Introduction

Modern biotechnology techniques can hasten the production of new genotypes and broaden the gene pool avaible for improvment of woody fruit species (Ainsley et al., 2001). However, fruit trees are amongst the most recalcitrant for in vitro culture, and regeneration of adventitious shoots from adult explants has proven difficult (Miguel et al., 1996). Conventinal breeding of woody fruit species is a slow and difficult process due to high levels of heterozygosity and long generation cycles ( Sriskandarajah et al., 1994). To minimize the problem of enormous genetic variation and to obtain genetically identical populations, vegetative propagation via layering or cutting is applied. However, these techniques are inefficient due to the large number of problems faced in this fruit species in in vivo rooting of cutting. Because of the difficulties encountered with rooting of cuttings of almond cultivars, grafting and budding onto seedling rootstocks is the usual method used for vegetative propagation (Yildirim et al., 2010). Microshoots that are difficult to root, or do not form roots at all in vitro, can be micrografted onto rootstocks to obtain rooted plantlets (Thimmappaiah Putra and Anil 2002). In vitro micrografting has been reported in many plants such as cashew (Ramanayake and Kovoor 1999; Mneney and Mantell 2001; Thimmappaiah Puthra and Anil 2002), pistachio (Abousalim and Mantel 1992; Onay et al., 2002; Onay et al., 2007), olea europaea (Revilla et al., 1996), almond (Channuntapipat et al., 2003), Prunus avium L.(cherry) var. (Amiri 2006). Micrografts, developed in the 1970s, involve the grafting of millimeter-size vegetative meristems. Micrografting is mostly used for obtaining virus-free plants, separating viruses in infections, breeding specific genotypic combinations, and for studying graft incompatibility between scions and rootstocks (Burger 1985; Navarro 1988). In vitro micrografting may provide several advantages such as elimination of viruses, rejuvenation of mature tissues, year round plant production, enhance compatibility studies and correlative relation between rootstocks and scions, breeding for specific genotypic combinations to increase plant productivity, and extention of ecological limits of a particular plant species (Richardson et al., 1996; Hartmann et al., 1997). Compared to traditional grafting with in vitro grafting procedure, micrografting has several potential advantages over the traditional procedure; it is much more rapid, requires much less space, particularly disease-free planting materials and producing genetically uniform plantles. In in vitro micrografting studies, ensuring good contact between the microscion and the rootstock was essential for the graft unions to form successfully. Also, the optimization of culture conditions and media is significant for micrografting studies. The objective of the present study was to investigate influence of BAP for establishment of the microscions cultures and micrografting procedure was to develop for almond cultivar 'Nonpareil'.

Materials and methods

Establishment of the rootstocks

The seeds from naturally grown wild almond trees found in Diyarbakir Province of southeastern Turkey were used as explants for the establishment of the rootstocks. The explants were surface-sterilized by immersion in a 10% (w/v) commercial bleach solution (NaOCI) for 30 min after presterilisation processes, which included washing with running water for 5-10 minutes and then, rinsing with 70% (w/v) ethanol for 30 seconds. The explants were rinsed with sterilized water 5 times for 5 minutes to remove the NaOCI, then, kernel with embryos were cultured in Magenta GA-7 vessels containing 50 ml MS (Murashige and Skoog 1962) medium including 1.0 mg l^sup -1^ BAP (Isikalan et al., 2008).

Preparation of the microscion

Young offshoots were collected during the months of March- April from 7-years-old trees of almond cultivars, Nonpareil growing in the Botanical garden of the University of Harran, Sanliurfa, Turkey. To evaluate the effect of various cytokinins on shoots development, leaves from offshoots were eliminated, and were cut into 8-12 cm in length pieces, each having an apical shoot. The explants were washed with running water for 5-10 minutes, then they were dipped in 70% ethanol for 30 second, surface-sterilized in 10% (w/v) commercial bleach solution (NaOCI) for 20 min, and rinsed five times with sterile distilled water (5 min per rinse). Sterile apical tips (1.5-2.0 cm in length) were cultured into the MS medium containing 0.5, 1.0, 1.5, 2.0, 4.0 mg l^sup -1^ BAP separately. Moreover, aseptic shoots were cultured on media containing different concentrations of BAP (0.5, 1.0, 1.5 mg l^sup -1^) combined with 0.2 and 0.4 mg l^sup -1^ IBA separately. In vitro regenerated shoots were micropropagated and subcultured every 3 weeks. After 2 or 3 weeks, elite shoot tips were used as microscions for in vitro grafting experiments. The results were analyzed in a completely randomized block design. Significance was determined by analysis of variance, with the least significant (p ≤ 0.05) differences among mean values were estimated using Duncan's New Multiple Range Test.

Media and culture conditions

In this study, all media were supplemented with 3% sucrose (w/v), solidified with 5.45 g l^sup -1^ agar (Sigma), and media were adjusted to pH 5.8 prior to autoclaving (120°C for 20 min), and in vitro cultures were maintained at 25 ± 2°C with light at 40 µmol m-2 s^sup -1^ photosynthetic photon flux density (PPFD) ("cool white" fluorescent lamps, 75 W) a photoperiod of 16 h.

Grafting procedure and maintenance of micrograft

Micrografting was achieved by grafting the microscions onto the seedlings in vitro-germinated. Seedlings (2 weeks-old) germinated under in vitro conditions were used as rootstock (Fig. 1A). These were decapitated, and a 2 mm vertical incision was made from the top of each rootstock. Microscions were prepared by cutting the microshoots evenly into 1.5-2.0 cm in length, and each bottom end into a wedge ('V') shape. In vitro grafting was performed under sterile conditions. The micrografted seedlings were cultured into the MS medium containing 1.0 mg l^sup -1^ BAP and IBA with a control group which did not contain any PGRs.

Acclimatization

Successfully grafted plantlets were removed from the culture vessels, and rinsed with tap water to remove remaining agar from the root system. Subsequently, they were transplanted into individual commercial plastic pots filled with a autoclaved mix of sand, perlite and soil [1:1:1 (v/v)], covered with a beher glass to maintain a 90±5% relative humidity. During the first 2 weeks, the beher glass were gradually punctured to allow air exchange for plant acclimatization. Grafted plantlets were maintained at 25 ± 2°C with light at 40 µmol m^sup -2^ s^sup -1^ photosynthetic photon flux density (PPFD) ("cool white" fluorescent lamps, 75 W) a photoperiod of 16 h.

Results

Effect of BAP on shoot multiplication of microscions

For micropropagation of microscions, apical tips were cultured on MS medium (Fig. 1B) which was supplemented with different concentrations of BAP. From the results presented in Table 1, it appears that the number of shoots changed depending on different concentrations of BAP. When the number of shoots was compared, there were statistically significant differences among concentrations tested. Also, it is clear that the addition of BAP (1.0 mg l^sup -1^) to the medium increased the shoot number and enhanced them vigorously. The average number of shoots per explant decreased significantly (P<0.05) whereas, increasing BAP concentration from 1.0 mg l^sup -1^ to the high concentrations (2.0 or 4.0 mg l^sup -1^). The lowest shoot formation was observed on media supplemented with 4.0 mg l^sup -1^BAP (2.91±1.26). The maximum number of shoots (6.66±1.27) was obtained from MS medium with 1.0 mg l^sup -1^ BAP (Fig 1C), followed by 4.70±1.35 shoots from 1.5 mg l^sup -1^ BAP. At the same time, maximum shoot length (1.95±0.25) was observed in the presence of 1.0 mg l^sup -1^ BAP (Table 1). In the absence of growth regulators, it was observed as 0.58±0.14 new shoot formation. The results showed that using BAP for the new shoot formation was absolutely necessary.

Effects combination of BAP and IBA on shoot multiplication of microscions

Results in Table 2 showed that using of IBA alone lead to decrease at length and in number of shoots. However, the length and number of shoots are determined to increase when used together with BAP and IBA. For development of microscions, the best result was obtained from MS medium supplemented with 1.0 mg l^sup -1^ BAP + 0.2 mg l^sup -1^ IBA (Table 2). However, only the use of BAP proved to be more beneficial than the combination of IBA and BAP for development of microscions. Consequently, for multiplication and development of scions, the best plant growth regular was determined as 1.0 mg l^sup -1^ BAP (6.66 ± 1.27 - 1.95±0.25), when Table 1 and Table 2 were compared.

Effects of BAP and IBA on micrograft development

Grafted plantlets under in vitro conditions were cultured on MS medium containing 1.0 mg l^sup -1^ BAP and 1.0 mg l^sup -1^ IBA with a control group, separately (Fig. 1D). At all treatments tested, initial traces of graft unions were observed between scions and rootstocks (2 weekly) within 7- 10 days after graft procedure. After 2 weeks, cultured graft unions were 90% on medium with BAP and IBA (1.0 mg l^sup -1^) except for the control group. It was also noted that there was a color change in plantlets on the hormone-free medium and leaves became light-green, white or brown, while healthy shoots were obtained on medium suplemented with 1.0 mg l^sup -1^ BAP.

After 25 days of culture, new shoot formation was observed from the medium supplemented with 1.0 mg l^sup -1^ BAP, while the best response for root formation and elongation was obtained from the medium with 1.0 mg l^sup -1^ IBA (Table 3). At the same time, it was determined that there were no significant differences between IBA and BAP for graft union rate. For shoot number, the best result was obtained on the medium supplemented with 1.0 mg l^sup -1^of BAP and an average of 2.75 shoots was formed from each explant. Although similiar results were obtained on graft union rate at all tested group, the medium supplemented with 1.0 mg l^sup -1^ BAP was determined as favorable one for development grafted plantlets (Fig. 1E-a,b). In vitro micrografted plantlets were successfully acclimatized and no problems were encountered with the establishment of micrografted plants in vivo (Fig. 1F).

Discussion

Micrografting is a technique that potentially can combine the advantages of rapid in vitro multiplication with the increased productivity that results from grafting superior rootstock and scion combinations (Gebhardt and Goldbach 1988). In general, micropropagation is difficult from mature tissues or organs of all woody trees. However, previous publications indicate that almond is difficult to micropropagate efficiently from mature tissues also (Caboni 1994; Akbas et al., 2009). Each plant species that was propagated in vitro has different plant growth regulator requirements and concentrations. Due to these reasons, optimization of culture conditions and media were important for micropropagation studies. In this research, MS medium containing 1.0 mg l^sup -1^ BAP was chosen as the optimum medium for multiplication and development shoot tips of Nonpareil. Gürel and Gülsen (1998) also reported that BA was absolutely necessary for in vitro propagation of shoot tips of almond (Nonpareil and Texas) during culture initiation and proliferation stages. On the contrary, Channuntapipat et al. (2003) reported that AP medium containing 0.049 µM IBA and 3 µM BA was effective for propagation shoot tips of Nonpareil 15-1. Gürel and Gülsen (1998) reported that the best result for shoot development and growth of almond was obtained from the combination of 0.1 mg l^sup -1^ IBA and 1.0 mg l^sup -1^ BAP. In contrast, our results showed that the use of only BAP on medium proved to be more beneficial than the combination of IBA and BAP for shoot development. The difficulty in the rooting of almond occurs both in vivo and in vitro especially when shoots come from mature trees. Although several trials were performed on almond in order to overcome this problem by using different auxin types, concentrations, basal salt composition and fenolic compounds, the rooting frequency especially of paper shell almond cultivars, still needs to be improved as rooting levels up to 75% were achieved in hard shell cultivars (Caboni et al., 1997). Thus, development of a suitable micrografting technique can provide a solution to overcome rooting difficulties of plant species like in vitro proliferated almond tissues and explants. The technique of micrografting was used in this study because this prodecure is a safe and an alternative method for producing genetically uniform, disease-free planting materials and micropropagation of fruit trees. In the present study, an effective micrografting technique was developed and 90% success was obtained for Nonpareil. Channuntapipat et al. (2003) obtained 50-65% success from micrografting of Nonpareil 15-1 and Ne Plus Ultra. Ghorbel et al. (1998) micrografted apical buds from in vitro shoots of the almond cultivar Achak on to rootstocks derived from zygotic embriyos of the same cultivars with 60- 80% success. In this research, an efficient procedure was developed for micrografting of Nonpareil and 90% success was obtained for graft union rate. The MS medium supplemented with 1.0 mg l^sup -1^ BAP was determined as optimum medium for development grafted plantlets. This procedure provided more micrografted seedling on a little area in a short time. Furthermore, this technique can be adopted in breeding of specific genotypes or other species that are difficult to root. Likewise, when considering the application of genetic transformation to the almond, it is also important to develop protocols for efficient regeneration of plants from adult trees.

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Author affiliation:

Çigdem Isikalan1*, Süreyya Namli1, Filiz Akbas2, Bekir Erol Ak3

1 Dicle University, Faculty of Science, Department of Biology, 21280, Diyarbakir-Turkey

2 Batman University, Faculty of Science and Arts, Department of Biology, Batman-Turkey

3 Harran University, Faculty of Agriculture, Department of Horticulture, Urfa-Turkey

* corresponding author: ahrar@dicle.edu.tr