Plant Materials
In this research seeds and seedlings were used during the experiment.
Two hundred seeds from the following citrus rootstocks were sown and germination rates were precisely determined during four weeks. Seedling populations comprising 100 plants from each rootstock were obtained.
Gou Tou Sour Orange (Citrus aurantium L. var. Gou Tou): A rootstock of Chinese origin, apart from being resistant to tristeza disease, there is no information about soil wants or resistance to other diseases. In Florida plants grafted onto Gou Tou rootstock have larger crown structure compared to those grafted on other bitter oranges [31]. Gou Tou sour orange is reported to reduce yield in grapefruits [16]. Gou Tou sour orange is tolerant of Phytophthora citrophthora and Phytophthora parasitical diseases [17].
C-35 citrange (Poncirus trifoliata (L.) Raf. X Citrus sinensis. Osb. 'Ruby'): A rootstock obtained by hybridization of Ruby Blood orange and trifoliate orange. It is tolerant of gummosis (Pytophthora citrophthora (Smith and Smith) Leon.) and Tristeza diseases and resistant to nematodes. Resistance to cold is equivalent or slightly better than Carrizo citrange. Trees have moderate size and those grafted on Troyer have 25% smaller crown. Good compatibility with sandy, sandy-clayey and clayey soils; however, is more susceptible to limey soils than Carrizo citrange [31].
Troyer citrange (Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osb.): A sweet orange and trifoliate orange hybrid. Generally, it has trifoliate properties, with more compliant characteristics in terms of environmental conditions and compatibility with varieties so it is used more often in recent years and in most cases is chosen as alternative to sour orange rootstock. Proliferation with seed and grafting is easy, growth is moderate, yield is high, maturation and fruit setting are early, effects on fruit quality are high and economic lifespan is at moderate levels [23].
Alemow (macrophylla) (Citrus macrophylla Wester): Important features are resistance to salinity and boron. Generally, it has good compatibility with all varieties. However, it is used as rootstock for lemon and lime mainly due to susceptibility to Tristeza and Xyloporosis diseases. It appears to be tolerant of Exocortis and Psorosis diseases. Varieties grafted on it grow rapidly and set early fruit. However, quality of fruit is negatively affected. It is susceptible to cold.
Flying Dragon (Poncirus trifoliata var. Monstrosa): The trifoliate clone Flying Dragon was found in America in 1915. This rootstock ensures tight crown formation for lime, grapefruit and tangelo, and has the effect of dwarfing mandarin and orange. It is very sensitive to calcium and chlorosis. It develops excessively slowly on mild sandy soils. It is resistant to tristeza (CTV) virus and Phytophthora spp. (root neck rot). It is sensitive to Exocortis. The Eureka group showed incompatibility with lemons. Due to showing dwarfing effect on all citrus types and varieties, it is appropriate for dense planting, ensuring convenient harvesting of grafted varieties. It has positive effects on fruit quality, like the trifoliate rootstock. The body having zigzag form and many thorns makes grafting difficult [2]. The Flying Dragon rootstock which is resistant to cold and humid conditions and sensitive to high-lime soils, does not have a tendency to form nucellar plants at high rates [1, 10].
Sunki mandarin (Citrus sunki (Hayata) hort ex. Tanaka): It is very widely used as rootstock in China. It is tolerant of Tristeza and Xyloporosis, but sensitive to Exocortis. Studies have reported Sunki is susceptible to Phytophthora brown rot. This rootstock is tolerant of salt, has moderate resistance to low temperatures, and can withstand chlorosis in limey soils. It is a polyembryonic rootstock. [16] reported adaptation to limey soils was good, and that it was tolerant to iron chlorosis. Fruit yield, fruit juice amounts and sugar content in fruit juice was equivalent or superior to fruit obtained from trees grafted on bitter orange [31].
Yuzu (Citrus junos Sieb. ex Ten.): It is a common rootstock in the southern regions of China. From China, production spread to Japan and it forms an important commercial rootstock in Japan. Proliferation from seeds is easy, with slow growing features. It is a rootstock with high fruit quality and yield. It is resistant to phytophthora, fungus and nematodes. It is tolerant of tristeza, dwarfing and spalling diseases. It has moderate levels of resistance to limey and salty soils. It has high resistance to low temperatures and polyembryony tendency.
Taiwanica (Citrus taiwanica Tan. and Shim.): It is a rootstock that is easily proliferated from seeds, and has moderate levels of tree growth, fruit quality and yield. It is resistant to phytophthora disease, very susceptible to fungal disease and susceptible to nematode damage. It is tolerant of tristeza, dwarfing and spalling diseases. It has moderate levels of resistance to limey soils, with weak resistance of saline soil conditions. It has moderate resistance to low temperatures and is a rootstock with very high tendency for polyembryony.
Yuma Citrange (P. trifoliata × C. sinensis): A trifoliate orange hybrid. It matures in the months of October-November. It is a rootstock susceptible to iron deficiency. It has smaller fruits than Citrumelo. It forms trees of moderate size, with trifoliate leaves, and crown volume in global structure. It has low tendency toward polyembryony, and forms zygotic plants at high rates [12]. It is a very suitable rootstock for grapefruit. In terms of features like susceptibility to disease and nematodes, fruit quality and adaptation to soil types, Yuma citrange is similar to Carrizo and Troyer citranges.
Chinese bitter orange (C. myrtifolia Rafinesque): Chinese bitter orange, susceptible to CTV, matures from January-March. Leaves are small and don’t have pointed tips. Fruit are small and rounded, with variability in seed numbers from low to high, and it forms small tress. The peel of the fruit has moderate roughness, with color ranging from orange to dark orange. It originated in China.
Citremon (Poncirus trifoliata (L.) Raf. X Citrus lemon (L.) Burm): Most of these hybrids show abnormal small leaf features. They die in the germination stage or a short while after. Large leaved plants survive, fruit has many seeds and rough structure, trees show rapid development like lemon.
Determination Of Germination Rates
Seeds were provided from open pollinated mature fruits of Gou-Tou sour orange, C-35 citrange, Troyer citrange, Taiwanica, Citremon, Yuzu, Sunki mandarin, Flying Dragon trifoliate orange, Yuma Citrange, Macrophylla and Chinese orange rootstocks trees in Citrus orchards in Adana – Turkey. 200 seeds from each genotype were sown in growing media containing of vermiculite No:3 in greenhouse. Seedlings were counted 15 days later after seed germination then seedlings with three developed leaves were transferred into the plastic pots containing peat moss. The seedlings grown well without any blemishes were used for cytometry analysis.
Isolation And Staining Of Nuclei
To release cell nuclei, approximately 50 mg of fresh tissue form each seedling leaf was mixed with Tahiti lime (Citrus latifolia Tan.) leaf pieces, which were used as a control, and chopped into small pieces with a sharp razor in a sterile Petri dish containing 300 µl of nuclei buffer (pH7.4) of the following composition: 0.14 M NaCl, 0.003 M KCl, 0.012 M NaH2PO4, 0.002 M KH2PO4, 0.1% Triton 100, 50 µg of RNAse and 100 µl of dithiothreitol. For measurements of absolute DNA values, Tahiti lime leaf tissues were included as an internal standard, as previously described by [19, 20]. Tahiti lime was described as triploid and nuclear DNA content was found to be 1.17 pg/2C by [19, 20]. The suspension was filtered through a 50 µm pore nylon filter into microcentrifuge tubes. After filtration, 100 ll (1 mg/ml) of propidium iodide was added for staining of the DNA. Then the suspensions were incubated for approximately 5 min at room temperature. After incubation, each sample was run on a flow cytometer [33].
For estimation of DNA content of nuclei, the relative fluorescence of nuclei was measured by using a CA-III Flow Cytometer (Partec GmbH, Münster, Germany) with an Argon laser light source operating at a wavelength of 488 nm. Histograms and cytograms were evaluated on DPAC Software (Partec GmbH, Münster, Germany). From 2000 to 5000 nuclei were counted per flow cytometry measurement. The nuclear DNA contents of different seedlings were calculated by comparison of relative positions for G0−1 peaks corresponding to the sample nuclei and the nuclei isolated from Tahiti lime or mungbean, respectively. This permits accurate determination of the unknown DNA content [33].
Data analysis and estimation of nuclear genome size: The nuclear DNA contents of the different rootstock seedlings were calculated by comparison of the relative positions for the G0−1 peaks corresponding to the sample nuclei and the nuclei isolated from Tahiti lime, respectively. This permits accurate determination of the unknown DNA content. Calculation was made according to the formula:
Q = R x(E/S)
where Q = unknown DNA content (pg/2C), R = standard 2C DNA content (1.17 pg), E = sample G0−1 peak mean, and S = standard G0−1 peak mean.
Statistical Analysis
Statistical analyses were carried out with the genome results obtained from each seedling. Significance of genome size variation among the seedlings was determined from analysis of variance by using SAS statistical analysis software. Analysis of variance was computed using the General Linear Model (GLM).