INTRODUCTION

Citrus is one the most important group of plants in earth with a long history of cultivation originated from tropical and subtropical regions of southeast and spread toward Europe, Africa, America and Oceania (Wu et al., 2018). The genus Citrus belongs to the order of Geraniales, family Rutaceae, subfamily Aurantiodeae. The Rutaceae family includes about 160 genera and 1650 species consisting of trees and shrubs evergreen that grow and fruit at latitudes between 40º north and 40 º south; fruits are very rich in vitamin C, consumed fresh and transformed into juices, wedges, preserves, jellies and jams, and provide by products for cosmetics and medicinal products (Inglese and Sortino, 2019). Annual production of citrus in the world in 2018 was around 150 million ton where oranges accounted for 50%, mandarins 22%, lemons and limes 12% and other citrus fruits 6%. In Colombia, production in 2018 was estimated in 75000 ton distributed in 20% oranges, 13% mandarins, 14% limes and lemons and 52% other citrus fruits (FAO, 2020). In Córdoba department, citrus fruit production in 2018 was 1150 ton with more than 90% was orange production (Agronet, 2021). Citrus plants for planting commercial crops are usually propagated by grafting the desired cultivar onto selected rootstocks using “T” budding; rootstocks are usually selected for stress tolerance and growth habit, while grafts are usually selected for fruit quality and production (Wasielewsky and Balerdi, 2018; Albrecht et al., 2017; Baron et al., 2019). At the present there is a worldwide shortage in the supply of planting material due to new strict regulations to produce and commercialize citrus plants; these regulations include upgraded infrastructure to prevent pest arrivals and test to confirm negative diagnose for pathogens such as CTV (Citrus tristeza virus), CEVd (Citrus Exocortis Viroid) and HLB (Huanlongbing) (Vashisth et al., 2020; ICA, 2019) in both rootstock and grafts. Micrografting is an in vitro technique used to propagate plants by positioning an isolated shoot tip meristem on top of a decapitated rootstock; it combines the advantages of rapid in vitro multiplication with the ability to combine the genotypes desirable characteristics in a single plant (Suárez et al., 2005). Success of micrografting is associated with genotype, shoot tip source, rootstock culture and age, nutrient medium composition, sucrose concentration and graft technique. In vitro seed germination for micrografting can be affected for PGR (Plant Growth Regulator) supply in the medium; GA₃ (Gibberellic acid) is one of the most studied (Miceli et al., 2019; Iftikhar et al., 2019; Rout et al., 2017); while wedge and slit are the most common techniques used for micrografting fruit crops species (Rehman and Gill, 2015). In citrus, micrografting has been employed mostly as a technique to recover disease-free plants to be used as a source for massive propagation (Navarro et al., 1975; Kapari-Isaia et al., 2002; Singh, 2018). In the present research, the conditions for in vitro rootstock germination, plant management for shoot tip source and shoot tip meristem placement onto the decapitated rootstock were evaluated in the micrografting of two different, Valencia orange and Tahiti lime, citrus species.

MATERIALS AND METHODS

Plant material

Seeds for rootstock production were extracted from mature fruits obtained from field grown Cleopatra mandarin trees planted at the Universidad de Córdoba – Berastegui Campus (8º40´26” N 75º46´44” W). Fruits were washed twice with distilled water, hand-squeezed and seeds separated with a plastic sieve. Seeds were profusely washed with sterile-distilled water, air-dried overnight on filter paper and stored in sterile closed glass flasks during 4 weeks in a conventional fridge at 8 °C. Shoot tips were obtained from 2-year old grafted plants of Tahiti Lime and Valencia orange obtained from an authorized citrus plant distributor (Reg. ICA 25290-06V) and maintained in a shade house (50%) with 5-min fog irrigation once a day.

In vitro rootstock germination

After storage, a total of 300 seed were scarified by seed coat removal, surface disinfected in a 1.05% active chlorine solution during 15 min and washed 3 times with sterile distilled water inside of a laminar flow hood. To evaluate the effect of GA3 (gibberellic acid) on seed germination, half of the seeds (150) were established in test tubes (150 x 25 mm) dispensed with 30 ml of ½ MS (Murashige and Skoog, 1962) medium supplied with (in mg L^-1) sucrose (30000), myo-inositol (100), thiamine HCl (0.4), GA3 (1) and TC-agar (6000). The remaining 150 seeds were established in medium of the same formulation with no GA3 addition. Test tubes were capped with plastic caps, sealed with Parafilm® and placed in racks at 20 °C with 12-hour photoperiod (40 µmol m^-2 s^-2) supplied with white cold fluorescent lamps. The experiment was a one-way factorial arrangement with 2 treatments (0 and 1.0 mg l^-1 GA₃) and 150 replicates each. The experimental units (300) were distributed with a complete randomized design. The number of seeds germinated was registered daily and the percentage of germination calculated

Shoot induction and growth in stock plants

To evaluate the effect of different treatments on shoot induction in 2-year-old grafted plants to be used as shoot tip source, plants were either pruned or sprayed with 1 mg l^-1 BAP (6-benzilminopurine). Terminal shoots, 4-5 cm from the apex, were removed in 6 plants of Valencia Orange and six plants of Tahiti Lime with a pruner. Simultaneously, the same number of different plants from both cultivars were sprayed with a solution at 1 mg l^-1 BAP. Plants were maintained in a 50% shade house, with a 5-min fog irrigation a day. The experiment was of a two-way factorial (2 citrus cultivars and 2 GA₃ concentrations) with 4 treatments and 6 replicates per treatment for a total of 24 experimental units distributed with a complete randomized design. After 4 weeks, the number of new shoots was registered and analyzed with an ANOVA (α = 0.05) and means were separated with the Tukey test (α = 0.05).

Micrografting and plantlet recovery

The effect of three different shoot tip positions (Topping, in contact with central cylinder, chip budding, sided, and slant) on the success of micrografting of Valencia orange and Tahiti lime on in vitro germinated “Cleopatra” mandarin rootstock were evaluated. Shoot tips were obtained from 2-year old grafted plants maintained in a shade house with fog irrigation once a day. Stem sections (3-5 cm long) were surface disinfected in a 1.05% active chlorine solution and washed with three changes of sterile distilled water inside of a laminar Flow Hood. Rootstocks were individually germinated in germination medium consisting of semisolid MS (Murashige and Skoog, 1962) salt medium supplied with (in mg l^-1) sucrose (30000), myo-inositol (100), thiamine HCl (0.4) and TC-agar (6000). Rootstocks were decapitated at 2-3 cm height, and the shoot tips (<5 mm long) were placed as indicated for the shoo tip position. Micrografted plants were individually maintained in baby food jars dispensed with 30 ml of germination medium. Recipients were covered with 2 layers of heavy-duty aluminum foil and sealed with Parafilm®. Cultures were stored at 20 ºC with 12-hour photoperiod (40 µmol m^-2 s^-2) supplied with white cold fluorescent lamps. The experiment consisted of a two-way factorial (2 citrus genotypes x 3 shoot tip positions) with 6 treatments and 50 replicates per treatment for a total of 300 experimental units distributed with a complete randomized design. After 10 weeks, the number of grafts with active shoot growth was registered and the success percentage of the specific micrografting technique was calculated. Successfully micrografted plants were individually transplanted to ex vitro conditions in T53 tube recipients (12.5 cm x 3.4 cm) filled with peat as substrate. During the first week, plants were fog-irrigated 3 times a day, 5 min each. After 1 week, irrigation frequency was reduced to twice 5 min a day each, and finally after 2 weeks, plants were irrigated once a day with a 5 min irrigation. The number of plants that fully adapted to ex vitro conditions was registered for each treatment.

RESULTS AND DISCUSSIONS

In vitro rootstock germination

Cleopatra mandarin seeds germination began 10 after in vitro establishment and continue until day 20th (Figure 1A). A total of 125 (83.3%) seeds established in medium supplied with 1 mg l_-1 GA₃, germinated while 139 (92.7%) of the seeds established in medium without GA3 germinated (Table 1). In both cases, maximum germination rate per day occurred at day 20th, and no further seed germination was observed beyond this day.

Citrus seed germination has been reported to be low in percentage and relatively time consuming (Prajapati et al., 2017; Hussain et al., 2017). Pregermination treatments such as GA₃ (Gibberellic acid) and scarification have been used in an attempt to increase germination rates and decrease the time for seed germination in several plant species (D´Este et al., 2019; Sayyad-Amin and Shahsavar et al., 2019; Marler et al., 2019). GA₃ is a plant growth regulator well known to induce physiological response in plant processes such as germination, plant growth and development, and photosynthesis (Miceli et al., 2019; Iftikhar et al., 2019; Rout et al., 2017; Neelambari et al., 2018; Shekafandeh et al., 2017). Results of citrus seeds treated with GA₃ to improve seed germination have been inconsistent. Al-Janaby et al. (2016) achieved significant increase in germination up to 71.3%. of “Cleopatra” mandarin seeds treated with scarification and 500 mg L_-1GA₃. Khopkar et al. (2017) reported that “Pummelo” (Citrus grandis L. Osbeck) freshly collected seeds treated with 50 ppm GA₃ and heat at 50°C for 24 h resulted in significant increase in germination as well as more leaf area and root volume in seedlings compared to non-treated seeds. In contrast, Khan et al. (2002) evaluated the effect of 50, 300 and 500 ppm GA₃ on germination percentage and germination rate seeds of grapefruit (Citrus paradise Macf.), Kinnow mandarin (Citrus reticulata Blanco) and rough lemon (Citrus lemon L.) finding no effects of GA₃ on final germination percentage compared to control treatments. Al-Musawi and Al-Moussawi (2020) reported a gradual decrease in germination when acid lime seeds were treated with several concentrations (750 mg l_-1 to 3000 mg l_-1) of GA₃ compared to water soaking as control treatment. Chaudhary et al. (2019) reported a reduction in time for seed germination and increased percentage of germination when seeds of Kagzi lime (Citrus aurantifolia Swingle) were soaked in 500 ppm GA₃ for 12 hours. In the present research, seeds germinated in basic MS without GA₃ germination percentage was close to 10% higher compared to those germinated in medium added with GA₃.

Table 1. Effect of GA3 on in vitro germination of Cleopatra


Mandarin seeds.

Shoot induction and growth in stock plants

Shoot grew from axillar buds 1 week after pruning or BAP spraying. The ANOVA allowed to detect statistical differences (Pr< 0.0001) in the number of new shoots produced as a result of the treatments. Valencia Orange plants produced more shoots than Tahiti Lime plants regardless of the treatment (Table 2). For both cultivars, pruning resulted in higher number of induced and grown shoots than spraying with BAP. Tahiti Lime pruned plants induced 2x more new shoots than BAP sprayed plants, while Valencia Orange pruned plants produced 4x more shoots than Tahiti Lime plants treated with BAP (Table 2).

Table 2. Effect of pruning and BAP on average new shoot formation in stock plants of Valencia Orange and Tahiti lime

*Number with the same letter are not different according to Tukey (α 0 0.05)

Treatment	Shoots
Valencia orange prunning	60.50 A*
Valencia orange BAP 1.0 mg l-1	49.25 B
Tahiti lime prunning	31.25 C
Tahiti lime BAP 1.0 mg l-1	14.50 D

BAP (6-benzilaminopurina) is a synthetic PGR that promotes cell division and multiplication, promotes axillary shoot proliferation and reduce apical shoot growth; additionally, is commonly used in micropropagation (Akhtar et al., 2020). BAP between 1 and 5 mg l_-1 is reported to significantly increase lateral shoot production in Gmelina arborea Roxb seedlings during stage 0 of micropropagation (Acosta, 2011). Amelia et al. (2020) reported that 5 mg l_-1 BAP increased leaf number and shoot longitude in Maleluca alternifolia seedlings. On the other hand, pruning is a citrus crop activity intended to improve tree architecture, removal of unproductive and damaged parts, favor air flow and increase fruit quality (Chueca et al., 2021; Robinson, 2020; Vincent y Ritenour, 2020; Astiari et al., 2019). In the present research, new shoot promotion, by pruning or BAP spray to increase availability of plant material for grafting procedures, was evaluated observing that pruning produces more plant material (Shoots) and, eventually, reduce costs by eliminating PGR use.

Micrografting and plantlet recovery

Of the 300 plants that were in vitro grafted, 21 (14%) showed scion growth eight weeks after grafting was performed (Figure 1B). Graft success was observed only when shoot tip was placed in a slant position at the decapitated surface of the rootstock for both, Valencia orange and Tahiti lime shoot tips (Table 3). Shoot tip positioned at the top in contact with the central cylinder of the decapitated rootstock, or sided as a chip budding in contact with the cortex of the decapitated rootstock showed no success. It was observed that for some graftings, the decapitated rootstock area produced a callus tissue that overgrowth the grafted shoot tip (Figure 1C). Micrografted plants of Valencia Orange and Tahiti lime were transferred to ex vitro conditions; after 8 weeks of transplant, 7 (50%) plants of Valencia Orange and 3 (47%) of Tahiti lime survived and fully adapted to ex vitro conditions (Figure 1D).

Table 3. Effect of three methods of micrografting on Valencia orange and Tahiti lime

Figure 1.In vitro micrografting of citrus plants. A = In vitro germinated seeds, B = Micrografted plant of Valencia Orange, C = Overgrowth of callus tissue at the decapita- ted surface of rootstock, D = Micrografted plant adapted to ex vitro conditions.

Micrografting is a clonal propagation technique which involves the placement of a shoo tip meristem on top of a decapitated rootstock cultured in vitro; this technique is commonly used for recovering pathogen-free plants of citrus species (Hussain et al., 2014; Chand et al., 2013; Singh et al., 2018). In the present study, success level for micrografting ranged from 14% to 28%, slant positioned shoot tip was the only micrografting technique that allowed grafted plants recovery, and 50% of the successful grafts survived when transplanted to ex vitro conditions. Effect of shoot tip position on the decapitated rootstock seems to be genotype associated. Shridar and Venugopal (2019) reported highest success in pistachio when shoot tips were positioned in slant followed by wedge method; in contrast, for cherry plants cleft grafting resulted in better results. Micrografting technique seems to depend on several variables for successful graft union and plantlet recovery. Holding grafts and rootstock closely united until graft fusion is completed improve success level, and materials such as nylon band, aluminum foil tubes and filter paper have proven to be convenient (Obeidy and Smith, 1991). Naz et al. (2007) reported that increasing sucrose concentration in the semisolid MS (Murashige and Skoog) culture medium from 3% to 5% improved micrografting success from 21% to 33% in both, Kinoow mandarin and Succari oranges. Likewise, Parzaei et al. (2018) observed maximum micrografting success percentage when lime (Citrus aurantifolia) shoots were micrografted onto four rootstock genotypes cultured in MS medium supplied with 7.5% sucrose. Singh et al. (2018) reported that micrografting of Kashi mandarin resulted in maximum (56.8%) response when the rootstocks were cultured in semisolid MS medium fortified with 0.5 mg L_-1 BAP and 0.1 mg L_-1indole-3-acetic acid (IAA) along with 5% sucrose. Kanwar et al. (2019) found that culturing rootstocks of Carrizo citrange with 3 mg l_-1 2,4-D (2,4-diclorophenoxyacetic acid) resulted in maximum success (70%) and growth of grafted plants of sweet orange (Citrus sinensis) Cv. Red Blood. Success levels of micrografting obtained in the present research are low for massive plant propagation purposes; however, they are consistent with levels used as a technique for recovering disease free plant material (Shridar and Venugopal, 2019; KapariIsaia et al., 2002). Strategies such as modified medium components (Sucrose, PGRs, additives), size of shoot tips, type of recipient and height of rootstock decapitation will be addressed in future studies as a way to improve success levels.

CONCLUSIONS

The results of the present research allowed to conclude that germination of Cleopatra mandarin seeds in vitro was not improved by GA₃ addition in the medium, manual pruning induced a larger number of vegetative shoots in mother plants than BAP sprays and shot tip placement in slant position allowed recovery of micrografted plants for both, Valencia orange and Tahiti lime citrus species; however, more studies are recommended to improve micrografting success levels to be used as a mass plant propagation technique.

Acknowledgments

The authors express their gratitude to the following: Universidad de Córdoba Project FCA 03-17, the lab assistance of Carmen Polo Tordecilla and statistical support of Dairo Javier Perez Polo, I.A. are gratefully acknowledged.

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Additional information

Conflict of Interest: The authors declare that it is an original work and there was no conflict of interest of any kind in the elaboration and publication of the manuscript.