Abstract
The neem tree, Azadirachta indica A. Juss., is an important multipurpose tree globally used in agriculture with recalcitrant and chilling-sensitive seeds. Here, we studied the encapsulation in sodium alginate of in vitro-derived nodal segments of neem as a method to preserve in vitro culture material of this species for laboratory exchange and for the medium-term storage. A good gel complexation was achieved using 3% sodium alginate and 70 mM CaCl2·2H2O. An average bead conversion of 75%, 0.5 cm of shoot length and 2 leaves were recorded after 4 weeks of culture, irrespective of the polymerisation duration (5 to 20 min). The growth of nodal segments was completely inhibited when they were kept at 4 °C or at 8 °C for 4 weeks, while those encapsulated and stored at 12 °C for 4 weeks, sprung up only 20% under optimal conditions, 23 °C. However, bud sprouting of encapsulated nodal segments stored at 12 °C for 4 weeks increased to 75% under optimal conditions by incubating the nodal segments with 25 µM acetylsalicylic acid (ASA) for 4 weeks prior to encapsulation. We observed that this ASA pre-treatment stimulated the antioxidative defenses of the explant, especially ascorbate peroxidase, catalase, peroxidase, dehydroascorbate reductase and glutathione reductase enzymes that could have improved the viability of the buds stored at 12 °C. Additionally, ASA seemed to protect the cell membranes, as observed by the lipid peroxidation results. All these data together led us to suggest the use of ASA in short- and medium-term storage of in vitro-derived nodal segments of neem.
Key message
Neem nodal segments, encapsulated in alginate beads and stored at 12 °C recovered their viability when were pre-treated with acetylsalicylic acid, which improved buds’ antioxidant defences.
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References
Ahmad N, Anis M (2010) Direct plant regeneration from encapsulated nodal segments of Vitex negundo. Biol Plant 54(4):748–752. https://doi.org/10.1007/s10535-010-0134-8
Allan EJ, Stuchbury T, Mordue AJ (1999) Azadirachta indica A juss. (Neem tree): in vitro culture, micropropagation, and the production of Azadirachtin and other secondary metabolites. In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, 43. Medicinal and aromatic plants XI. Springer-Verlag, Berlin, pp 11–41
Arora K, Sharma M, Srivastava J, Ranade SA, Sharma AK (2010) Rapid in vitro cloning of a 40-year-old tree of Azadirachta indica A. Juss (Neem) employing nodal stem segments. Agrofor Syst 78(1):53–63. https://doi.org/10.1007/s10457-009-9230-1
Asada K (1999) The water-water cycle in chloroplasts: Scavenging of active oxygens and dissipation of excess photons. Ann Rev Plant Biol 50:601–639. https://doi.org/10.1146/annurev.arpplant.50.1.601
Barba-Espín G, Clemente-Moreno MJ, Álvarez S, García-Legaz MF, Hernández JA, Díaz-Vivancos P (2011) Salicylic acid negatively affects the response to salt stress in pea plants: effects on PR1b and MAPK expression. Plant Biol 13:909–917. https://doi.org/10.1111/j.1438-8677.2011.00461.x
Bhushan A, Gupta RK (2017) In vitro shoot regeneration and heterosis studies in tomato (Solanum lycopersicon Mill.). Int J Plant Res 30:359–364. https://doi.org/10.5958/2229-4473.2017.00097.0
Block W (2003) Water status and thermal analysis of alginate beads used in cryopreservation of plant germplasm. Cryobiology 47:59–72. https://doi.org/10.1016/S0011-2240(03)00069-5
Chand S, Singh AK (2004) Plant regeneration from encapsulated nodal segments of Dalbergia sissoo Roxb., a timber-yielding leguminous tree species. J Plant Physiol 161(2):237–243. https://doi.org/10.1078/0176-1617-01036
Chaturvedi R, Razdan MK, Bhojwani SS (2004) In vitro morphogenesis in zygotic embryo cultures of neem (Azadirachta indica A. Juss.). Plant Cell Rep 22(11):801–809. https://doi.org/10.1007/s00299-004-0768-0
Clemente-Moreno MJ, Díaz-Vivancos P, Barba-Espín G, Hernández JA (2010) Benzothiadiazole and L-2-oxothiazolidine- 4-carboxylic acid reduced the severity of Sharka symptoms in pea leaves: effect on the antioxidative metabolism at subcellular level. Plant Biol 12:88–97. https://doi.org/10.1111/j.1438-8677.2009.00204.x
Danso KE, Ford-Lloyd BV (2003) Encapsulation of nodal cuttings and shoot tips for storage and exchange of cassava germplasm. Plant Cell Rep 21(8):718–725. https://doi.org/10.1007/s00299-003-0594-9
Driver JA, Kuniyuki AH (1984) In vitro propagation of Paradox walnut rootstock. HortScience 19:507–509. https://doi.org/10.7235/Hort.2012.12009
Fujita M, Fujita Y, Noutoshi Y, Takahashi F, Narusaka Y, Yamaguchi-Shinozaki K, Shinozaki K (2006) Crosstalk between abiotic and biotic stress responses: a current view from the points of convergence in the stress signaling networks. Curr Opin Plant Biol 9(4):436–442. https://doi.org/10.1016/J.Pbi.2006.05.014
Gairi A, Rashid A (2005) Direct differentiation of somatic embryos on cotyledons of Azadirachta indica. Biol Plant 49(2):169–173. https://doi.org/10.1007/s10535-005-0173-8
Gangopadhyay G, Bandyopadhyay T, Poddar R, Gangopadhyay S, Mukherjee KK (2005) Encapsulation of pineapple micro shoots in alginate beads for temporary storage. Curr Sci 88(6):972–977
Grossman K (1990) Plantgrowth retardants as tools in physiological research. Physiol Plant 78(4):640–648. https://doi.org/10.1111/j.1399-3054.1990.tb05254.x
Gupta SD, Kumar A, Agarwal A (2019) Impact of light-emitting diodes (LEDs) on the growth and morphogenesis of encapsulated shoot buds of Curculigo orchioides Gaertn., an endangered medicinal herb. Acta Physiol Plant 41(4):50. https://doi.org/10.1007/s11738-019-2840-y
Habing WH, Jakoby WB (1981) Assays for differentiation of glutathione S-transferases. Methods Enzymol 77:398–405. https://doi.org/10.1016/S0076-6879(81)77053-8
Hung CD, Trueman SJ (2012) Alginate encapsulation of shoot tips and nodal segments for short-term storage and distribution of the eucalypt Corymbia torelliana x C. citriodora. ACTA Physiol Plant 34(1):117–128. https://doi.org/10.1007/s11738-011-0810-0
Jarret RL (1997) Effects of chemical growth retardants on growth and development of sweetpotato (Ipomoea Batatas (L.) Lam) in vitro. J Plant Growth Regul 16(4):227–231. https://doi.org/10.1007/Pl00007001
Joshi MS, Thengane SR (1996) In vitro propagation of Azadirachta indica A. Juss (neem) by shoot proliferation. Ind J Exp Biol 5:480–482
Karpinski S, Wingsle G, Karpinska B, Hallgren JE (2002) Low-temperature stress and antioxidant defense mechanism in higher plants. In: Inze D, van Montagu M (eds) Oxidative stress in plants. Taylor and Francis Publishers, London, pp 69–103
Katouzi SSS, Majd A, Fallahian F, Bernard F (2011) Encapsulation of shoot tips in alginate beads containing salicylic acid for cold preservation and plant regeneration in sunflower (Helianthus annuus L.). Aust J Crop Sci 5(11):1469–1474
Khan MI, Ahmad N, Anis M, Alatar AA, Faisal M (2018) In vitro conservation strategies for the Indian willow (Salix tetrasperma Roxb.), a vulnerable tree species via propagation through synthetic seeds. Biocatal Agric Biotechnol 16:17–21. https://doi.org/10.1016/j.bcab.2018.07.002
Korsukova AV, Borovik OA, Grabelnych OI, Voinikov VK (2015) The tebuconazole-based protectant of seeds “bunker” induces the synthesis of dehydrins during cold hardening and increases the frost resistance of wheat seedlings. J Stress Physiol Biochem 11(4):118–127
Kulus D, Zalewska M (2014) In vitro plant recovery from alginate encapsulated Chrysanthemum × grandiflorum /Ramat./ Kitam shoot tips. Propag Ornam Plants 14(1):3–12
Kulus D (2018) Genetic resources and selected conservation methods of tomato. J Appl Bot Food Qual 91:135–144. https://doi.org/10.5073/JABFQ.2018.091.00X
Kulus D (2019) Application of synthetic seeds in propagation, storage and cryopreservation of Asteraceae plant species. In: Faisal A, Alatar A (eds) Synthetic seeds germplasm regeneration, preservation and prospects. Springer Science & Business Media, Dordrecht, pp 155–179
Kumar D, Mishra DS, Chakraborty B, Kumar P (2013) Pericarp browning and quality management of litchi fruit by antioxidants and salicylic acid during ambient storage. J Food Sci Technol 50(4):797–802. https://doi.org/10.1007/s13197-011-0384-2
Kumar D, Mishra DK (2014) Variability in permeability and integrity of cell membrane and depletion of food reserves in neem (Azadirachta indica) seeds from trees of different age classes. J For Res 25(1):147–153. https://doi.org/10.1007/s11676-014-0440-7
Littell RC, Stroup WW, Freund RJ (2002) SAS for linear models, 4th edn. SAS Institute Inc, Cary, p 466
López-Delgado HA, Scott IM (1997) Induction of in vitro tuberization of potato microplants by acetylsalicylic acid. J Plant Physiol 151(1):74–78. https://doi.org/10.1016/S0176-1617(97)80039-9
López-Delgado HA, Jimenez-Casas M, Scott IM (1998) Storage of potato microplants in vitro in the presence of acetylsalicylic acid. Plant Cell Tiss Org Cult 54(3):145–152. https://doi.org/10.1023/A:1006110118669
Lukatkin AS, Brazaityte A, Bobinas C, Duchovskis P (2012) Chilling injury in chilling-sensitive plants: a review. Zemdirbyste 99(2):111–124
Lyons JM (1973) Chilling injury in plants. Annu Rev Plant Physiol 24(1):445–466. https://doi.org/10.1146/annurev.pp.24.060173.002305
Marino G, Negri P, Cellini A, Masia A (2010) Effect of carbohydrates on in vitro low temperature storage of shoot cultures of apricot. Sci Hortic 126:434–440. https://doi.org/10.1016/j.scienta.2010.08.008
Moradi S, Baninasab B, Gholami M, Ghobadi C (2017) Paclobutrazol application enhances antioxidant enzyme activities in pomegranate plants affected by cold stress. J Hortic Sci Biotechnol 92(1):65–71. https://doi.org/10.1080/14620316.2016.1224605
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant 15:473–497. https://doi.org/10.1111/j.1399-3054.1962.tb08052.x
Muslihatin W, Jadid N, Safitri CE, Kuncoro EP (2018) In vitro germination of Moringa oleifera synthetic seed on different composition of medium. Biosci Res 15(3):1982–1991
Mutlu S, Karadagoglu O, Atici O, Nalbantoglu B (2013) Protective role of salicylic acid applied before cold stress on antioxidative system and protein patterns in barley apoplast. Biol Plant 57(3):507–513. https://doi.org/10.1007/s10535-013-0322-4
Naik SK, Chand PK (2006) Nutrient-alginate encapsulation of in vitro nodal segments of pomegranate (Punica granatum L.) for germplasm distribution and exchange. Sci Horti 108:247–252. https://doi.org/10.1016/j.scienta.2006.01.030
Nair DS, Reghunath BR, Soni KB, Alex S (2019) Cryopreservation of encapsulated axillary buds of Clitoria ternatea (L). Cryoletters 40(1):28–35
Noctor G, Foyer CH (1998) Ascorbate and glutathione: keeping active oxygen under control. Ann Rev Plant Physiol Plant Mol Biol 49:249–279. https://doi.org/10.1146/annurev.arplant.49.1.249
Ojaghian MR, Almoneafy AA, Zq C, Xie G-L, Zhang J (2013) Application of acetyl salicylic acid and chemically different chitosans against storage carrot rot. Post Biol Tech 84:51–60. https://doi.org/10.1016/j.postharvbio.2013.04.006
Padilla IMG, Fernández-García N, Olmos E, Burgos L, Piqueras A (2015) Effects of growth retardants on sprouting and development of apricot (Prunus armeniaca L.) and neem (Azarchta indica A. Juss.) nodal buds. Plant Cell Tiss Org Cult 122(2):285–297. https://doi.org/10.1007/s11240-015-0765-8
Quraishi A, Koche V, Sharma P, Mishra SK (2004) In vitro clonal propagation of neem (Azadirachta indica). Plant Cell Tiss Org Cult 78(3):281–284. https://doi.org/10.1023/B:TICU.0000025647.58548.3d
Rai MK, Jaiswal VS, Jaiswal U (2008) Alginate-encapsulation of nodal segments of guava (Psidium guajava L.) for germplasm exchange and distribution. J Hort Sci Biotechnol 83(5):569–573. https://doi.org/10.1080/14620316.2008.11512425
Rout GR (2005) In vitro somatic embryogenesis in callus cultures of Azadirachta indica A. Juss.: a multipurpose tree. J For Res 10(4):263–267. https://doi.org/10.1007/s10310-004-0130-y
Rihan HZ, Kareem F, El-Mahrouk ME, Fuller MP (2017) Artificial seeds (principle, aspects and applications). Agron-Basel 7(4):71. https://doi.org/10.3390/agronomy7040071
Sacandé M, Buitink J, Hoekstra FA (2000) A study of water relations in neem (Azadirachta indica) seed that is characterized by complex storage behaviour. J Exp Bot 51(344):635–643. https://doi.org/10.1093/jexbot/51.344.635
Sedghi M, Basiri HK, Sharifi RS (2013) Effects of salicylic acid on the antioxidant enzymes activity in sunflower. Ann West Univ Timişoara 16(2):67–72
Shahin-Uz-Zaman M, Ashrafuzzaman M, Haque MS, Luna LN (2009) In vitro clonal propagation of the neem tree (Azadirachta indica A. Juss.). Afr J Biotechnol 7(4):386–391
Sharma S, Shahzad A, Mahmood S, Saeed T (2015) High-frequency clonal propagation, encapsulation of nodal segments for short-term storage and germplasm exchange of Ficus carica L. Trees 29:345–353. https://doi.org/10.1007/s00468-014-1114-y
Singh AK, Sharma M, Varshney R, Agarwal SS, Bansal KC (2006) Plant regeneration from alginate-encapsulated shoot tips of Phyllanthus amarus Schum and Thonn, a medicinally important plant species. In Vitro Cell Dev Biol Plant 42:109–113. https://doi.org/10.1079/IVP2005735
Soliman MH, Alayafi AAM, El Kelish AA, Abu-Elsaoud A (2018) Acetylsalicylic acid enhance tolerance of Phaseolus vulgaris L. to chilling stress, improving photosynthesis, antioxidants and expression of cold stress responsive genes. Bot Stud 59:6. https://doi.org/10.1186/s40529-018-0222-1
Srivastava P, Singh M, Mathur P, Chaturvedi R (2009) In vitro organogenesis and plant regeneration from unpollinated ovary cultures of Azadirachta indica. Biol Plant 53(2):360–364. https://doi.org/10.1007/s10535-009-0067-2
Varshey A, Anis M (2014) Synseed conception for short-term storage, germplasm exchange and potentialities of regeneration genetically stable plantlets of desert date tree (Balanites aegyptiaca Del.). Agrofor Syst 88(2):321–329. https://doi.org/10.1007/s10457-014-9685-6
You J, Chan Z (2015) ROS regulation during abiotic stress responses in crop plants. Front Plant Sci 6:1092. https://doi.org/10.3389/fpls.2015.01092
Zhang WP, Jiang B, Lou NL, Lu MH, Yang M, Chen JF (2011) Impact of salicylic acid on the antioxidant enzyme system hydrogen peroxide production in Cucumis sativus under chilling stress. Z Naturforsch C J Biosci 66(7–8):413–422
Zhang Y, Jiang J, Yang YL (2013) Acetyl salicylic acid induces stress tolerance in tomato plants grown at a low night-time temperature. J Hort Sci Biotech 88(4):490–496. https://doi.org/10.1080/14620316.2013.11512996
Zhao JJ, Feng NF, Wang XX, Cai GR, Cao MY, Zheng DF, Zhu HD (2019) Uniconazole confers chilling stress tolerance in soybean (Glycine max L.) by modulating photosynthesis, photoinhibition, and activating oxygen metabolism system. Photosynthetica 57(2):446–457. https://doi.org/10.32615/ps.2019.059
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IMGP thanks The Spanish National Research Council-European Social Fund I3P program for a postdoctoral contract.
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This work was funded by the Spanish Ministry of Education and Science with European Social Funds.
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Communicated by Maurizio Lambardi.
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Padilla, I.M.G., Barba-Espín, G., Hernández, J.A. et al. Acetylsalicylic acid improved antioxidative status and cold storage of encapsulated nodal segments of neem (Azadirachta indica A. Juss.). Plant Cell Tiss Organ Cult 144, 261–270 (2021). https://doi.org/10.1007/s11240-020-01828-z
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DOI: https://doi.org/10.1007/s11240-020-01828-z