Pollination following grafting introduces efficiently Ocimum basilicum L. genes into Nicotiana tabacum L.

Tobacco is an important cash crop in the world. However, the genetic basis is comparatively narrow among the modern Nicotiana tabacum cultivars, limiting its potential for quality improvement. To introduce genes conferring desirable chemical constituents from medicinal plants, a distant hybridization test was conducted between N. tabacum and Ocimum basilicum L. Seedlings of wild type Nicotiana sylvestris and N. tabacum cultivar 78-04 respectively acted as rootstock and scion. During the flowering season, hand pollination between 78-04 as pistillate parent and O. basilicum as pollen parent was carried out under 22-25°C temperature and 70-80% of relative humidity in the greenhouse. Seed sets of 55% were obtained in 78-04, and about 400 seeds per capsule were produced. But both non-grafted and self-grafted 78-04 plants rarely resulted in fruits by hand pollination and those obtained were without seed. Similar results were obtained in different material combination. The interfamilial F 1 hybrids acquired showed distinct variation with various morphological characteristics, and their hybrid nature was confirmed by isozyme and random amplified polymorphic DNA (RAPD) analyses. This result indicated that pollination following grafting can facilitate gene exchange and recombination at the interfamilial level and efficiently overcome barriers of sexual incompatibility between N. tabacum and O. basilicum . Our research not only extends the genetic basis of tobacco but also will provide valuable germplasm for improvement of varieties.

commerce used tobacco in most parts of the world, while the latter is grown extensively in parts of Eastern Europe and Asia Minor. Numerous types of tobacco are def ined by different criteria such as method of curing (flue-, air-, sun-and fire-cured tobacco) and morphological and biochemical characteristics (i.e., aromatic fire-cured, bright leaf tobacco, Burley tobacco, Turkish or oriental tobacco) (Gholizadeh et al., 2012). Tobacco cultivars are thought to have arisen from interspecific hybridization between Nicotiana sylvestris (2n = 24, subgenus Petunioides) and Nicotiana tomentosiformis (2n = 24, subgenus Tabacum). Some studies have shown that there is a narrow genetic background in Nicotiana species and a high genetic similarity between cultivated tobacco, limiting its potential for improving the quality (Lewis & Nicholson, 2007). In order to broaden the genetic base, new gene pools need to be incorporated into tobacco varieties. Since the chemical constituents of tobacco are very important to the cigarette industry, tobacco breeders become more interested in developing tobacco cultivars possessing the distinctive aroma and potentially reduced harmful characteristics. Cui et al. (2007) and Li et al. (2008) have tried to transfer chemical components from medicinal plants.
To develop varieties with wide adaptability, higher yield potential and suitable chemical constituents for cigarette industry, plant breeding techniques have been applied to N. tabacum for approximately seven decades. However, the genetically engineered cultivars are not currently accepted by the industry and the public (Moon & Nicholson, 2007). Generally, crossing barriers occur due to the result of incompatibility and incongruity, although wide hybridization has great potential for transferring desirable genes to crops. Up to now, very few instances have been successful in interfamilial hybridization by conventional sexual crosses except protoplast fusion (Kisaka et al., 1997). In this paper, a novel method (viz. pollination following grafting) could effectively overcome the interfamilial incompatibility between N. tabacum and O. basilicum. The F 1 hybrids obtained were confirmed by morphology, isozyme and random amplif ied polymorphic DNA (RAPD) analyses. This method is being applied in our laboratory to develop new types of tobacco containing active ingredient of O. basilicum. The new characteristics would be valuable germplasm for improving tobacco varieties.

Grafting method
Ten different genotypes of tobacco were seeded in cell flats (cell size 3 × 3 × 10 cm 3 ) filled with peat-lite mixture. According to different combinations, seedlings were grafted at 21 d after sowing by needle graft following the procedure described by Yasinok et al. (2009). And non-grafted and self-grafted plants were used as control. After 25 d, plants were transferred to a cultivation chamber under controlled experimental conditions with relative humidity of 60-80%, 25/15°C temperature (day/night) and 16/8 h photoperiod. The plants were grown in pots (50 cm upper diameter, 24 cm lower diameter, 40 cm in height), with one pot containing one plant (Fig. 1b,c,d).

Pollination method
During the flowering season, the hybridization between parental genotypes was done under controlled greenhouse conditions by pollinating the flowers of N. tabacum (scion) with the pollen of O. basilicum. Fresh O. basilicum pollen from just dehisced anthers was collected on a soft brush in the morning, and directly dusted on the stigmas of the emasculated tobacco flowers. The process was repeated 2 or 3 times. These hand-pollinated flowers were bagged to prevent other unwanted pollination and labeled individually with tags until harvest. The number of pollinated flowers, capsules with seeds, seeds produced and 1,000-seed weight was recorded. Seeds harvested from the mature capsules were sowed the following year and grown in the greenhouse. Putative hybrid plantlets were referred to as F 1 plants, and their phenotypes were compared with those of their parents and the hybrid nature was verified by isozyme and RAPD analyses.

Isozyme analysis
Fresh leaves of the parents and five putative F 1 hybrid plants derived from the cross combination [(N. sylvestris + 78-04) × O. basilicum] were sampled on 20 d after sowing. Plant materials were ground in 0.1 M Tris-HCl (pH 8.3) using a ratio of 1:2 (fresh weight of leaf to volume of buffer) and were centrifuged at 12,000 rpm for 10 min. The supernatant was electrophoresed with polyacrylamide gel electrophoresis (PAGE) in 3% spacer gel-10% separating gel. For  esterase analysis, the gel was stained with 90 mL phosphate buffer (pH 6.4) containing 90 mg fast blue B salt, 6 mL 2% alpha-naphthyl acetate, 3 mL 2% betanaphthyl acetate (both dissolved in 1 mL acetone and diluted to 100 mL with 80% ethanol). For peroxidase isozyme analysis, the gel was stained with 100 mL staining buffer containing 20 mL 2% benzidine, 70.4 mg vitamin C, 20 mL 0.6% H 2 O 2 and 60 mL distilled water (Xu et al., 2003).

Crossability between N. tabacum and O. basilicum
Large plump capsules were obtained from openpollinated flowers of the non-grafted and self-grafted plants. Hand pollination of non-grafted and selfgrafted tobacco rarely resulted in fruits and those obtained were without seed. But grafting followed by pollination produced up to 426 normal seeds per capsule (Table 3). The cross combination [(N. sylvestris A method for introducing Ocimum basilicum genes into Nicotiana tabacum 1071   (Table 4). Similar results were obtained in different material combination.

Morphology characteristic of F 1 hybrid
Among different cross combinations [(N. sylvestris + 78-04) × O.basilicum] was outstanding for hybrid seed sets and seed yield per capsule (about 400). One hundred seeds were planted the next year in the greenhouse, and putative hybrid plants generated obvious phenotypic variation, as observed at flowering time. In general, the morphological traits of the F 1 hybrids looked like N. tabacum. Figs. 2a-l present such agronomic performance as leaves, inflorescences and flowers, etc., which could be caused by the introduced DNA from O. basilicum. Most importantly, they were apparently completely fertile. Eight individual plants resembled O. basilicum, especially in the foliar morphology. Twenty four hybrid plants developed to a height approximately equal to that of 78-04, but distinct changes appeared as the light-colored flowers, leaves shape and the big capsules (Figs. 3a-j). This will facilitate mass selection for desired plant types and leaf characteristics from segregating hybrid populations.

Isozyme analysis
As shown in Figs. 4a,b, analyses with esterases and peroxidases were able to distinguish between N. tabacum and O. basilicum hybrids and parents and confirm the hybridity of five F 1 plants. The characteristic bands of both parents were not simply represented in the hybrids. Probably due to combination among enzyme subunits or differential gene expression in a different genetic background, the F 1 hybrids showed new bands.

RAPD analysis
Out of 20 RAPD primers assessed, two primers (S28 and S265) gave reproducible results and showed different band patterns specific to N. tabacum and O. basilicum. These major paternal bands were also found to be present in the five hybrid seedlings (Figs. 5a,b). The RAPD profiles from the two primers gave evidence for hybrid nuclear genome.

Discussion
Owing to the cross-incompatibilities, genetic introgression between distantly related species is very difficult via conventional ways. The graft is an effective agricultural approach to improve the stress resistance and quality of crops (Ruiz et al., 2005). The main feature of this method was the grafting of an immature scion on to a more mature stock before pollination.  Because the nutritional components offered by the rootstock were different from those existing in the scions, they might constitute a kind of stress to the scions . But between varieties and species, rootstocks might have different influence on scion' physiological metabolism or there might exist difference in affinity of every grafted combination. Despite the low seed set, a large number of unique interfamilial hybrids were still obtained using this method. Currently, the underlying molecular and biochemical mechanisms remain unknown, but this method can effectively introduce the genes of Gaertn. (Portulacaceae) into N. tabacum (Wei et al., 2010). N. tabacum is highly self-pollinating. Backcrossing is often used in cultivar development, and nuclear genes controlling disease resistance, morphological characteristics, or biochemical traits are frequently the focus of backcrossing (Lewis & Kernodle, 2009). Wei et al. (1998) study indicate that many germinating seeds were harvested from plants of both the BC 1 and open pollination of the F 1 hybrid. Environmental factors can influence the crossability. A positive effect of high temperature in overcoming incompatibility and incongruity has been detected and applied in plant breeding by pollinating at high temperatures (Van Tuyl et al., 1982;Okazaki & Murakami, 1992). But present study shows grafting followed by A method for introducing Ocimum basilicum genes into Nicotiana tabacum 1073 Figure 2. Representative variation of F 1 hybrids obtained from interfamilial hybridization between N. tabacum cv. 78-04 as pistillate parent and Ocimum basilicum L. as pollen parent. Some F 1 plants had only 1-4 flowers with altered morphology such as the short corolla and the exserted stigma (a, b, c, d) and part of them showed curling and twisting leaves with very dark green (e, f, g, h). Others had only 4-8 leaves and apparently dwarf, suggesting a shortened vegetative growth period and advanced flowering time (i, j, k, l). In different combinations, the number of seeds per capsule of the F 1 hybrids ranged from 0 to 426, which indicates that wide crosses also depend on the cross partners. Cytogenetic analyses have shown that chromosome number is heterogeneous in these hybrids (Chen, 2005;Li et al., 2006). In recent years, genetic variability in Nicotiana increasingly gained attention because of investment in Nicotiana genomics research, interest in development of tobacco products with reduced harmful characteristics, and concentration on using Nicotiana species for plant-based production of commercially useful proteins (Lewis & Nicholson, 2007). Unlike most agronomic crops, tobacco is cultivated for its leaves rather than its reproductive parts. Some F 1 hybrid material displayed unfavorable agronomic traits in plant type, plant height, leaf size, leaf shape and number of leaves, etc. This might be a result of pleiotropic effects of the gene of interest per se or because of linkage drag effects caused by genes linked to the character of interest (Lewis & Rose, 2010). Research showed that a tremendous amount of phenotypic variability exists among strains of N. tabacum, but nucleotide variation, as revealed by RAPD is comparatively low. The major advantage of RAPD technology lies in exploration of large genomic portions without prior sequence information and requires small quantity of DNA. The bands which are specific for the pollen parent and occur in the hybrids are good markers to confirm the hybridity. Those non-parental bands may be generated from the differential gene expression in a different genetic background and may also be created by heteroduplex formation (Kiran et al., 2009).
It is well-known that some plants, especially those belonging to the Lamiaceae family, possess a wide range of biological and pharmacological activities (Danesi et al., 2008). O. basilicum has been cultivated from ancient times both as an ornamental plant and as a major essential oil crop. Although essential oils in different basil cultivars are variable, the prevalent chemical components are phenylpropanoids, such as estragole, eugenol, methyl-eugenol and methyl-cinnamate, and monoterpenes, such as linalool, geranial, neral and eucalyptol (De Masi et al., 2006). Scientific studies have also shown that bioactive constituents in basil oil are antioxidant, anticancer, antimicrobial, antiviral, antifungal, repellent, insecticidal, or nematicidal (Tsai et al., 2011). Many reports have been given about the agronomic performance of hybrids, but little or no information is available on chemical constituents of hybrids when compared with their parents. Our previous study indicated that the volatile compound of the hybrids, as assessed by gas chromatography-mass spectrometer (GC-MS), largely resembled that of the A method for introducing Ocimum basilicum genes into Nicotiana tabacum 1075 parents (Wei et al., 2008), which may be a favourable change in chemical quality. These components of O. basilicum are secondary metabolites produced under genetic regulation. This suggests that the biosynthesis pathway of essential oil of O. basilicum was transferred to the tobacco. Meanwhile, some new medicine and flavor components were found such as α-terpineol, famesene, and γ-gurjunene (Wei et al., 2008). In summary, our research indicates that pollination following grafting could efficiently overcome barriers of sexual incompatibility between N. tabacum and O. basilicum and introgress the valuable genes from O. basilicum into the gene pool of cultivated tobacco. The results will be of value in broadening the genetic basis of N. tabacum and creating new types of tobacco.