Environmental and Experimental Biology (2017) 15: 217–224 Original Paper DOI: 10.22364/eeb.15.22 In vitro micrografting of lucumo (Pouteria lucuma), Sapotaceae Jeanine Córdova-Risco, Consuelo Rojas-Idrogo, Guillermo E. Delgado-Paredes* Facultad de Ciencias Biológicas, Universidad Nacional Pedro Ruiz Gallo, Ciudad Universitaria, Juan XXIII No 391, Lambayeque, Perú *Corresponding author, E-mail: [email protected] Abstract Effects of plant growth regulators [gibberellic acid (GA3) and indole-3-butyric acid (IBA)], holding grafts (aluminum foil and cooper wire), pre-treatment [immersion in 1.5 mg L–1 6-benzylaminopurine (BAP)] of scions, and genotype combination (rootstock × scion) were investigated on micropropagation of rootstocks and scions and micrografting of lucumo (Pouteria lucuma) cv. ‘Beltrán’ and ‘Seda’. In vitro germinated commercial lucumo seedlings developed from seeds were used as rootstocks and scions. The rootstocks were cultured –1 on Murashige and Skoog (MS) culture medium supplemented with different concentrations (0.0, 0.1 and 1.0 mg L ) of GA3. The data showed that in the cv. ‘Beltrán’ the maximum shoot height (6.1 ± 1.2 cm) and nodes formed (7) were observed in the presence of 0.1 –1 –1 –1 –1 mg L GA3. The effects of two different MS media containing 0.1 mg L GA3 (MM-1) and 0.1 mg L GA3 and 0.4 mg L IBA (MM-2) were studied on development of micrografted plantlets. Propagated shoots tips, were micrografted ontoin vitro germinated commercial lucumo seedlings. The results indicated that the best graft rate were obtained in the genotypes combination ‘Seda’ × ‘Beltrán’ (rootstock× scion), holding grafts with cooper wire, and pre-treatment of scions with immersion in 1.5 mg –1L BAP, reaching a survival rate of 87.5%; however, in both MM-1 and MM-2 culture media the survival rate of grafting was similar (52.6 and 54.1%, respectively). Key words: lucumo, rootstocks, shoot elongation, scions. Abbreviations: BAP, 6-benzylaminopurine; GA3, gibberellic acid; IBA, indole-3-butyric acid; MS, Murashige and Skoog medium. Introduction adventitious shoots from adult explants has proven to be difficult (Miguel et al. 1996). In woody fruit trees, vegetative In Peru, the Sapotaceae family comprises 10 genera propagation by cuttings, is used to minimise the enormous (Manilkara, Sideroxylon, Ecclinusa, Micropholis, Pouteria, genetic variation caused by sexual propagation; however, as Sarcaulus, Chrysophyllum, Diploön, Elaeoluma and rooting is of great difficulty, as observed in almondPrunus ( Pradosia). Genus Pouteria comprises nearly 50 species, dulcis) cultivars, micrografting has been the most widely and is the largest genus in the family and an important used method for vegetative propagation (Yildirim et al. component of lowland rain forest (Brako, Zarucchi 1993). 2010). In this sense, micrografting or in vitro grafting is a Pouteria caimito is one of the most widespread species and relatively new technique for plant propagation which, in Pouteria lucuma is another widely cultivated species native the classic definition of Hartmann et al. (2002), “involves in wet montane and cloud forest at 1500 to 3000 m alt; the the placement of a meristem or shoots tip explant onto globose fruit, 6 to 12 cm diameter, contain a rather dry a decapitated rootstock that has been grown aseptically yellowish-brown flesh, much used for flavouring ice-cream from seed or micropropagated cultures”. Micrografting was and cakes (Pennington et al. 2004). initially used in herbaceous plants, but then was used in Conventional propagation and breeding the lucumo is particular on woody species and especially on fruit species; by sexual seed and grafting, but these methods have some it was modified and improved for increasing the graft limitations caused by the following reasons: perennial nature, success by Murashige et al. (1972) and Navarro et al. (1975). long generation cycle, self-incompatibility, low success rate In general, the technique has great potential for fruit plant of hand pollination, long duration for seed maturation, clonal improvement and large-scale multiplication, production difference of flowering time and fruit bearing capability of of pest and systemic disease-free (such as virus, viroids, some clones, high levels of heterozygosity, and others, as bacteria and fungi) plants, safe international germplasm it was observed in Camellia sinenis (Mondal et al. 2005). exchange and multiplication of plants with problems in Therefore, tissue culture is certainly a better alternative for root system formation (Hussain et al. 2014). conventional propagation and reduction in growth and In micrografting of almond (Amygdalus communis) development time; however, fruit trees are amongst the cv. ‘Nonpareil’, the effect of several growth regulators on most recalcitrant for in vitro culture, and regeneration of microscions micropropagation was studied, obtaining Environmental and Experimental Biology ISSN 2255-9582 217 J. Córdova-Risco, C. Rojas-Idrogo, G.E. Delgado-Paredes the highest shoot regeneration (6.66) and shoot length Scion source (1.95 cm) with Murashige and Skoog (MS) culture The in vitro seedlings used as scion were obtained from medium (Murashige, Skoog 1962) supplemented with P. lucuma seeds cv. ‘Beltrán’ and ‘Seda’, commercial trees. 6-benzylaminopurine (BAP) 1.0 mg L–1 (Işıkalam et al. P. lucuma seeds were selected and sterilized following the 2011). Using micrografting, grapevine (Vitis vinifera) cv. same protocol used to disinfect rootstocks, and individually ‘Jing Xiu’ was used as scion and Vitis amuensis cv. ‘Shuang inoculated into test tubes (25 × 150 mm) containing 25 –1 you’, a new wild grape variety, as rootstock; from the eight mL MS medium supplemented with 0.1 mg L GA3. Sterile micrografting methods tested, three of them presented apical tips and axillar buds (1.5 to 2.0 cm in length) were successful root formation (formation rate > 60%), when obtained directly from the apical bud and nodal segments, the shoot tip was selected as scion material (Zhu et al. respectively. 2007). In another study, the rejuvenation of mature lentisk In ex vitro experiments, young offshoots were collected (Pistacia terebinthus) by in vitro micrografting method from 15-year-old trees of lucumo cv. ‘Seda’ growing in the was developed for the production of plantlets, and genetic Botanical Garden of the Universidad Nacional Pedro Ruiz stability of 3-, 6-, and 24-times subcultured clones of both Gallo, Lambayeque, Peru; as before we used apical tips male and female genotypes was assessed and compared obtained from rootstocks germinated in substrate S1 for with the mother plants using fluorescent-based amplified the latter experiments. These explants were washed with fragment length polymorphism analysis (Onay et al. 2016). running water for 10 min, surface-sterilised in 10 mL of In general, in vitro micrografting has been reported in commercial bleach solution (5.25% NaOCl) 1.0 L–1 in many plants such as cashew (Anacardium occidentale) sterile distilled water for 10 min, and rinsed five times with (Thimmappaiah et al. 2002), pistachio (Pistacia vera) (Onay sterile water. et al. 2007), olive (Olea europaea) (Revilla et al. 1996), almond (Amigdalus communis) (Channuntapipat et al. In vitro micrografting procedure and maintenance of 2003) and cherry (Prunus avium) (Amiri 2006). Reviews on micrograft micrografting have been published by Jonard et al. (1983), Under aseptic conditions, shoots of uniform length (2.0 Jonard (1986), Roistacher et al. (1976), Navarro (1988), cm) and diameter (1 to 2 mm), aged 8 weeks, were selected Parkinson et al. (1990), Monteuuis (2012) and recently by from in vitro cultures and used as rootstocks and scions in Hussain et al. (2014). micrografting. The rootstock was decapitated, and a 5 mm The objective of the present study was to investigate vertical incisión was made from the top of each rootstock, the most reliable micrografting technique, and the optimal and the scion was cut at 45° angle (wedge shape) from the grafting materials for lucumo. We specifically investigated base. The joined part was ligated with aseptic aluminum the influence of BAP, holding grafts, culture medium and foil (1.0 × 0.5 cm) or with aseptic wires of cooper. To genotypes over the micrografting of lucumo. prevent dehydration of in vitro grafts, before micrografting, the cutting area of the scion was immersed in BAP solution Materials and methods (1.5 mg L–1). The micrografted seedlings were cultured into –1 two different MS medium containing 3.53 g L CaCl2 H2O Obtaining in vitro and ex vitro rootstocks –1 –1 and 0.1 mg L GA3 (MM-1) and 3.53 g L CaCl2 H2O, 0.1 –1 –1 The in vitro seedlings used as rootstocks were obtained mg L GA3 and 0.4 mg L IBA (MM-2). from Pouteria lucuma seeds cv. ‘Beltrán’ and ‘Seda’, The procedures for the ex vitro micrografting were commercial trees found in Huaral Province, Lima, Peru. similar to the ones used for the in vitro micrografting. The Mature seeds were washed with detergent and water, and number of successful micrografts was recorded 12 weeks sectioned to obtain the explant with the embryo and part after micrografting and the percentage of success was of the endosperm. The explant were surface-sterilised by recorded. immersion in a 5.25% (w/v) commercial bleach solution for 2, 3 and 5 min in several treatments and then washed with Media and culture conditions running sterilised water for 3 to 5 minutes to remove the In this study, in all experiments the basal MS medium NaOCl (Table 1), before being individually inoculated into was supplemented with the vitamins myo-inositol 100 mg glass vessels (10 × 6 cm) containing 50 mL MS (Murashige, L–1 and thiamine HCl 1.0 mg L–1 and 6.0 g L–1 agar. The Skoog 1962) medium including 0.0, 0.1 and 1.0 mg L–1 pH of all media was adjusted to 5.8 ± 0.1, with KOH and gibberellic acid (GA3).