الفهرس 
Seedling selection under salinityOnly 14 pages are availabe for public view
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Abstract
This work was carried out during 2011- 2015 in the laboratories of Cell Res. Dept. (CRD), Field Crops Res. Institute (FCRI), Agric. Res. Center (ARC), Giza, as well as, in the greenhouse of Horticulture Dept, Fac. of Agric., Ain Shams Uni., Egypt, to study the variation among canola varieties in response to salt tolerance through tissue culture propagation. This work was aimed to establish a regeneration protocol for canola varieties, production of salt-tolerance plants through selection of salt-tolerance callus via somaclonal variation and subsequent regeneration of plantlets from such callus, identifying the genetic variations among the used varieties and the newly developed salt tolerant genotypes produced via somaclonal variation using RAPD and ISSR banding patterns.
The important results could be summarized as follow:
1. selection of the highest and lowest salt tolerant canola varieties
Four concentrations of NaCl, i.e. 4000, 8000, 12000 and 16000 ppm as well as the control treatment were used to evaluate the level of salinity tolerance for 22 canola varieties and to choose the most salt-tolerant varieties and most susceptible ones (two genotypes for each group) At the end of the experiment some germination indices correlated to seed vigour were evaluated. Results indicated that germination percentage and germination velocity were significantly affected by all concentrations of NaCl, as well as the germination rate. All studied traits were decreased with increasing salinity concentrations beyond 8000 ppm NaCl, with a sharp DROP at 12000 ppm NaCl concentration and prevention of germination of canola seeds at the concentration of 16000 ppm NaCl.
All canola varieties were affected significantly by the high salt concentrations. Bingo and Torpe varieties markedly surpassed all varieties concerning to germination percentage and germination velocity traits; meanwhile, Conny and Siberian varieties recorded the lowest values.
2. Establishment of canola regeneration protocol
Four canola varieties were selected according to their salinity tolerance: two salt tolerant varieties (Bingo and Torpe) and two salt susceptible ones (Conny and Siberian). Theses varieties were used for establishing a regeneration protocol for canola.
2.1. Callus induction
Cotyledon and hypocotyl explants were cultured on MS-medium supplemented with three concentrations of 2,4 dichlorophenoxy acetic acid ( 1.0, 1.5, 2.0 mg/l 2,4-D, as well as, control) to enhance callus initiation. Results showed that 2.0 mg/l 2,4-D concentration was the best medium for cotyledon explants. Torpe variety recorded the highest frequency (89.00% equal to 1.059) followed by Siberian variety (64.00% equal to 1.028). On the other hand, the lowest callus initiation percentage was (38.33% equal to 0.879) for Conny genotype. At using hypocotyl explants, 2.0 mg/l 2,4-D concentration reflected a high percentage of medium formed callus (49.00% equal to 1.094), by contrast, the callus was more depressed for Torpe variety (4.25% equal to 0.769) compared to other varieties. Siberian variety recorded the highest callus initiation percentage (53.50% equal to 1.040) compared to other varieties. The initiated calli for all varieties reached a fixed size (about 1-3 mm) and then stopped its growth thereafter. Thus the calli were not sufficient for shoot regeneration.
2.2. Shoot regeneration
This experiment was carried out on callus induction to determine the best protocol for canola regeneration including the best hormones combination. The combinations of hormones were: MS medium without any hormone (control), MS + 1.0 mg/l Benzyl adenine (BA) + 1.0 mg/l Kinetin (Kin), MS + 3.0 mg/l BA + 0.5 mg/l Indole acetic acid (IAA), MS + 4.0 mg/l BA + 0.5 mg/l Naphthalene acetic acid (NAA) and MS + 5.0 mg/l BA + 0.05 mg/l NAA. Generally, results cleared that the best regenerated shoot medium was achieved by the supplementation of 5 mg/l BA + 0.05 NAA (41.67 equal to 0.944), while, control medium and MS-medium supplemented with 1 mg/l BA + 1.0 mg/l Kin recorded the lowest values of shoot induction: (11.67% and 25.84% equal to 0.779 and 0.861), respectively. the varieties differed in their response, e.g. Torpe genotype show the highest regeneration ability (42.67% equal to 0.949) followed by Siberian and Bingo varieties and the lowest percentage obtained from Conny variety (16.67% equal to 0.809). The best results of shoot regenerated percentage occurred when calli tissue of Torpe variety were grown on MS-medium supplemented with 5 mg/l BA + 0.05 NAA, where recorded (63.33% equal to 1.054) followed by the same variety with 3 mg/l BA + 0. 5 IAA, were recorded (53.33% equal to 1.000).
2.3. Root formation
Three centimeters in length regenerated shoots were transferred to different rooting media which were contained half strength MS-medium with B5 vitamins, 1.5% (w/v) sucrose, 0.6% (w/v) agar and supplemented with 1.0 mg/l IBA or 0.5 mg/l NAA or hormone free MS medium. The results showed that MS medium supplemented with 1 mg/l IBA was better and faster than the other media concerning to root formation, it gave (85.71% equal to 0.713) root formation percentage. Torpe variety recorded the highest frequency of root induction percentage, while Conny variety recorded the lowest value (61.90% equal to 0.711), respectively.
3. In vitro selection of salt tolerate calli
Calli of four canola varieties were increased in mass through five subcultures and consequently were used to select the most salt tolerant ones. Healthy callus was used for each replicate and transferred to a freshly prepared MS medium supplemented with 2 mg/l 2,4-D and contained five different concentrations of NaCl (0, 4000, 8000, 12000 and 16000 ppm). The callus survival and shoot frequency were measured for each salt concentration after two weeks and six weeks, respectively. The results indicated that the reduction below the control treatment in callus survival percentage varied according to the concentration of NaCl with the maximum reduction when 16000 ppm NaCl was applied. The highest reduction was noticed in Conny variety: 53.33% equal to 0.922, while, Torpe variety recorded the highest value: 80.74% equal to 1.193. Torpe variety overcome the others in calli survival percentages under 8000, 12000 and 16000 ppm NaCl concentrations, where it recorded (88.89, 59.26 and 55.56% equal to 1.173, 1.070 and 1.013, respectively). Under the highest concentration of NaCl (16000 ppm) there was no survived calli produced from Bingo and Conny varieties, while, callus of Siberian variety hold out under this concentration despite that it was within the salt susceptible group (22.22% equal to 0.843).
The results, of shoot frequency initiated from tolerant calli, cleared that when salt concentration was increased up to 12000 and 16000 ppm NaCl, the shoot percentage was decreased up to 22.22 and 5.56% equal to 0.838 and 0,743, respectively. Torpe variety recorded the highest value of shoot percentage followed by Bingo variety, it recorded 48.89% equal to 0.983 and 26.66% equal to 0.889, respectively. Whereas, Conny followed by Siberian varieties were recorded the lowest values 11.11% equal to 0.862 and 25.00% equal to 0.889, respectively. At 16000 ppm NaCl concentration only tolerant calli of Torpe variety was able to initiat shoot under this concentration up to 22.22% equal to 0.843.
4. Plant acclimatization
Plantlets that showed a well-developed root system (through four to five weeks) and became ready to be acclimatized were transferred to sterilized mixture of beat moss and vermiculite (1:1) in 25 cm plastic pots after washed them under running tap water under green house conditions. Only five salt tolerant plantlets developed from Torpe variety and eight salt tolerant plantlets developed from Siberian variety succeeded to complete their life cycle and reached maturity stage and produced seeds. These regenerated genotypes were referred as T1R0, T2R0, T3R0, T4R0, T5R0 for the five regenerated plants developed from Torpe variety that tolerate NaCl concentration up to 16000 ppm, as well as, S1R0, S2R0, S3R0, S4R0, S5R0, S6R0, S7R0, S8R0 for the eight regenerated plants developed from Siberian variety that tolerate NaCl concentration up to 12000 ppm.
5. Molecular analysis
5.1. RAPD analyses
Five primers out of twenty RAPD primers produced scorable banding patterns for the four canola varieties, as well as, the newly salt tolerant genotypes developed from Torpe and Siberian varieties, which were referred as T1R1, T2R1, T3R1, T4R1, T5R1 for the five regenerated plants developed from Torpe variety that tolerate NaCl concentration up to 16000 ppm, as well as, S1R1, S2R1, S3R1, S4R1, S5R1, S6R1, S7R1, S8R1 for the eight regenerated plants developed from Siberian that tolerate NaCl concentration up to 12000 ppm.
The highest similarity value between the four canola varieties (0.630) were recorded between Bingo and Torpe canola varieties. However the lowest similarity value was observed between Bingo and S8R1 it was 0.280. On the other hand, concerning to the newly developed canola regenerates, the genetic distance between the salt tolerant regenerates recorded 0.820, 0.822, 0.817 between T2R1 and T4R1, T3R1 and T5R1, T4R1 and T5R1, respectively. While the similarity values between Siberian canola variety and S1R1, S2R1 and S3R1 recorded 0.833 and 0.837.
A dendrogram for the genetic relationship among the four canola variety and the thirteen newly developed regenerates as they were separated into two major groups. The first group included Bingo variety the most susceptible variety, while the second group was divided into two subgroups, The first subgroup included the newly developed canola genotype developed through somaclonal variation on Nacl concentration of 12000ppm (S4R1), and the other subgroup included two mains sub-subgroups, one of them gathered the Siberian canola varieties and almost all its newly developed regenerates, however the other sub-subgroups contained Torpe variety and almost all its newly developed regenerates.
The clustering results and the genetic variation within and between the studied genotypes and the degree of gene differentiation suggest a relatively high genetic diversity that can be used in canola breeding programs. Considering the diversity of the genotypes, the cultivars with a wide genetic distance may be used as parents to crosses for taking advantage of heterosis and for making mapping populations in the QTL mapping studies. In this study, the RAPD molecular data provided a lot of information.
5.2. ISSR analyses
Five primers out of fifteen ISSR primers produced scorable banding patterns. ISSR data were used to estimate the genetic similarity values among the four canola variety and the thirteen newly developed regenerates by using UPGMA computer analysis.
For ISSR represented markers with the highest detectable extent of genetic variation; the lowest genetic distances (0.649) were determined between S3R1 and T4R1, the first is developed from Siberian and tolerate salt up to 12000ppm, while the second one is developed from Torpe and tolerate NaCl concentration up to 16000 ppm. On the other hand, the highest distance (0.933) was detected between T4R1 and T2R1, both were developed from Torpe variety through tissue culture via somaclonal variation and tolerate salt concentration up to 16000 ppm.
The highest similarity values between the four canola varieties (0.842) were recorded between Bingo and Torpe canola varieties, followed by 0.779, 0.753, 0.744 and 0.734 that observed between Torpe and Siberian, Bingo and Siberian, Conny and both of Bingo and Torpe and Conny and Siberian canola varieties, respectively.
A dendrogram for the genetic relationship among the four canola variety and the thirteen newly developed regenerates based on ISSR analyses was performed. They were separated into two major groups that were separated in many subclusters. One of these subclusters was gathered the all newly developed regenerates from Torpe variety, which tolerate the salt concentration of NaCl up to 16000 ppm. On the other hand, the second group was divided into many subgroups, one of them included the newly developed canola genotype developed through
Nahid A. A. Morsi,Ph.D.,2015
somaclonal variation on Nacl concentration of 12000 ppm (S5R1, S6R1, S7R1 and S8R1, while another subcluster gathered Sibrian canola variety and some of the newly regenerates developed from him S1R1, S2R1 and S3R1, which tolerate the NaCl concentration up to 12000 ppm.