Optimization of the Sterilization and Establishment Steps for Almonds 2-22 Genotype

Document Type : Research Article


1 Department of Horticultural Sciences, Faculty of Agriculture, Tarbiat Modares University, Tehran, Iran

2 Horticultural Department of Seed and Plant Improvement Institute (SPII), Karaj, Iran



The almond is one of the most important nut crops in many countries, including Iran. On the other hand, due to the difficult rooting in conventional propagation methods such as cuttings, its propagation has faced challenges. Therefore, this study was aimed at optimizing in vitro culture conditions for the promising 2-22 genotype as a high-yield and late-blooming cultivar. To this end, after collecting apical and lateral buds of 2-22 genotype, surface sterilization and establishment treatments were applied to explants. The second-order equation is selected for the central compound design (CCD) with two variables (HgCl2 and NaClO) to obtain a good fit in the sterilization treatment.  The results showed that 1.8 % NaClO for 12 min + 0.1 % HgCl2 for 3 min and 1.5 % NaClO for 8 min with 81.25%, and 100% of healthy seedlings were the best treatment in contamination control and explant Viability, respectively. The experimental establishment was conducted as a factorial experiment using a completely randomized design (CRD) with four replications. The establishment treatments indicated that the WPM medium was more effective than the MS medium and Knop medium. The hormonal composition of 1 mgL-1 BA + 0.05 mgL-1 IBA had the best results in the percentage of the establishment (72.25%), number of foliage (7.24), and shoot length (13.77 mm).


Abbasi F, Khadivi A, Taghizadeh M and Valizadehkaji B (2018) Micropropagation of Prunus scoparia, a suitable rootstock for Almond under drought conditions. International Journal of Fruit Science. 19 (2), 221–230
Aghaei N, Yadollahi A (2012) Micropropagation of GF 677 rootstock. Agricultural Science. 4(5), 131-138
Ahmad T, Abbasi NA, Hafiz IA, Ali A (2007) Comparison of sucrose and sorbitol as main carbon energy sources in micropropagation of peach root stock GF677. Pakistanian Journal of Bottany. 39(4), 1269-1275
Ansar A, Touqeer A, Nadeem AA, Eshfaq AH (2009) Effect of different media and growth regulators on in vitro shoot proliferation of olive cultivar 'Moraiolo'. Pakistanian Journal of Botany. 41,783-795
Ansari A, Gharaghani A (2019) A comparative study of genetic diversity, heritability and inter-relationships of tree and nut attributes between Prunus scoparia and P. elaeagnifolia using multivariate statistical analysis. International Journal of Horticultural Science and Technology. 6,137-150.
Antonopoulou C, Dimassi K, Therios I, Chatzissavvidis C, Papadakis I (2007) The effect of Fe-EDDHA and of ascorbic acid on in vitro rooting of the peach rootstock GF677 explants. Acta Physiologia Plantarum. 29, 559-561
Arab MM, Yadollahi A, Eftekhari M, Ahmadi H, Akbari M, Khorami SS (2018) Modeling and optimizing a new culture medium for in vitro rooting of G× N15 Prunus rootstock using artificial neural network-
genetic algorithm. Scientific Reports 8:e9977. doi: 10.1038/s41598-018-27858-4
Arab MM, Yadollahi A, Shojaeiyan A, Shokri S, Ghojah SM (2014) Effects of nutrient media, different cytokinin types and their concentrations on in vitro multiplication of G × N15 (hybrid of almond ×
peach) vegetative rootstock. Genetic Engineering & Biotechnology. 12(2), 81-87
Babaoglu M, Yorgancilar M, Akbudak MA (2001) Doku Kulturu: Basic Laboratory Techniques. Selcuk University Foundation Publications, Konya. Pp, 1-35.
Battistini A, Paoli G (2002) Large-scale micropropagation of several peach rootstocks. Acta Horticulturae. 592, 29-33.
Da Silva JAT, Kulus D (2014) Chrysanthemum biotechnology: discoveries from the recent literature. Folia Horticulture. 26, 67-77
Da Silva JAT, Kulus D, Zhang X, Zeng S Ma, G Piqueras A (2016) Disinfection of explants for saffron (Crocus sativus) tissue culture. Environmental and Experimental Biology 14, 183–198doi: 10.22364/eeb.14.25
Dixon RA, Gonzales RA (1996) Plant cell culture. A practice approach. Oxford University Press. Pp, 278. 127-138.
Dobranszki J, Teixeira JA (2010) Micropropagation of apple. A review Biotechnology Advances. 28, 462-488
Erfani M, Miri M, Imani A (2017) In vitro shoot proliferation and rooting of Garnem rootstock as influenced by basal media, plant growth regulators, and carbon sources. Plant Cell Biotechnology and Molecular Biology. 18, 101-109
Eshghi Khas M, Abbasifar A, Valizadeh Kaji B (2020) Optimization of in vitro propagation of purple passion fruit (Passiflora edulis), an important medicinal and ornamental plant. International Journal of Horticultural Science and Technology. 7, 305-314
Farsi M, Fatahi Moghadam MR, Zamani Z, Hassani D, Ahmadi A (2016) The histology of minigrafting of Persian walnut trees cv. chandler. International Journal of Horticultural Science and Technology. 3(2), 167-177
 Fotopoulos S, Sotiropoulos TE (2005) In vitro rooting of PR 204/84 rootstock (Prunus persica×P. amygdalus) as influenced by mineral concentration of the culture medium and exposure to darkness for a period. Agronomy Research. 3, 3-8
George EF (1996) Plant propagation by tissue culture: in Practice. Exegetics Ltd. London. 447– 470
Grouh MSH, Vahdati K, Lotfi M, Hassani D, Pirvali Biranvand N (2011) Production of haploids in Persian walnut through parthenogenesis induced by gamma-irradiated pollen. Journal of the American Society of Horticultural Sciences. 136(3): 198–204.
Hasan SZ, Ahmad T, Hafiz IA, Hussain A (2010) Direct plant regeneration from leaves of Prunus rootstock GF-677 (Prunus amygdalus × P. persica). Pakistan Journal of Botany. 42(6), 3817-3830
Hassankhah A, Vahdati K, Lotfi M, Mirmasoumi M, Preece J, Assareh MH (2014) Effects of ventilation and sucrose concentrations on the growth and plantlet anatomy of micropropagated Persian walnut plants. International Journal of Horticultural Science and Technology. 1(2), 111-20
Hesami M, Naderi R, Yoosefzadeh-Najafabadi M (2018) Optimizing sterilization conditions and growth regulator effects on in vitro shoot regeneration through direct organogenesis in Chenopodium quinoa. BioTechnologia. 99, 49–57.
http:// FAO. (2018- 2019): Food and Agricultural Organization.
http://INC (2018-2019). The International Nut and Dried Fruit Council Foundation.
knop W (1895) Landwirtsh. Ver.s. sfn.70-140
Kose Canli FA (2015) In vitro propagation and rooting of ‘Garnem’ (P. persica × P. dulcis). 5(1), 25-30
Lloyd G, McCown, B (1980) Commercially feasible micropropagation of mountain laurel, Kalmia latifolia by use shoot-tip culture. Int. Plant Propagators' Soc. Proceed.30,421-427
Matt A, Jehle JA (2005) In vitro plant regeneration from leaves and internodes sections of sweet cherry cultivars (Prunus avium L.). Plant Cell Reports. 24,468-476
Mihaljevic I, Dugalic K, Tomas V, Viljevac M, Pranjic A, Cmelik Z, Puskar B, Jurkovic Z (2013) In vitro sterilization procedures for micropropagation of ‘OBLAČINSKA’ Sour Cherry. Agricultural Sciences. 58, 117-126
Modgil M, Gupta R, Thakur M (2010) In vitro rooting and hardening in apple rootstock EMLA111- influence of some factors. Acta Horticulture. 865, 339-344
Murashige T, Skoog FA (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum. 15,473–497
Pourkhaloee A, Khoshkhui M (2015) Pupae are excellent explants with low microbial contamination and high regeneration frequency for micropropagation of Freesia × hybrida Bailey'Argenta. International Journal of Horticultural Science and Technology. 2(1), 97-109.
Rezaei A, Hosseipour B (2015) In vitro propagation and rooting of Garnem rootstock. Plant Cell Biotechnology and Molecular Biology. 16(1-2), 41-47
Ruzic DJV, Vujovic TI (2008) The effect of cytokinin types and their concentration on in vitro multiplication of sweet cherry cv. Lapins (Prunus avium L.). Horticultural Science. 35, 12-21
Taghizadeh M, Solgi M (2014) The application of essential oils and silver nanoparticles for sterilization of bermudagrass explants in in vitro culture. International Journal of Horticultural Science and Technology. 1(2), 131-40
Tatari M, Musavi A (2013) Optimization of in vitro culture of Tetra, Nemagard and GF677. Journal of Crops Improvement. 15(3), 103 -115
Ugur R (2020) Development of in vitro sterilization protocol for DO-1 (Prunus domestica) rootstock. Applied Ecology and Environmental Research. 18(2), 2339-2349.