growth without STN De Block (1990) Portulaca grandiflora Hook Optimum SMM: MS + 4µM BA + 8µM Kin. STN = 80–90% (0.1–0.4mM B); ~ 75–100% (3, 6, 12, 18, 24 and 30mM CG); 50–85% (3, 6, 9, 12, 24 and 30mM CaCl2) Insufficient Ca2+ and BO− 3 Use of 18mM CaCl2 reduced STN to 40% Srivastava and Joshi (2013) Prunus armeniaca L. cv. Helena, Lorna Optimum SMM + RIM: QL + 1.78µM BA + 0.2µM IBA. STN was observed in the rooting phase (~ 65% for ‘Lorna’; ~ 75% for ‘Helena’) No reason provided Adding 0.2mg/l BA reduced STN to ~ 5% in ‘Lorna’ (~ 25% for ‘Helena’), but this also reduced rooting efficiency. High rooting ability of both cultivars maintained with reduced STN when 5–20mg/l BA added Pérez-Tornero and Burgos (2000) Prunus armeniaca Lam Optimum SMM: WPM + 0.5mg/l BA. STN = UQ, only weighted Low NH+ 4 and NO− 3 . Low mesos (Ca2+, Mg2+, K+) Critical threshold for CaCl2·2H2O: 2.94x. If CaCl2·2 H2O > 2.94x interaction with KH2PO4, so it should be higher than 1.12x. Recommended NO− 3 level: > 45mM. Considering STN and other growth factors, optimum range of NO− 3 is > 25mM and ≤ 35mM and optimum NH+ 4 / Ca2+ ratio is ≤ 0.8 Kovalchuk etal. (2017a, b, 2018) Pyrus communis cvs. Old Home × Farmingdale 87, Horner 51, Winter Nelis; P. dimorphophylla; P. ussuriensis cv. Hang Pa Li Optimum SMM: MS + 4.44µM BA. STN = UQ, but genotype-dependent and characterized as a function of significant interactions between multiple factors General trends: low mesos (Ca Ca2+, K+, Mg2+) and N caused STN. P. communis: low mesos + low Fe and N; P. dimorphophylla: high NH4NO3, mesos + Fe with low KNO3; P. ussuriensis: low NH4NO3, KNO3 and mesos + high Fe and micros caused STN STN was reduced by increasing the mesos (P. communis), using low NH4NO3, KNO3 and high mesos (P. dimorphophylla), and using high KNO3 and low mesos (P. ussuriensis). STN frequently occurred simultaneously with other physiological problems such as callus induction, hyperhydricity, hypertrophy, fasciation and formation of hooked leaves Reed etal. (2013) Planta (2020) 252:47 1 3 47 Page 16 of 35 (WPM; Lloyd and McCown 1980). De Block (1990) also noted that the occurrence of STN might have been caused by a decrease in medium pH, possibly as a result of ammonium ( NH+ 4 ) uptake by shoots. Relative to Murashige and Skoog (1962) (MS) medium, WPM has almost the same Ca2+ content (≅ 3mM in WPM), about a quarter the concentration of NH+ 4 (20.61 vs 5.00mM) and nitrate ( NO− 3 ) (39.41 vs 9.71mM), about two-thirds the content of K+ (20.05 vs 12.61mM), about a quarter of the NH+ 4 /Ca2+ ratio, but more than 1.5-times higher Ca2+/K+ ratio (Suppl. Table2). MS was employed in 68.6% of the studies listed in Table1 while 21.4% used WPM. This suggests that the use of these basal media is not recommended, especially for trees and woody shrubs. This is curious if one considers that WPM was designed specifically for Ericaceous woody plants. The most popular theory for the cause of STN is related to nutrient deficiency and imbalance. Another is the impact and imbalance of PGRs. These possibilities are explored in greater detail next. Nutrient deficiencies Calcium deficiency The most commonly ascribed reason for STN is Ca deficiency (32.8% of studies in Table1). Table1 indicates that one of the most popular methods to relieve STN has been to increase Ca2+ concentration in the culture media (35.9% of studies in Table1). In pistachio, Barghchi and Alderson (1985) suggested that STN was caused by Ca and boron (B) deficiency, but only on some shoots that had not rooted. Dolcet-Sanjuan and Claveria (1995) reduced STN by lowering the concentration of Ca2+ (as calcium chloride, CaCl2·2H2O) in medium to one-third of the level in MS, and by reducing the subculture period from 4–6weeks to 3weeks. Kovalchuk etal. (2017a) used a CART (classification and regression tree analysis) decision tree to model the incidence of STN in wild apricot (Prunus armeniaca L.) shoot cultures based on previous response surface methodology (RSM). They noted that no STN developed in wild apricot shoots when CaCl2·2H2O was < 2.94mM, which is the precise concentration of CaCl2·2H2O in WPM medium (Suppl. Table2). Furthermore, the Reed etal. (2013) study of pear (one of several connected studies), which was based on MS medium, noted an increase in STN with low mesos (CaCl2·2H2O, MgSO4·7H2O, KH2PO4) but also the involvement of ammonium nitrate. Wang and van Staden (2001) doubled the concentration of CaCl2 in WPM to 6mM to reduce the incidence of STN in tree peony (Paeonia × suffruticosa Andrews) cultures. Machado etal. (2014) found that the incidence of STN was halved when the level of CaCl2·2H2O was increased threefold from 440 to 1320mg/l (from 3.96mM to 11.89mM) in true lavender (Lavandula angustifolia Mill.) shoot multiplication medium. Christensen etal. (2008) completely eliminated STN in Chinese hibiscus (Hibiscus rosa-sinensis L.) shoot cultures after increasing CaCl2 concentration in MS from 2.99mM to 9mM, independent of the N6-benzyladenine (BA) concentration used (0.22 or 2.2µM). STN was observed in cultures of potato (Solanum tuberosum L.) ‘Dark Red Norland’ when insufficient (68µM) CaCl2 was provided, resulting in a loss of apical dominance and enhanced axillary branching, a response that did not occur when there was sufficient (1360µM) Ca2+ in medium (Busse etal. 2008). The level of CaCl2 was one of the factors that affected the level of STN in Indian lilac (Azadirachta indica A. Juss) cultures (Arora etal. 2010). In potato ‘Dark Red Norland’, Ozgen etal. (2011) ascribed the increase in STN, as a result of low Ca2+ levels in medium, to injury of the shoot tip and subsequent loss of apical dominance, thereby stimulating axillary shoot formation. In Indian redwood (Soymida febrifuga (Roxb.) A. Juss.) cultures, the simultaneous use of calcium nitrate and calcium pantothenate (vitamin B5) at intermediate concentrations could eliminate the incidence of STN (Chiruvella etal. 2011, 2014). Mubina etal. (2018) eliminated STN by doubling the MS-based levels of CaCl2 and KNO3 in chickpea (Cicer arietinum L.) shoot regeneration medium. Nutrient deficiencies or excesses sensu lato accounted for 9.4% of the studies in Table1. Thirugnanasampandan etal. (2009) found that an adjustment of CaCl2 and MgSO4 in sarasaparilla (Smilax zeylanica Vent.) shoot regeneration medium prevented STN. In lentil (Lens culinaris Medikus), increasing Ca2+ (up to 750mg/l, i.e., 6.75mM) and BA concentration (0.2–0.6mg/l, i.e., 0.89–2.66µM) in MS and B5 (Gamborg etal. 1968) basal media decreased the incidence of STN (Ye etal. 2002). That decision that was based on earlier research by Parh etal. (1998). Wetzstein etal. (1989) noted STN during the acclimatization stage of pecan nut (Carya illinoensis (Wangenh.) K. Koch) and not invitro, reducing its incidence by applying a foliar spray of 0.4% calcium nitrate. Another way to increase Ca concentration in plant culture medium is using calcium gluconate (6.3% of studies in Table1), usually from the start of shoot induction or multiplication stages (McCown and Sellmer 1987). The application of Ca-gluconate during invitro culture of hybrid aspen (Populus alba × Populus tremula) and poplar (Populus trichocarpa × P. deltoides) eliminated STN in 50% of the shoots (De Block 1990). However, if 3mM Ca-gluconate was combined with 0.5mg/l (2.5µM) 2-(N-morpholino) ethanesulfonic acid (pH 5.8), a buffer, then STN was totally eliminated. This may be because Ca-gluconate uptake into cells has a different pathway, via the glucose uptake system, and this form of Ca2+ does not involve the release of toxic chloride if CaCl2 is used, allowing medium pH to be Planta (2020) 252:47 1 3 Page 17 of 35 47 stabilized and thus ion exchange and uptake to occur at an optimum pH range of 5.6–5.9 (Pasqua etal. 2002). However, the supply of additional Ca2+ via CaCl2 can also increase the concentration of chloride (Cl−) ions, similar to the use of NaCl, and this may be toxic to plant tissues (McCown and Sellmer 1987). In wild apricot, Pérez-Tornero and Burgos (2000) found that the addition of calcium nitrate or Cagluconate decreased the incidence of STN but also lowered rooting ability. Shoot growth rate may be balanced by Ca2+ supply to shoots to avoid STN. This balance might depend on species and cultivars, the concentration of other nutrients in the medium that might modify Ca uptake, as well as the tissue or plant’s developmental stage. The form of Ca2+ may also affect STN since the same ion (Ca2+) content (Suppl. Table2) can be supplied by different additives (salts or organic forms), but with different uptake mechanisms (Thor 2019) and thus various effects on STN (Table1). The organic form has a dual uptake mechanism: (1) after dissociation of the Ca2+ ion through the highly regulated Ca2+ uptake system which is strongly affected by the culture conditions (pH, relative concentration of other cations and anions, etc.); (2) without dissociation, the organic form of calcium is taken up directly into the cytoplasm via the uptake system but the organic part is under completely different regulation (White and Broadly 2003). Boron deficiency Unlike Ca deficiency, where the effect of STN occurs on younger leaves in the growing meristem and develops basipetally, STN caused by B deficiency (6.3% of studies in Table1) affects older leaves and spread upwards, or acropetally, as was reported in pistachio (Abousalim and Mantell 1994). Martinelli (1988) indicated the same problem in zebrawood (Pistacia integerrima J.L. Stewart ex Brandis) and Mt. Atlas mastic tree (Pistachia atlantica Desf.). Similarly, Parfitt and Almehdi (1994) found STN in hybrid pistachio UCB-1 (P. atlantica × P. integerrima), independent of the basal medium used, suggesting that the condition was not based on nutrients. Abousalim and Mantell (1994) confirmed these findings, noticing STN in P. vera cv. Mateur shoot cultures, but partially resolved this by adding calcium (Ca2+) or boron ( BO− 3 ). Barghchi and Alderson (1996) used the same approach (see details in Table1) but could also reduce STN using liquid medium. There is an interaction between BO− 3 and Ca2+ uptake (Fox and Albrecht 1958): (1) a high BO− 3 concentration can improve the uptake of Ca2+; (2) boron helps the movement of Ca2+ in plants. However, Abdulnour etal. (2000) described that high BO− 3 concentrations could adversely affect Ca2+ uptake, even causing toxicity if BO− 3 levels were as high as 0.4mM, as in the case of devil’s claw (Harpagophytum procumbens (Burch.) DC. ex Fig. 2 Incidence of shoot tip necrosis (STN) in in vitro cultures of walnut (Juglans regia L.) Paradox rootstock during micropropagation in Driver and Kuniyuki walnut medium (DKW; Driver and Kuniyuki, 1984) (unpublished results). (1) if 3-week-old shoots were used, the incidence of STN was high (20–30%), most likely because tissue is soft (non-lignified), but the use of 4-week-old shoots, which are more lignified, have a lower incidence of STN, even reduced to 0%; (2) initial “Vlach” [a selection of Paradox (J. hindsii x J. regia)] material is from a 110-year-old mother tree, located near Modesto (CA, USA) for which invitro cultures were originally established by John Driver in 1985; (3) walnut tends to be somewhat recalcitrant to rooting, so occasionally high concentrations of IBA (8–10 mg/l) are added to rooting medium. If IBA is transported to the shoot tip, especially soft shoots that may take up excessive amounts of IBA, this may result in the death to the shoot tip, a condition we coin as “IBA burn”, which is visually similar to STN. However, this does not take place if more mature shoots are used and this can be achieved by increasing the subculture interval from 3 to 4 weeks. Black arrows indicate STN. Scale bars indicate 3cm (top), 2cm (middle) and 2cm (bottom) Planta (2020) 252:47 1 3 47 Page 18 of 35 Meisn.) (Bairu etal. 2009a). Boron deficiency often appears to occur in invitro cultures of Pistachia species. However, the proper balance of nutrients should be assessed due to their interaction. Nitrogen deficiency: nitrogen form andquantity Grigoriadou etal. (2000) found that the occurrence of STN in pear was cultivar dependent and strongly related to the basal medium used. In their study, the application of Quoirin and Lepoivre medium (1977) resulted in the highest rate of STN (64%) in the case of ‘Highland’, while they observed that most shoots were affected by STN on half-strength MS medium in ‘Williams’ (31%). The former medium contains about a quarter the level of NH+ 4 , a quarter of the NH+ 4 / NO− 3 ratio, and only about 14% of the NH+ 4 /Ca2+ ratio compared to MS medium. However, the rate of STN was only 10% in ‘Highland’ and 14% in ‘Williams’ when shoots were cultured on WPM, in which the NH+ 4 / NO− 3 ratio is the same as in MS medium but the total level of N and the NH+ 4 /Ca2+ ratio is only one-quarter of that in MS. In shoot cultures of wild apricot (Kovalchuk etal. 2017a), the use of RSM showed that some STN occurred in control shoot cultures in WPM. However, the influence of NH+ 4 and NO− 3 was much stronger, i.e., when the concentration of these nutrients was low, STN was higher (Kovalchuk etal. 2017b). Ultimately, the recommended level of NO− 3 was > 45mM (Kovalchuk etal. 2018). Excessive NH+ 4 and NO− 3 in two pear rootstock cultures (12.3 and 13.2mM for OHF; 22 and 20.9mM for Pyrodwarf) resulted in STN (Jamshidi etal. 2016). In contrast, a shortage of N in dunns white gum (Eucalyptus dunnii Maiden) cultures resulted in STN, and the minimum level of N required was 27.7g/kg (Oberschelp and Gonçalves 2018). The total N content and/or the NH+ 4 / NO− 3 ratio differ in several media commonly used for the micropropagation of various plant species (Suppl. Table2; Phillips and Garda 2019). These can cause variation in the growth and developmental responses of invitro shoots. From the above results, the occurrence of STN appears to depend mainly on the quantity and form of N, the NH+ 4 /Ca2+ ratio, and the quantity of mesos elements [mainly Ca2+, magnesium (Mg2+) and potassium (K+)] in medium (Reed etal. 2016; Kovalchuk etal. 2017a, b). Interaction ofother ions onSTN: theion‑confounding effect Unlike the above studies, which concluded that one of the main reasons for STN was Ca deficiency, some studies did not show any effect of Ca2+ on STN (4.7% of studies in Table1). When Piagnani etal. (1996) applied CaCl2 at 3, 9 or 18mM, this did not reduce the incidence of STN in two sweet chestnut cultivars. In fact, 18mM CaCl2 reduced rooting. When Grigoriadou etal. (2000) increased the level of Ca2+, this did not decrease the incidence of STN in pear. Thomas (2000) observed that the balance of Ca2+ and Mg2+ ions in roots and shoots was responsible for STN. Unlike the trend in most of these studies, Offord and Tyler (2009) found that the addition of Ca2+ to half-strength MS medium almost doubled STN in an endangered Australian shrub, pink pimelea (Pimelea spicata R.Br.). Recently, the implementation of knowledge-based design of experiment (DOE) techniques has been widely used for understanding and improving the performance of complex invitro systems (for example, Wada etal. 2015; Kovalchuk etal. 2017a). Niedz and Evens (2016) reviewed the greatest advantage of DOE in simultaneously minimizing the quantity of data while maximizing data quality based on considering only low order interactions in multi-factor studies (“hierarchical ordering”) on the basis of “sparsity of effects” wherein just a few factors would drive the system efficiently (Box and Meyer 1986). The use of DOE by Reed etal. (2013) enabled them to conduct a unique experiment to simultaneously study the effect of all macroand microelements of MS medium on a wide range of physiological disorders in diverse pear germplasms. They divided mineral nutrients of MS medium into five independent groups with the advantage of reducing the required treatment numbers from 3125 (55) to just 43 treatments. Noticeably, their findings asserted that STN is a genotype-dependent disorder that is affected by an imbalance of nutrients in culture media. Therefore, deficiencies in mesos (CaCl2·2H2O, MgSO4·7H2O, and KH2PO4) or nitrogen (either NH4NO3 or KNO3) commonly contributed to STN. Wada etal. (2013, 2015) followed the same approach to improve the quality of many invitro pear genotypes by readjusting nutrients in MS medium, such as increasing mesos (CaCl2, MgSO4, KH2PO4) with increased nitrogen, to eliminate all physiological disorders. In their studies, STN was more evident with lower Ca2+ content than MS-based concentrations although lower concentrations of some mesos, including in the MS medium control, may have accounted for the disorders, although no general trend was observed. In addition to the level of CaCl2, Arora etal. (2010) reported that other nutrients, principally Ca(NO3)2, Na2SO4, and K2SO4 in basal MS medium, also affected the level of STN in Indian lilac (Azadirachta indica A. Juss.) cultures. The next challenge of tissue culture studies are ion-confounding problems (Niedz and Evens 2006, 2007), wherein salts are subjected as factors in an experimental design and analysis rather than ions by themselves, whilst ions drive the system. For instance, many authors have frequently tried to alleviate STN in different species by increasing the amount of MS-CaCl2 because this unique salt contains the Ca2+ ion. CaCl2 in MS medium releases 2.99mM Ca2+ plus 6mM Cl− into solution (Suppl. Table2). Therefore, it is Planta (2020) 252:47 1 3 Page 19 of 35 47 inconclusive to attribute the problem of STN exclusively to Ca2+ deficiency while the role of Cl− is completely overlooked. Numerous examples of this inconclusiveness can be found in the literature (Barghchi and Alderson 1996; Piagnani etal. 1996; Bairu etal. 2009a, 2009b; Ozgen etal. 2011; Machado etal. 2014; Poothong and Reed 2014; Surakshitha etal. 2019). Nevertheless, it has recently been proved that Cl− (> 4.67mM) has a positive effect on reducing STN symptoms in pistachio (Nezami-Alanagh etal. 2019). To the best of our knowledge, the latter study was the first report of the beneficial effect of Cl− on controlling STN in plants. Ca-gluconate has been reported as a way to alleviate STN in herbal medicinal plants (Srivastava and Joshi 2013), woody shrubs (Amalia etal. 2014), fruit trees (Abousalim and Mantell 1994; Pérez-Tornero and Burgos 2000), and other trees (De Block 1990; Pasqua etal. 2002). As far as we know, the only report to assess the individual role of the gluconate− ion (C6H11O7 −) in plant growth and development was Nezami-Alanagh etal. (2017). Using artificial intelligence models, a significant negative influence of gluconate− concentration (range 0.0–6.02mM) on two growth parameters (shoot length and total fresh weight) during pistachio micropropagation was determined. Thus, we strongly advise to cautiously use gluconate in medium formulations for plant micropropagation. Moreover, we also encourage the use of any method (statistical, response surface methodology, chaid or artificial intelligence) that allows the simultaneous study, on one hand, of the effect of a single ion, and on the other hand, of interactions between several factors. Plant growth regulators affect STN Another popular theory to explain the cause of STN is the effect of the level and type of PGRs in the medium. STN has been linked to the level of PGRs in 23.4% of the studies in Table1. However, an increase in PGRs may alleviate some nutrient deficiencies (Preece 1995). This fortifies the notion that nutrient deficiency is the major cause of STN. STN in apple (Malus × domestica Borkh.) was attributed to low endogenous hormone content (Kataeva etal. 1991). According to Kataeva etal. (1991), in the absence of roots, where cytokinins (CKs) are mainly synthesized, endogenous CK concentrations in shoots decrease. This affects the synthesis of auxin in the shoot apical meristem, stimulating STN. In sweet chestnut and oak, the absence of CK (BA) in rooting medium, or the presence of a low concentration of BA, induced STN, although the application of BA to cut ends of shoots prior to rooting increased axillary shoot production (Vieitez etal. 1989). When Piagnani etal. (1996) added 5µM BA to sweet chestnut shoot tips, STN was eliminated, but a mixture of 5µM BA and 3mM CaCl2 delayed STN. A CK × Ca2+ × BO− 3 interaction on STN was observed in grape (Vitis vinifera L.) cv. Red Globe where supplementary CaCl2 and H3BO3 were needed to suppress STN, even after the level of BA had been optimized (Surakshitha etal. 2019). Thomas (2000) observed that CK concentration had no signficant effect on STN. Surakshitha etal. (2019) did not observe this effect in grape; instead, the level of STN depended on BA concentration. When BA concentration was increased from 8.9µM (0% STN) to 17.8µM, cane apple (Arbutus unedo L.) cultures displayed 8.7% STN (Gomes etal. 2010). Pérez etal. (1985) reduced STN in filbert (Corylus avellana L.) by adding indole-3-butyric acid (IBA) to medium at a low concentration (10 or 25µM), or by reducing the period of exposure to IBA. In apricot, dipping shoot tips in a solution of BA (1.78–3.11µM, depending on the cultivar) prior to culture in rooting medium alleviated STN while kinetin had no effect (Pérez-Tornero and Burgos 2000). The mere presence of 2.5µM BA in MS medium induced STN in moringa (Moringa oleifera Lam.) (Hassanein etal. 2018). In pistachio micropropagation, STN was significantly reduced when BA was added at high concentrations (5.77 < BA < 6.66µM) to basal media (Nezami-Alanagh etal. 2019). In contrast, in blackberry (Rubus sp. ‘Dirkson Thornless’), rhododendron (Rhododendron ‘P.J.M. Hybrids’) and Chinese hibiscus, when Compton and Preece (1988) increased BA concentration to 10µM, STN increased (details in Table1). Norton and Norton (1985) also noticed STN in Gaultheria sp. and Rhododendron sp. (Ericaceae) when any concentration of BA was used, although 17 other Ericaceae species did not show STN. As mentioned above, Podwyszyńska and Goszczyńska (1998) found that when indole-3-acetic acid (IAA) was present in medium, the incidence of STN increased in rooting cultures of dwarf rose (Rosa gymnocarpa Nutt. ‘Starina’). Lin etal. (2011) also observed STN in Korean pasque flower (Pulsatilla koreana) shoots on MS-based rooting medium containing BA and IAA. Serres etal. (1990) observed STN in American chestnut (Castanea dentata [Marsh.] Borkh.) in rooting medium containing IBA, and only the top node was affected, allowing lower axillary shoots to form shoots and thus not influencing explant survival. Bairu etal. (2009a) found that the inclusion of BA increased STN in devil’s claw, even more so when an auxin (IAA) was also added. However, the inclusion of meta-topolin (mT) or meta-topolin riboside (mTR; more background in Aremu etal. (2012)) could reduce— but not eliminate—the incidence of STN. Kinetin stimulated STN in Rosa clinophylla Thory cultures (Misra and Chakrabarty 2009). In buchu (Coleonema pulchellum I.Williams) shoot-inducing cultures, STN only occurred when thidiazuron (TDZ) was applied at 13.6µM in MS basal medium, or in response to 300µM casein hydrolysate or mebendazole, 40µM glutamine, or 40µM glutamine in combination with 4.5µM TDZ (Baskaran etal. 2014). STN was also observed Planta (2020) 252:47 1 3 47 Page 20 of 35 in grape ivy (Cissus rhombifolia Vahl, syn. Cissus alata Jacq.) shoot cultures grown in the presence of 4.5µM TDZ, but not in response to 4.4µM BA (Dewir etal. 2018). The use of 2µM TDZ, or even the lack of TDZ, induced STN in 100% of white saxaul (Haloxylon persicum (Bunge ex Boiss and Buhse)) shoot cultures. The latter was also associated with stem fasciation, a common response to high concentrations of TDZ (Dewir etal. 2018). Intermediate concentrations (0.5 or 1µM) of TDZ reduced the incidence of STN by 10–14% (Kurup etal. 2018). The incidence of STN was reduced when 0.1 or 0.2mg/l (0.8µM) mT was added to the shoot multiplication medium of Scots elm (Ulmus glabra Huds.) shoots (Mirabbasi and Hosseinpour 2014). When Marín etal. (2016) replaced BA with 5µM mT in pistachio shoot culture medium, STN was reduced to 20% of cultures. The application of 15mg/l (40.7µM) adenine sulfate prevented STN in nannaari (Hemidesmus indicus (L.) R.Br.) (Nagahatenna and Peiris 2007). When Naaz etal. (2014) added 100mg/l (271.3µM) adenine sulfate to BAsupplemented MS medium (WPM resulted in higher levels of STN), STN was reduced to 10% in jambolan (Syzygium cumini (L.) Skeels.) shoot cultures. Several other studies assessed the ability of PGRs to reduce STN. Podwyszyńska and Goszczyńska (1998) significantly reduced the incidence of STN in dwarf rose rooting medium containing IAA by adding 2.5–10mg/l (14.7–58.8µM) silver nitrate (AgNO3), and by increasing the level of MS-based Ca2+ 1.5-fold (increasing the level of MS-based Mg2+ twofold was optional). AgNO3 is an effective ethylene inhibitor (Purnhauser etal. 1987). Vieitez etal. (2009) reduced the incidence of STN in northern red oak (Quercus rubra L.) cultures by supplementing medium with 3mg/l (17.6µM) AgNO3. Martínez etal. (2017) found that AgNO3 at 20µM reduced the incidence of STN in evergreen oak (Quercus ilex L.) cultures. Park etal. (2016) found that the production of ethylene in rose (Rosa hybrida cv. Tineke) shoot multiplication medium increased the level of STN. They proved this by applying different levels of an ethylene promoter, 1-aminocyclopropane-1-carboxylic acid (ACC), to medium. Ahmed and Palta (2017a) reduced the incidence of STN in Ca2+-deficient (6.7 or 27.75mg/l (60.3–250µM) CaCl2) potato shoot induction medium by adding 1 or 2μM NAA, or 300–500μM lysophosphatidylethanolamine (a phospholipid). Curiously, Ahmed and Palta (2017b) found that agars with different levels of Ca2+ significantly affected the level of STN: Acros agar was Ca2+ deficient (22.92mg/l (0.5718mM)) while Fischer Scientific agar was slightly Ca2+ deficient (84.36mg/l (2.1mM)) relative to the control (MS Ca level = 3000μM or 120.23mg/l). However, supplementation with 27.75–221.96mg/l (0.25–1.99mM) CaCl2 reduced or eliminated STN in five potato cultivars (see details in Table1). If auxin is used in excess, especially in juvenile pistachio cultures at the rooting stage, STN may develop (Fig.2). The ability of endogenous and exogenously added PGRs to alter the level of STN in response to PGR type and concentration, especially during the rooting phase, suggests their important role in STN. To limit or prevent STN, an adequate level of BA and TDZ should be applied, while the application of mT and/or its derivatives may be beneficial. Broadly, altering the type or level of exogenously applied PGRs in plant invitro cultures might not impact STN exclusively, but might also impact many mechanisms, while different genera or species might respond differently (Cardoso etal. 2018). Auxins should not be used at excessive concentrations while ethylene production should be inhibited as much as possible. Excessive ethylene production in plant invitro cultures can be avoided by applying auxins at a suitable concentration, by increasing aeration of culture vessels (Kumar etal. 1998), using aerated containers, or it can be inhibited by applying ethylene inhibitors such as AgNO3 (Teixeira da Silva 2013). Other factors andinteractions impacting theincidence ofSTN Timing ofmeasurements andsubculture length Grigoriadou etal. (2000) noted that the level of STN was much higher at 4weeks than at 2weeks, suggesting that sampling time influenced the quantitative outcome. This issue was not raised in most other studies on STN but is an important issue to consider when dealing with plant tissue cultures (Teixeira da Silva and Dobránszki 2013). Srivastava and Joshi (2013) found that STN was 62% after 2weeks, but 90% after 4weeks in rose moss (Portulaca grandiflora Hook.) cultures. The same time-dependent incidence of STN was observed in tissue cultures of five pear cultivars (Thakur and Kanwar 2011). The time-sensitive outcome of STN was also observed by Kishore etal. (2015) in pointed gourd (Trichosanthes dioica Roxb. var. Swarna Alaukik). They observed higher STN (83%) during shoot multiplication at 42days than at 14 (16%), 21 (44%), 28 (61%), and 35 (72%) days on MS medium containing 3% sucrose, 0.8% agar, 0.02% carbendazim and 37.17µM kinetin. Ahmed and Palta (2017a) observed 56% STN in Ca2+-deficient (60μM CaCl2; 52% STN with 250μM CaCl2) shoot induction medium of potato cv. Dark Red Norland when sampled at 15days, but 75% STN after 25days (62% STN with 250μM CaCl2). In other words, reported STN levels were higher in older cultures. Similarly, Ahmed and Palta (2017b) found higher levels of STN in the majority of five potato cultivars (i.e., a genotype-specific response) when two Ca2+-deficient agar brands were used in shoot induction medium and sampled at Planta (2020) 252:47 1 3 Page 21 of 35 47 23days relative to 15days. Thakur and Kanwar (2011) also observed STN during invitro rooting on semisolid and liquid medium in five pear cultivars: 6%, 28%, 39%, 49%, and 64% of cultures displayed STN at 14, 21, 28, 35, and 42days (details in Table1). Sudha etal. (1998) attributed a long culture period, in excess of 8months, to the incidence of STN in arka (Holostemma annulare (Roxb.) K. Schum.). Amin and Jaiswal (1988) also attributed STN to excessive subculture length in guava (Psidium guajava L.) for cv. Chittidar during shoot tip (derived from mature plants) culture on MS medium with 4.4µM BA. Papadatou etal. (1990), however, did not observe any STN when seedling-derived shoot tips of the same guava cultivar was used on Rugini olive medium (Rugini 1984) with 8.8µM BA. Delaying the subculture period longer than 2 weeks induced STN in rose and miniature Chinese rose (Rosa chinensis minima (Sims) Voss.) (Hsia and Korban 1996). Ca2+ concentration that exceeded 6mM negatively impacted Pistacia vera shoot growth and increased shoot chlorosis, but a reduction of the subculture period from 4–5weeks to 3weeks reduced the incidence of STN (Dolcet-Sanjuan and Claveria 1995). Tilkat etal. (2008) also found 3weeks to be suitable for reducing STN in pistachio cultures. Alderson etal. (1987) suggested that increasing the frequency of subcultures, thus reducing the subculture period, could reduce the incidence of STN in dwarf Russian almond (Prunus tenella Batsch). A longer subculture length was also associated with hyperhydricity, which is frequently caused by the accumulation of ethylene in cultures (Park etal. 2004). These results are not surprising. One cause of STN is the deficiency of nutrients, so the chance of nutrient deficiencies within a subculture increases over time as nutrients become depleted (Ramage and Williams 2002). The timing of sampling can influence the reported outcome of STN, although the likelihood of STN is higher in older cultures and may be related to changes in the nutrient content of tissue culture medium over time. Genotype‑specific responses Mythili and Thomas (1999) successfully micropropagated two female cultivars (Swarna Alaukik and Swarna Rekha) and one male line of pointed gourd on MS medium but noted a decline in transferable nodes in Swarna Alaukik due to leaf chlorosis if subculture was delayed by 8weeks. In contrast, no symptoms of STN were observed in pointed gourd accession IIVRPG-102 (Kumar etal. 2016), suggesting that STN could be a genotype-specific response or due to the presence of carbendazim, as was also reported by Kishore etal. (2015). Thakur and Kanwar (2011) observed STN between the 6th and 8th week at the shoot regeneration stage in three pear rootstocks (P. pyrifolia [Burm F.] Nakai, P. pashia Buch. Ham. and P. serotina Rehd.), and two scion cultivars ‘Patharnakh’ (P. pyrifolia [Burm F.] Nakai) and ‘Punjab Beauty’ (P. pyrifolia x P. communis), but the level of STN was dependent on genotype. Thakur and Kanwar (2011) found a genotype dependence in response to Ca and B supplementation. When 3µM Ca2+ (up from 1.5µM) and 200µM BO− 3 were used, this completely alleviated the incidence of STN in the wild cultivar (from 9.12% to 2.60%) but had no significant effect nor did it prevent STN in the remaining four cultivars. In London plane tree (Platanus acerifolia (Ait.) Willd), Alegre etal. (2015) found a clear influence of genotype on the incidence of STN during shoot multiplication, with a wide range (~ 20–69%) of affected cultures that was genotype dependent. Thus, the susceptibility of a plant to develop STN might be both species and cultivar dependent. Choice ofbasal medium Bosela and Michler (2008) also noticed that the choice of basal medium affected the level of STN in Eastern black walnut (Juglans nigra L.). However, this was also dependent on the invitro developmental stage and the CK used, with higher levels of STN observed in the presence of Driver and Kuniyuki walnut medium (DKW; Driver and Kuniyuki 1984) and zeatin. Similarly, in unpublished results, STN was observed invitro cultures of walnut Paradox rootstock during micropropagation in DKW medium (Fig.2). Shoots were first multiplied on DKW basal medium supplemented with 1mg/l (4.4µM) BA, 0.1mg/l (0.49µM) IBA and 30g/l sucrose and subcultured every 3weeks. Three-weekold shoots, in preparation for rooting, were first placed in the dark for 5days at 24°C. STN was observed in rooting medium consisting of DKW free of cytokinins (BA), but including 10mg/l (44µM) IBA and 50mg/l (146µM) sucrose. After 5days in rooting medium, auxin-induced shoots were placed in a greenhouse and exposed to high relative humidity (> 95%). These induced shoots rooted and acclimatized concurrently ex vitro. Curiously, García etal. (2011) observed quite the opposite in pistachio where DKW medium resulted in lower levels of STN than in MS or WPM media. They attributed STN to the three times higher levels of Ca2+ in DKW (relative to MS and WPM). Moreover, some authors previously recommended the inclusion of calcium gluconate to prevent STN (Abousalim and Mantell 1994). However, Nezami-Alanagh etal. (2017) fund that gluconate− had an adverse effect on invitro pistachio plant growth. In high-bush blueberry (Vaccinium corymbosum L.), the use of MS medium induced STN, especially when 0.5mg/l zeatin was used with higher concentrations (> 1mg/l) of IBA, but when this was replaced by Anderson’s rhododendron medium (Anderson 1984), STN was eliminated (Ružić etal. 2012). Anderson’s Planta (2020) 252:47 1 3 47 Page 22 of 35 rhododendron medium, relative to MS medium, contains about one-quarter the concentration of K+, NH+ 4 and NO− 3 (Suppl. Table2). Martin etal. (2007) tested several factors, including PGRs, carbohydrate sources, and AgNO3, in the media of subcultured necrotic shoots to try and improve the incidence of STN in invitro banana (Musa spp.) cultures. Normal shoots were recovered only with the addition of 50–100mg/l (0.45–0.9mM) of CaCl2. When full-strength MS medium was used, STN was observed in Zeyheria montana Mart. cultures, but not when halfor quarter-strength MS was used (Cardoso and Teixeira da Silva 2013). Similarly, full-strength MS medium induced STN in Barbados nut (Jatropha curcas L.) cultures, but not half-strength MS (Daud etal. 2013), an outcome that Dangi etal. (2014) also observed for bahera (Terminalia bellerica (Gaertn.) Roxb.). Using basal CK medium that had diluted levels of MS microand macronutrients (Cellárová etal. 1992), Moura (1998) found 15% and 23% STN in shoot initiation and elongation stages, respectively, of leafy St. John’s wort (Hypericum foliosum Aiton). The use of WPM induced more STN than MS in the multiplication of wych elm (Ulmus glabra Huds.) shoots (Mirabbasi and Hosseinpour 2014). Consequently, the choice of appropriate basal medium can be a solution in itself. Further, altering the level of certain ions, especially Ca2+, can also help to reduce STN. However, changing a single medium constituent might affect the uptake or utilization of other nutrients, while agar source and type may affect micronutrients, as discussed elsewhere in this review. Thus, this solution should be viewed cautiously. Moreover, several species responded well to reduced MS salts. Antioxidants Amalia etal. (2014) also noticed some (unquantified) reduction in STN of raspberry (Rubus idaeus L.) shoots when 50 or 100mg/l (0.284–0.568mM) of ascorbic acid was used, but not as effectively as the use of 1g/l Ca-gluconate. The reduction in STN was also genotype dependent. Misra etal. (2010) were also able to reduce STN in Barbados nut cultures by adding antioxidants, either 25mg/l (81.3µM) of reduced glutathione or 10mg/l (56.7µM) of ascorbic acid. Jaiswal etal. (2013) observed STN in Indian kino tree (Pterocarpus marsupium Roxb.) cultures. They eliminated STN by adding 568µM ascorbic acid, 260µM citric acid, 605µM ammonium sulfate, and 217µM adenine sulfate to MS basal medium. By adding 1% activated charcoal to root proliferation medium, Sánchez etal. (1997) reduced the incidence of STN from 89 to 30% in sweet chestnut clone 90,025 and from 38 to 13% in clone Pr5. The addition of antioxidants to basal medium during shoot multiplication might be an effective way to reduce or prevent STN. Humidity, aeration, andhyperhydricity: isthere alink toSTN? High humidity and weak ventilation in culture vessels can cause abnormalities, including hyperhydricity (Lai etal. 2005), or STN (Fig.3a). These abnormalities may in turn be caused by increased ethylene production (Isah 2015). A decrease in humidity within culture vessels can be achieved by improving the ventilation of vessels, or by increasing the agar concentration in basal medium. The former can encourage gas exchange, thereby decreasing ethylene concentration within the vessel (reviewed in Isah 2015). In their summary, Bairu etal. (2009b) concluded that better aeration decreased STN. Barghchi and Alderson (1983, 1985, 1996), in addition to stating that STN was caused by Ca deficiency, also proposed that STN was linked to high humidity in a culture vessel. They found that high humidity reduced plantlet transpiration rate, causing a “low mobility of calcium ions in the xylem”, i.e., reduced nutrient flow to meristematic regions in growing shoot tips. Several authors found that high relative humidity and low transpiration caused by closed culture vessels decreased Ca2+ flow during transpiration, causing Ca deficiency (Sha etal. 1985; Singha etal. 1990; Abousalim and Mantell 1994) (Fig.3a). Ca2+ transport is inhibited by apoplast flooding in which apoplastic air spaces are blocked as a result of water clogging (van den Dries etal. 2013). Bhalla and Mulwa (2003) noted that when Ca2+ in medium exceeded 6mM in macadamia nuts (Macadamia F. Muell.), STN symptoms increased. They found that this was as a result of poor culture vessel aeration and high relative humidity and was not linked to Ca2+ level in the medium. McCown and Sellmer (1987) suggested that when culture vessels that increase gas exchange or reduce relative humidity are used, hyperhydricity as well as STN were reduced (Fig.3b), while the use of Gelrite instead of agar improved shoot growth, but increased the incidence of hyperhydricity. Although Matu etal. (2006) did not specifically link aeration problems or hyperhydricity with the incidence of STN in staff tree (Maytenus senegalensis (Lam.) Exell) tissue culture, they described this condition as “a major problem”. They improved shoot growth by substituting Gelrite for agar as the gelling agent during shoot multiplication. Offord and Tyler (2009) noted that increased ventilation by employing vented lids for greater transpiration, STN in pink pimelea (Pimelea spicata R. Br.) cultures increased from 38 to 73% on MS medium and from 18 to 56% on half-MS medium, but hyperhydricity was observed in both ventilated and unventilated treatments. Compared to cultures on solid medium containing DKW macroelements, MS microelements, 3% sucrose and 0.44µM BA, cultures of dahlia (Dahlia x hybrida) in liquid culture eliminated STN (De Klerk and ter Brugge 2011). Vibha etal. (2014) reached the same conclusion for North Indian rosewood Planta (2020) 252:47 1 3 Page 23 of 35 47 (Dalbergia sissoo Roxb.) cultures, reducing hyperhydricity by adding ammonium sulfate to the medium. In quince (Cydonia oblonga Mill.), Singha etal. (1990) found that long culture periods and infrequent subcultures resulted in both STN and hyperhydricity, as well as leaf necrosis. However, the application of 3 to 18mM Ca2+ and increasing agar concentration from 0.6% to 1.2% reduced the incidence of these two physiological disorders, but also lowered shoot proliferation and shoot fresh and dry weight. McCown and Sellmer (1987) suggested that some poplar genotypes developed hyperhydricity in response to media with high nitrogen (N) levels. Balla and Kirilla (2006) noted STN in invitro cultures of peach interspecific rootstocks at the rooting phase. One possible reason was the development of hyperhydricity at temperatures exceeding 22°C (Balla and Mansvelt 2012). Kataeva etal. (1991) found that the absence of BA in medium resulted in no hyperhydricity, but in high levels of STN, in unrooted apple and tea (Camellia sinensis (L.) Kuntze) shoots and in rooted poplar (Populus tremula L. × P. alba L.) and gerbera (Gerbera jamesonii Bolus ex Hooker f.) plantlets (Table1). However, when BA was added at 4.4µM into media for apple, hyperhydricity increased to 4% in cotton-covered vessels (18% in foil-covered vessels), even more at 22.1µM (18% in cotton-covered vessels and 73% in foil-covered vessels), and even more at 22.1µM with 5.3µM NAA (58% in cotton-covered vessels and 80% in foil-covered vessels). Had the levels of STN in these four plant species been defined, this would have been an important assessment of the possible link between STN and hyperhydricity. We recommend the use of culture vessels with improved ventilation and reduced hyperhydricity to reduce the accumulation of ethylene. This would improve Ca2+ flow, ultimately reducing the incidence of STN. Other factors Several studies in the literature have reported the incidence of STN in response to factors that are not linked to nutrients, PGRs, or other factors discussed previously. Lall etal. (1997) observed that exposure of invitro Mrs Flanagan’s impatiens (Impatiens flanaganiae Hemsl.) plantlets to high light intensity (280µmolm−2·s−1) for 7weeks induced necrosis in terminal parts, but it was not clear if this was STN. However, Marks and Simpson (1999) also noticed a similar pattern of increased STN in invitro cultures of disanthus (Disanthus cercidifolius Maxim.) and Northern European hawthorn (Crataegus oxyacantha ‘Paul’s Scarlet’), but not of three Rhododendron cultivars. In their study, plants were exposed to moderate or high light intensity (55 or 106µmolm−2s−1) and tested against low light intensity (11 or 26µmolm−2s−1), in culture (Table2). In American chestnut genotypes B’ville, Iowa #2 and VDW, wounding of cuttings did not significantly affect the rate of STN, but promoted rooting, although the level of STN and rooting was intricately dependent on the level of auxin and cytokinin (Xing etal. 1997). Khalafalla and Daffalla (2008) found that scion length and rootstock age impacted the incidence of STN in grafted gum arabic (Acacia senegal Fig. 3 Schematic diagram depicting how high humidity and reduced transpiration in closed tissue culture vessels may induce shoot tip necrosis (STN). Such growth conditions can induce low levels of calcium (Ca) which in turn reduces cell motility and pectin synthesis, disrupting cell (cell wall or cell membrane) and tissue integrity, and reduce transpiration (Hepler 2005), potentially leading to STN. This biochemical hypothesis has still not yet been tested specifically for STN Planta (2020) 252:47 1 3 47 Page 24 of 35 (L.) Willd.) shoot tips. They registered 7% STN when scions were 2.5–3cm, 27% when they were 1.5–2cm, or 14% when rootstocks were 7days old (0% STN when rootstocks were 14days old). The incidence of STN in grape cv. Arka Neelamani also depended on the position of the explant on the stock plantlet and its initial weight (Thomas 2000). The establishment of STN is also influenced by light intensity during shoot multiplication, and other factors such as rootstock age. Possible mechanisms underlying STN Programmed cell death, necrosis, andstress It is possible that the underlying mechanisms to explain explant wounding and subsequent tissue browning may be similar. However, since we consider STN to be a physiological response of a living tissue on a developing invitro plant, rather than a cut explant, we will hereafter only consider the possible factors that might affect STN. The use of chemicals such as antioxidants (e.g., Misra etal. 2010), reduction in light intensity since high light intensity stimulates polyphenol oxidase (Krishna etal. 2008), or the inhibition of phenylpropanoid biosynthesis (Jones and Saxena 2013) may be viable ways to alleviate STN, similar to tissue browning after explant cutting during the establisment of an invitro culture. When oxidative stress can no longer be controlled, programmed cell death (PCD) develops (Gaspar etal. 2002), which may explain STN. Beckman (2000) further suggested that specialized cells induced cell suberization and lignification as a result of the accumulation of phenolic compounds, thereby strategically stimulating PCD. PCD develops as a function of H2O2, the “death signal”, and in response to other reactive oxygen species (Demidchik 2015). PCD affects several developmental events in plants, including sensescence where proteins, phospholipids and pigments may be degraded (Drury and Gallois 2006; Henmi etal. 2007; Misra etal. 2010; Kacprzyk etal. 2011). Ethylene, which can accumulate in closed tissue culture vessels invitro, is a strong inducer of leaf senescence and a trigger for PCD (Santner etal. 2009; Trobacher 2009; Park etal. 2016). Moreover, ethylene initiates a signaling pathway, including calcium transport, during the development of aerenchyma, which also plays a role in PCD (Jones 2001). It is still unclear if PCD is involved in, related to, or the cause of STN. Calcium andcalcium signaling Ca deficiency is one of the most commonly cited reasons for STN (Table1). In closed tissue culture vessels, the high relative humidity and reduced transpiration induced low Ca2+ levels, because Ca2+ cannot translocate but must be actively transported (Hepler 2005). This does not permit pectin to be synthesized, impeding the formation of the shoot meristem due to compromised cell integrity and membrane permeability (Martin etal. 2007; Naaz etal. 2014) (Fig.4). Moreover, Ca2+ serves as a universal secondary messenger in cellular signaling in plants, so the hormonal balance and Ca2+ supply during growth and development may affect each other. There is a correlation between Ca2+ and auxin signaling: auxin induces Ca2+ signals and vice versa, and Ca2+ controls the speed of transport of an auxin (Vanneste and Friml 2013). A high level of auxin might cause excessive ethylene production in jars and change the CK: auxin ratio. As was observed by Busse etal. (2008), STN in potato cultures, which resulted in a loss of apical dominance, was caused by low levels of Ca2+ in medium (Ozgen etal. 2011). This theory was confirmed by two experiments (Ozgen etal. 2011): the addition of a Ca2+ chelator, ethylene glycol tetra acetic acid (EGTA), to medium with sufficient Ca2+ (2720µM) induced the precise same symptoms as low Ca2+ levels, namely STN and axillary shoot formation; in that condition, the supplemental addition of 204µM strontium (Sr2+), which is a Ca2+ analog, restored apical dominance. Increasing Ca2+ in medium of several tree species has been shown to alleviate STN (McCown and Sellmer 1987). Cagluconate is an organic form of Ca that allows Ca2+ to be released into aqueous solutions, explaining why it has occasionally been used to alleviate STN, but it negatively affects shoot growth (Singha etal. 1990; Amalia etal. 2014). We suspect that the use of gas-permeable culture vessels, such as the Vitron or Miracle Pack (Teixeira da Silva etal. 2006), could reduce hyperhydricity, reduce the accumulation of ethylene, increase transpiration and consequently increase the transport of Ca2+ to the shoot apical meristem, although this hypothesis has yet to be tested on invitro plant cultures displaying STN. Since the level of the endogenously accessible Ca2+ depends not only on its content in the medium but also on its uptake, it is reasonable to expect that the content of other ions in the medium such as Mg2+, K+, some microelements or NH+ 4 , which can modify the uptake of Ca2+ from the medium based on nutrient interactions (Fageria 2001), may have an effect on STN. In this sense, Ca deficiency may be relative. The content of mesoelements such as Ca2+, Mg2+ and K+ in the medium modified the rate of the STN in different plant species (Reed etal. 2016; Kovalchuk etal. 2017a, b; details in Table1). In wild apricot shoot culture, the NH+ 4 /Ca2+ ratio should be optimally below 0.8, to minimize STN, specifically NO− 3 > 45mM and 25mM < NH− 3 ≤ 45mM + NH+ 4 /Ca2+ ≤ 0.8 for node 5 (Kovalchuk etal. 2018). In shoot cultures of different pear species, besides the role of various mesoelements like Ca2+, Mg2+, and K+, the roles of Fe2+ and the proper Planta (2020) 252:47 1 3 Page 25 of 35 47 concentrations of nitrogen compounds were reported to be involved in the occurrence of STN (Reed etal. 2013, details in Table1). In addition, Subbaiah etal. (2000) found that activation of protease, which played a role in PCD induced by anoxia in maize (Zea mays L.) roots, was Ca2+ dependent. The role of Ca2+ in plant stress response and signaling has been detailed in a review by Robertson (2013). Bairu (2008) found that the main problem related to Ca deficiency was not the level of Ca2+ in medium but its limited transport in plantlets due to excess BO− 3 . Moreover, Ca2+ transport in plants through the xylem sap requires transpiration, which is inhibited by high humidity in the culture vessel, thus the limited mobility of Ca2+ can play a role in the development of STN (Hirschi 2004). Plant growth regulators As indicated above, ethylene is a likely inducer of PCD and thus may be a direct cause of STN in unventilated culture vessels. Table1 indicates that PGRs have been heavily implicated in STN, mostly CKs during the shoot induction stage, but also the CK × auxin interaction during the root induction stage of shoots. For example, the absence or use of low concentrations of CKs was implicated as a reason for the presence of STN since roots are the main source of CKs (Chen etal. 1985), reducing cell division in the shoot apical meristem (Piagnani etal. 1996). The damage to shoot tips reduces the synthesis of auxin because shoot tips are the main site of auxin biosynthesis (Leopold 1975; Aloni etal. 2003; Hopkins and Hüner 2009). Exogenously added CKs can act with different efficiency depending on their structure, the plant species or even the cultivar (Dobránszki and Teixeira da Silva 2010). Application of the highly active mT or mTR (hydroxylated BA derivatives) can delay senescence and can eliminate abnormalities of invitro cultures, including a reduction of hyperhydricity and STN (Aremu etal. 2012). Similarly, Kumari etal. (2017) found that when mTR was used in the shoot regeneration medium of dwarf wild begonia (Begonia homonyma Steud.), the occurrence of shoot necrosis was reduced to about a half of other regenerants cultured on medium with BA or TDZ. After subculture of regenerated shoots onto elongation medium, STN occurred again at a low frequency (18%) if previously regenerants had developed on medium with mTR. Bairu etal. (2011) studied the background effects of CK on STN, including an analysis of both endogenous and exogenous CKs and their derivatives, in devil’s claw. They found a higher content of total CKs in necrotic shoots than in normal shoots in all studied cases. However, they also detected larger quantities of deactivated forms of CKs such as 9-glucolides in BA-treated and necrotic shoots relative to normal and mT-treated shoots, suggesting that the occurrence of STN may be due to a change of active CKs to other deactivated products, possibly reversibly, but that can be toxic. N7 and N9 conjugates, which are the inactive forms of BA, are biologically inactive and chemically quite stable, but their conjugation is not fully irreversible (Werbrouck etal. 1996). These conjugates usually accumulate at the base of invitro shoots, so the active form can be continuously released and cause disorders such as STN (Werbrouck etal. 1996; Strnad etal. 1997). Topolins are hydroxylated forms of BA with high activity in plant tissue culture but they have a different metabolism from that of BA and, therefore, side-effects caused by the release of the active form from inactive BA conjugates can be avoided (reviewed in: Dobránszki and Teixeira da Silva 2010). Table 2 Cause–effect (IF–THEN) rules created by neurofuzzy logic indicating the best combination of inputs to alleviate STN in pistachio invitro cultures Inputs with stronger effects have been highlighted by software (for additional details see Nezami-Alanagh etal. 2019) Rules Membership degree SubModel:1 1 IF EDTA− is low and K+ is low THEN Low (1.00) 2 EDTA− is low and K+ is high Low (1.00) 3 EDTA− is mid and K+ is low Low (1.00) 4 EDTA− is mid and K+ is high Low (1.00) 5 EDTA− is high and K+ is low High (1.00) 6 EDTA− is high and K+ is high High (1.00) SubModel:2 7 IF BA is low THEN High (1.00) 8 BA is high Low (1.00) SubModel:3 9 IF IF Cl− is low THEN High (1.00) 10 IF Cl− is high Low (1.00) SubModel:4 11 IF Genotype is Ghazvini THEN High (0.55) 12 Genotype is UCB-1 Low (0.93) SubModel:5 13 IF Na+ is low THEN Low (0.78) 14 Na+ is mid High (0.94) 15 Na+ is high Low (1.00) Planta (2020) 252:47 1 3 47 Page 32 of 35 abscission and shoot tip necrosis. Plant Sci 88:107–112. https :// doi.org/10.1016/0168-9452(93)90115 -G Lall N, Bosa A, Nikolova RV (1997) Morphological characteristics of impatiens flanaganiae Hemsl. grown under different light conditions. S Afr J Bot 63:216–222. https ://doi.org/10.1016/S0254 -6299(15)30747 -X Leopold AC (1975) Part II. Growth regulation. In: Leopold AC, Kriedemann PE (eds) Plant growth and development, 2nd edn. McGraw-Hill Book Company, USA, pp 109–222 Lin G-Z, Zhao X-M, Hong S-K, Lian Y (2011) Somatic embryogenesis and shoot organogenesis in the medicinal plant Pulsatilla koreana Nakai. Plant Cell Tiss Org Cult 106:93–103. https ://doi. org/10.1007/s1124 0-010-9897-z Linington IM (1991) Invitro propagation of Dipterocarpus alatus and Dipterocarpus intricatus. Plant Cell Tiss Org Cult 27:81–88. https ://doi.org/10.1007/BF000 48211 Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127. https ://doi. org/10.1111/j.1399-3054.1965.tb068 74.x Lišková D, Kollárová K, Kučerová D, Vatehová Z, Zelko I, Lux A, van Staden J (2016) Alternatives to improve long-term cultures of Harpagophytum procumbens in vitro. S Afr J Bot 104:55–60. https ://doi.org/10.1016/j.sajb.2015.10.008 Lloyd G, McCown B (1980) Commercially-feasible micropropagation of mountain laurel, Kalmia latifolia, by use of shoot-tip culture. Comb Proc Intl Plant Prop Soc 30:421–427 Machado MP, da Silva ALL, Biasi LA, Deschamps C, Filho JCB, Zanette F (2014) Influence of calcium content of tissue on hyperhydricity and shoot-tip necrosis of invitro regenerated shoots of Lavandula angustifolia Mill. Braz Arch Biol Technol 57:636– 643. https ://doi.org/10.1590/S1516 -89132 01402 165 Mackay WA, Tipton JL, Thompson GA (1995) Micropropagation of Mexican redbud, Cercis canadensis var. mexicana. Plant Cell Tiss Org Cult 43:295–299. https ://doi.org/10.1007/BF000 39959 Marks TR, Simpson SE (1999) Effect of irradiance on shoot development invitro. Plant Growth Regul 28:133–142. https ://doi. org/10.1023/A:10062 76724 956 Martin KP, Zhang CL, Slater A, Madassery J (2007) Control of shoot necrosis and plant death during micro-propagation of banana and plantains (Musa spp.). Plant Cell Tiss Org Cult 88:51–59. https ://doi.org/10.1007/s1124 0-006-9177-0 Martinelli A (1988) Use of “in vitro” techniques for selection and cloning of different Pistacia species. Acta Hortic 227:436–437. https ://doi.org/10.17660 /ActaH ortic .1988.227.85 Martínez MT, Corredoira E, Vieitez AM, Cernadas MJ, Montenegro R, Ballester A, Vieitez FJ, San José MC (2017) Micropropagation of mature Quercus ilex L. trees by axillary budding. Plant Cell Tiss Org Cult 131:499–512. https ://doi.org/10.1007/s1124 0-017-1300-x Marín JA, García E, Lorente P, Andreu P, Arbeloa A (2016) A novel approach for propagation of recalcitrant pistachio cultivars that sidesteps rooting by ex vitro grafting of tissue cultured shoot tips. Plant Cell Tiss Org Cult 124:191–200. https ://doi.org/10.1007/ s1124 0-015-0871-7 Mason GF, Guttridge CG (1974) The role of calcium, boron and some divalent ions in leaf tipburn of strawberry. Sci Hortic 2:299–308. https ://doi.org/10.1016/0304-4238(74)90039 -9 Mason GF, Guttridge CG (1975) The influence of relative humidity and nutrition on leaf tipburn of strawberry. Sci Hortic 3:339–349. https ://doi.org/10.1016/0304-4238(75)90048 -5 Matu ENN, Lindsey KLL, van Staden J (2006) Micropropagation of Maytenus senegalensis (Lam.) Excell. S Afr J Bot 72:409–415. https ://doi.org/10.1016/j.sajb.2005.11.005 McCown BH, Sellmer JC (1987) General media and vessels suitable for woody plant culture. In: Bonga JM, Durzan D (eds) Cell and tissue culture in forestry. Springer, The Netherlands, pp. 4–16. https ://doi.org/10.1007/978-94-017-0994-1_2 Millington WF (1963) Shoot tip abortion in Ulmus americana. Am J Bot 50:371–378. https ://doi.org/10.1002/j.1537-2197.1963. tb072 05.x Mirabbasi SM, Hosseinpour B (2014) Prevention of shoot tip necrosis, hyperhydricity and callus production associated with invitro shoot culture of Ulmus glabra. J Novel Appl Sci 3:683–689 Misra P, Chakrabarty D (2009) Clonal propagation of Rosa clinophylla Thory. through axillary bud culture. Sci Hortic 119:212–216. https ://doi.org/10.1016/j.scien ta.2008.07.028 Misra P, Toppo DD, Gupta N, Chakrabarty D, Tuli R (2010) Effect of antioxidants and associate changes in antioxidant enzymes in controlling browning and necrosis of proliferating shoots of elite Jatropha curcas L. Biomass Bioenergy 34:1861–1869. https :// doi.org/10.1016/j.biomb ioe.2010.07.027 Moura M (1998) Conservation of Hypericum foliosum Alton, an endemic Azorean species, by micropropagation. Vitro Cell Dev Biol Plant 34:244–248. https ://doi.org/10.1007/BF028 22715 Mubina N, Hoque M, Sarker R (2018) In vitro regeneration and over expression of pea DNA helicase 45 (PDH45) gene into the local cultivars of chickpea (Cicer arietinum L.) through Agrobacterium-mediated genetic transformation. Plant Tissue Cult Biotechnol 28:125–140. https ://doi.org/10.3329/ptcb.v28i1 .37204 Mulwa RMS, Bhalla PL (2000) Invitro shoot multiplication of Macadamia tetraphylla L. Johnson J Hortic Sci Biotechnol 75:1–5. https ://doi.org/10.1080/14620 316.2000.11511 192 Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497. https ://doi.org/10.1111/j.1399-3054.1962.tb080 52.x Mythili JB, Thomas P (1999) Micropropagation of pointed gourd (Trichosanthes dioica Roxb.). Sci Hortic 79:87–90. https ://doi. org/10.1016/S0304 -4238(98)00201 -5 Naaz A, Shahzad A, Anis M (2014) Effect of adenine sulphate interaction on growth and development of shoot regeneration and inhibition of shoot tip necrosis under invitro condition in adult Syzygium cumini L.—a multipurpose tree. Appl Biochem Biotechnol 173:90–102. https ://doi.org/10.1007/s1201 0-014-0797-2 Nagahatenna DSK, Peiris SE (2007) Invitro propagation of Hemidesmus indicus (L.) R. Br. (Iramusu) through nodal culture. Trop Agr Res 19:181–192 Nezami SR, Yadollahi A, Hokmabadi H, Eftekhari M (2015) Control of shoot tip necrosis and plant death during invitro multiplication of Pistachio rootstock UCB1 (Pistacia integrima × P.atlantica). J Nuts 6:27–35. https ://doi.org/10.22034 /jon.2015.51564 6 Nezami-Alanagh E, Garoosi G-A, Haddad R, Maleki S, Landín M, Gallego PP (2014) Design of tissue culture media for efficient Prunusrootstock micropropagation using artificial intelligence models. Plant Cell Tiss Org Cult 117:349–359. https ://doi. org/10.1007/s1124 0-014-0444-1 Nezami-Alanagh E, Garoosi G-A, Landín M, Gallego PP (2018) Combining DOE with neurofuzzy logic for healthy mineral nutrition of Pistachio rootstocks invitro culture. Front Plant Sci 9:1474. https ://doi.org/10.3389/fpls.2018.01474 Nezami-Alanagh E, Garoosi G-A, Landín M, Gallego PP (2019) Computer-based tools provide new insight into the key factors that cause physiological disorders of Pistachio rootstocks cultured invitro. Sci Rep 9:9740. https ://doi.org/10.1038/s4159 8-01946155 -2 Nezami-Alanagh E, Garoosi G-A, Maleki S, Landín M, Gallego PP (2017) Predicting optimal invitro culture medium for Pistacia vera micropropagation using neural networks models. Plant Cell Tiss Org Cult 129:19–33. https ://doi.org/10.1007/s1124 0-016-1152-9 Planta (2020) 252:47 1 3 Page 33 of 35 47 Niedz RP, Evens TJ (2006) A solution to the problem of ion confounding in experimental biology. Nat Methods 3:417–427. https ://doi. org/10.1038/nmeth 0606-417 Niedz RP, Evens TJ (2007) Regulating plant tissue growth by mineral nutrition. Vitro Cell Dev Biol Plant 43:370–381. https ://doi. org/10.1007/s1162 7-007-9062-5 Niedz RP, Evens TJ (2016) Design of experiments (DOE)—history, concepts, and relevance to invitro culture. Vitro Cell Dev Biol Plant 52:547–562. https ://doi.org/10.1007/s1162 7-016-9786-1 Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87. https ://doi.org/10.1126/scien ce.163.3862.85 Norton ME, Norton CR (1985) Invitro propagation of Ericaceae: a comparison of the activity of the cytokinins N6-benzyladenine and N6-isopentenyladenine in shoot proliferation. Sci Hortic 27:335–340. https ://doi.org/10.1016/0304-4238(85)90038 -X Oberschelp GPJ, Gonçalves AN (2018) Analysis of nutrient deficiencies affecting invitro growth and development of Eucalyptus dunnii Maiden. Physiol Mol Biol Plants 24:693–702. https ://doi. org/10.1007/s1229 8-018-0560-1 Offord CA, Tyler JL (2009) Invitro propagation of Pimelea spicata R.Br (Thymelaeaceae), an endangered species of the Sydney region. Aust Plant Cell Tiss Org Cult 98:19–23. https ://doi. org/10.1007/s1124 0-009-9534-x Ozgen S, Busse JS, Palta JP (2011) Influence of root zone calcium on shoot tip necrosis and apical dominance of potato shoot: simulation of this disorder by ethylene glycol tetra acetic acid and prevention by strontium. Hort Sci 46:1358–1362. https ://doi. org/10.21273 /HORTS CI.46.10.1358 Papadatou P, Pontikis C, Ephtimiadou E, Lydaki M (1990) Rapid multiplication of guava seedlings by invitro shoot tip culture. Sci Hortic 45:99–103. https ://doi.org/10.1016/0304-4238(90)90072 -M Parfitt DE, Almehdi AA (1994) Use of a high CO2 atmosphere and medium modifications for the successful micropropagation of pistachio. Sci Hortic 56:321–329. https ://doi. org/10.1016/0304-4238(94)90050 -7 Parh DK, Conner AJ, Jacobs JME, McNeil DL (1998) Shoot-tip necrosis and alleviation during invitro culture of Lens culinaris. SABRAO J Breed Genet 30:97–101 Park SW, Jeon JH, Kim HS, Park YM, Aswath C, Joung H (2004) Effect of sealed and vented gaseous microenvironments on the hyperhydricity of potato shoots invitro. Sci Hortic 99:199– 205. https ://doi.org/10.1016/S0304 -4238(03)00097 -9 Park JS, Naing AH, Kim CK (2016) Effects of ethylene on shoot initiation, leaf yellowing, and shoot tip necrosis in roses. Plant Cell Tiss Org Cult 127:425–431. https ://doi.org/10.1007/s1124 0-016-1066-6 Pasqua G, Manes F, Monacelli B, Natale L, Anselmi S (2002) Effects of the culture medium pH and ion uptake in invitro vegetative organogenesis in thin cell layers of tobacco. Plant Sci 162:947–955. https ://doi.org/10.1016/S0168 -9452(02)00048 -1 Penel C, van Cutsem P, Greppin H (1999) Interactions of a plant peroxidase with oligogalacturonides in the presence of calcium ions. Phytochemistry 51:193–198. https ://doi.org/10.1016/ S0031 -9422(98)00741 -9 Phillips GC, Garda M (2019) Plant tissue culture media and practices: an overview. Vitro Cell Dev Biol Plant 55:242–257. https ://doi.org/10.1007/s1162 7-019-09983 -5 Piagnani C, Zocchi G, Mignani I (1996) Influence of Ca2+ and 6-benzyladenine on chestnut (Castanea sativa Mill.) invitro shoot-tip necrosis. Plant Sci 118:89–95. https ://doi. org/10.1016/0168-9452(96)04423 -8 Podwyszyńska M, Goszczyńska DM (1998) Effect of inhibitors of ethylene biosynthesis and action, as well as calcium and magnesium on rose shoot rooting, shoot-tip necrosis and leaf senescence invitro. Acta Physiol Plant 20:91–98. https ://doi. org/10.1007/s1173 8-998-0049-6 Poothong S, Reed BM (2014) Modeling the effects of mineral nutrition for improving growth and development of micropropagated red raspberries. Sci Hortic 165:132–141. https ://doi. org/10.1016/j.scien ta.2013.10.040 Preece J (1995) Can nutrient salts partially substitute for plant growth regulators? Plant Tiss Cult Biotech 1:26–37 Purnhauser L, Medgyesy P, Czakó M, Dix PJ, Márton L (1987) Stimulation of shoot regeneration in Triticum aestivum and Nicotiana plumbaginifolia Viv. tissue cultures using the ethylene inhibitor AgNO3. Plant Cell Rep 6:1–4. https ://doi. org/10.1007/BF002 69725 Pérez C, Rodríguez R, Tamés R (1985) “In vitro” filbert (Corylus avellana L.) micropropagation from shoots and cotyledonary node segments. Plant Cell Rep 4:137–139. https ://doi. org/10.1007/BF005 71300 Pérez-Tornero O, Burgos L (2000) Different media requirements for micropropagation of apricot cultivars. Plant Cell Tiss Org Cult 63:133–141. https ://doi.org/10.1023/A:10064 30718 024 Quoirin M, Lepoivre P (1977) Improved media for invitro culture of Prunus sp. Acta Hortic 78:437–442. https ://doi.org/10.17660 /ActaH ortic .1977.78.54 Ramage CM, Williams RR (2002) Mineral nutrition and plant morphogenesis. Vitro Cell Dev Biol Plant 38:116–124. https ://doi. org/10.1079/IVP20 01269 Reed BM, DeNoma J, Wada S, Niedz R (2016) Determining optimum invitro mineral nutrition for diverse pear germplasm using response surface methodology. Acta Hortic 1113:79–84. https ://doi.org/10.17660 /ActaH ortic .2016.1113.11 Reed BM, Wada S, DeNoma J, Niedz RP (2013) Mineral nutrition influences physiological responses of pear invitro. Vitro Cell Dev Biol Plant 49:699–709. https ://doi.org/10.1007/s1162 7-013-9556-2 Robertson DN (2013) Modulating plant calcium for better nutrition and stress tolerance. ISRN Bot 2013:952043. https ://doi. org/10.1155/2013/95204 3 Rugini E (1984) Invitro propagation of some olive (Olea europea sativa L.) cultivars with different rootability, and medium development using analytical data from developing shoots and embryos. Sci Hortic 24:12–134. https ://doi.org/10.1016/03044238(84)90143 -2 Rugini E, Tarini P, Mari F (1986) Invitro control of shoot vitrification in almond (P. dulcis) and development of technique to eliminate apex necrosis and shoot base photo-oxidation in pistachio (Pistacia vera). HortScience 21:804 Ruiz JM, Rivero RM, López-Cantarero I, Romero L (2003) Role of Ca2+ in the metabolism of phenolic compounds in tobacco leaves (Nicotiana tabacum L.). Plant Growth Regul 41:173–177. https ://doi.org/10.1023/A:10273 58423 187 Ružić D, Vujović T, Libiakova G, Cerović R, Gajdošova A (2012) Micropropagation invitro of highbush blueberry (Vaccinium corymbosum L.). J Berry Res 2:97–103. https ://doi.org/10.3233/ JBR-2012-030 Sanchez MC, San-Jose MC, Ferro E, Ballester A, Vieitez AM (1997) Improving micropropagation conditions for adult-phase shoots of chestnut. J Hortic Sci 72:433–443. https ://doi.or g/10.1080/14620 316.1997.11515 531 Santner A, Calderon-Villalobos LIA, Estelle M (2009) Plant hormones are versatile chemical regulators of plant growth. Nat Chem Biol 5:301–307. https ://doi.org/10.1038/nchem bio.165 Saure MC (1998) Causes of the tipburn disorder in leaves of vegetables. Sci Hortic 76:131–147. https ://doi.org/10.1016/S0304 -4238(98)00153 -8 Planta (2020) 252:47 1 3 47 Page 34 of 35 Serres R, Read P, Hackett W, Nissen P (1990) Rooting of American chestnut microcuttings. J Environ Hortic 8:86–88. https ://doi. org/10.24266 /0738-2898-8.2.86 Sha L, McCown BH, Peterson LA (1985) Occurrence and cause of shoot-tip necrosis in shoot cultures. J Am Soc Hortic Sci 110:631–634 Singha S, Townsend EC, Oberly GH (1990) Relationship between calcium and agar on vitrification and shoot-tip necrosis of quince (Cydonia oblonga Mill.) shoots invitro. Plant Cell Tiss Org Cult 23:135–142. https ://doi.org/10.1007/BF000 35834 Srivastava A, Joshi AG (2013) Control of shoot tip necrosis in shoot cultures of Portulaca grandiflora Hook. Not Sci Biol 5:45–49. https ://doi.org/10.15835 /nsb51 9009 Strnad M, Hanuš J, Vaňek T, Kamínek M, Ballantine JA, Fussell B, Hanke DE (1997) Meta-topolin, a highly active aromatic cytokinin from poplar leaves (Populus x canadensis Moench., cv. Robusta). Phytochemistry 45:213–218. https ://doi.org/10.1016/ S0031 -9422(96)00816 -3 Subbaiah CC, Kollipara KP, Sachs MM (2000) A Ca2+-dependent cysteine protease is associated with anoxia-induced root tip death in maize. J Exp Bot 51:721–730. https ://doi.org/10.1093/jexbo t/51.345.721 Sudha CG, Krishnan PN, Pushpangadan P (1998) Invitro propagation of Holostemma annulare (Robx.) K. Schum., a rare medicinal plant. Vitro Cell Dev Biol Plant 34:57–63. https ://doi. org/10.1007/BF028 23124 Surakshitha NC, Soorianathasundaram K, Ganga M, Raveendran M (2019) Alleviating shoot tip necrosis during invitro propagation of grape cv. Red Globe Sci Hortic 248:118–125. https ://doi. org/10.1016/j.scien ta.2019.01.013 Teixeira da Silva JA (2012) Is BA (6-benzyladenine) BAP (6-benzylaminopurine)? Asian Austral J Plant Sci Biotech 6:121–124 Teixeira da Silva JA (2013) The effect of ethylene inhibitors (AgNO3, AVG), an ethylene-liberating compound (CEPA) and aeration on the formation of protocorm-like bodies of hybrid Cymbidium (Orchidaceae). Front Biol 8:606–610. https ://doi.org/10.1007/ s1151 5-013-1283-x Teixeira da Silva JA (2015) Negative results: negative perceptions limit their potential for increasing reproducibility. J Negative Results BioMed 14:12. https ://doi.org/10.1186/s1295 2-015-0033-9 Teixeira da Silva JA, Dobránszki J (2013) How timing of sampling can affect the outcome of the quantitative assessment of plant organogenesis. Sci Hortic 159:59–66. https ://doi.org/10.1016/j. scien ta.2013.05.00 Teixeira da Silva JA, Giang DTT, Tanaka M (2006) Photoautotrophic micropropagation of Spathiphyllum. Photosynthetica 44:53–61. https ://doi.org/10.1007/s1109 9-005-0158-z Thakur A, Kanwar JS (2011) Effect of phase of medium, growth regulators and nutrient supplementations on invitro shoot-tip necrosis in pear. NZ J Crop Hort Sci 39:131–140. https ://doi. org/10.1080/01140 671.2011.55925 4 Thirugnanasampandan R, Mutharaian VN, Bai VN (2009) Invitro propagation and free radical studies of Smilax zeylanica Vent. Afr J Biotechnol 8:395–400 Thomas P (2000) Microcutting leaf area, weight and position on the stock shoot influence root vigour, shoot growth and incidence of shoot tip necrosis in grape plantlets invitro. Plant Cell Tiss Org Cult 61:189–198. https ://doi.org/10.10 23/A:10064 25807 85 3 Thor K (2019) Calcium-nutrient and messenger. Front Plant Sci 10:440. https ://doi.org/10.3389/fpls.2019.00440 Tilkat E, Onay A, Yildirim H, Çetin Ozen H (2008) Micropropagation of mature male pistachio Pistacia vera L. J Hortic Sci Biotechnol 83:328–333. https ://doi.org/10.1080/14620 316.2008.11512 387 Trobacher CP (2009) Ethylene and programmed cell death in plants. Botany 87:757–769. https ://doi.org/10.1139/B09-041 Turland NJ, Wiersema JH, Barrie FR, Greuter W, Hawksworth DL, Herendeen PS, Knapp S, Kusber W-H, Li D-Z, Marhold K, May TW, McNeill J, Monro AM, Prado J, Price MJ, Smith (eds) (2018) International code of nomenclature for algae, fungi, and plants (Shenzhen Code) adopted by the Nineteenth International Botanical Congress Shenzhen, China, July 2017. Regnum Vegetabile 159. Koeltz Botanical Books, Glashütten. https ://doi. org/10.12705 /Code.2018 van den Dries N, Giannì S, Czerednik A, Krens FA, de Klerk G-JM (2013) Flooding of the apoplast is a key factor in the development of hyperhydricity. J Exp Bot 64:5221–5230. https ://doi. org/10.1093/jxb/ert31 5 Vanneste S, Friml J (2013) Calcium: the missing link to auxin action. Plants 2:650–675. https ://doi.org/10.3390/plant s2040 650 Vibha JB, Shekhawat NS, Mehandru P, Dinesh R (2014) Rapid multiplication of Dalbergia sissoo Roxb.: a timber yielding tree legume through axillary shoot proliferation and ex vitro rooting. Physiol Mol Biol Plant 20:81–87. https ://doi.org/10.1007/s1229 8-013-0213-3 Vieitez AM, Corredoira E, Ballester A, Muñoz F, Durán J, Ibarra M (2009) Invitro regeneration of the important North American oak species Quercus alba, Quercus bicolor and Quercus rubra. Plant Cell Tiss Org Cult 98:135–145. https ://doi.org/10.1007/ s1124 0-009-9546-6 Vieitez AM, Sánchez C, San-José C (1989) Prevention of shoot-tip necrosis in shoot cultures of chestnut and oak. Sci Hortic 41:151– 159. https ://doi.org/10.1016/0304-4238(89)90059 -9 Wada S, Niedz RP, DeNoma J (2013) Mesos components (CaCl2, MgSO4, KH2PO4) are critical for improving pear micropropagation. Vitro Cell Dev Biol Plant 49:356–365. https ://doi. org/10.1007/s1162 7-013-9508-x Wada S, Niedz RP, Reed BM (2015) Determining nitrate and ammonium requirements for optimal invitro response of diverse pear species. Vitro Cell Dev Biol Plant 51:19–27. https ://doi. org/10.1007/s1162 7-015-9662-4 Wang H, van Staden J (2001) Establishment of invitro cultures of tree peonies. S Afr J Bot 67:358–361. https ://doi.org/10.1016/S0254 -6299(15)31141 -8 Werbrouck SPO, Strnad M, Van Onckelen HA, Debergh PC (1996) Meta-topolin, an alternative to benzyladenine in tissue culture? Physiol Plant 98:291–297. https ://doi.org/10.103 4/j.1399-3054.1996.98021 0.x Wetzstein HY, Ault JR, Merkle SA (1989) Further characterization of somatic embryogenesis and plantlet regeneration in pecan (Carya illinoensis). Plant Sci 64:193–201. https ://doi.org/10.1016/01689452(89)90024 -1 White PJ, Broadly MR (2003) Calcium in plants. Ann Bot 92:487–511. https ://doi.org/10.1093/aob/mcg16 4 Xing Z, Satchwell MF, Powell WA, Maynard CA (1997) Micropropagation of American chestnut: increasing rooting rate and preventing shoot-tip necrosis. Vitro Cell Dev Biol Plant 33:43–48. https ://doi.org/10.1007/s1162 7-997-0039-1 Ye G, McNeil DL, Conner AJ, Hill GD (2002) Multiple shoot formation in lentil (Lens culinaris) seeds. N N Z J Crop Hort Sci 30:1–8. https ://doi.org/10.1080/01140 671.2002.95141 93 Zielińska A, Kępczyńska E (2013) Neural modeling of plant tissue cultures: a review. BioTechnologia 94:253–268. https ://doi. org/10.5114/bta.2013.46419 Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Planta (2020) 252:47 1 3 Page 35 of 35 47 Affiliations JaimeA.TeixeiradaSilva1,2· EsmaeilNezami‑Alanagh3,4· MaríaE.Barreal3 · MafatlalM.Kher5· AdhityoWicaksono6 · AndreaGulyás2· NorbertHidvégi2· KatalinMagyar‑Tábori2· NóraMendler‑Drienyovszki2· LászlóMárton2· MarianaLandín7 · PedroPabloGallego3 · JohnA.Driver8· JuditDobránszki2 1 Present Address: Miki-cho Post Office, 3011-2, P. O. Box7, Ikenobe, Kagawa-ken761-0799, Japan 2 Research Institute ofNyíregyháza, IAREF, University ofDebrecen, P. O. Box12, Nyíregyháza4400, Hungary 3 Department ofPlant Biology andSoil Science, Faculty ofBiology, University ofVigo, 36310Vigo, Spain 4 Pinar Biotech. Co., Ltd., East Azarbaijan Science andTechnology Park, Tabriz, Iran 5 School ofScience (SOS), GSFC University, P. O. Fertilizernagar, Vadodara391750, Gujarat, India 6 Division ofBiotechnology, Generasi Biologi Indonesia (Genbinesia) Foundation, Jl. Swadaya Barat No. 4, GresikRegency61171, Indonesia 7 Department ofPharmacology, Pharmacy andPharmaceutical Technology, Faculty ofPharmacy, University ofSantiago, SantiagodeCompostela, Spain 8 Driver Consulting Inc., 2601 Tim Bell Road, Waterford, CA95386, USA