الفهرس 
IntroductionOnly 14 pages are availabe for public view
 |  | from | 126 |  |  |
| | | from | 126 | | |
Abstract
In the present work, the effects of genotype, explants, type and media content from growth regulators on callus induction were studied. Two types of explants (hypocotyls and cotyledon leaves of seedlings 14 days old of seven genotypes (Peto-86, Castle Rock, Super Strain B, Heins 2710, Amcostar, Super Marmand and 12-M) were cultured on MS medium supplemented with different combinations and concentrations of growth regulators.
1. Callus induction:-
The results revealed that there are significant differences between genotypes for their ability for callus formation on the two types of explants on the different types of media. The highest genotype for callus formation was Super Marmand (77.10%) and the lowest genotype for callus formation was 12 M (56.3 %). The highest percentage of calli induction (79.2%) was produced from hypocotyls explants of Super Marmand and the lowest percentage (49%) was obtained from hypocotyls explants of 12-M genotype. The results of this work revealed highly significant differences between the mean of callus formation from the two types of explants of the different genotypes. M2 medium (83%) could be recommended as appropriate medium for callus induction for all genotypes. Concerning the type of explants the results showed that cotyledon leaf explants (70.39%) were better than hypocotyls explants (66.67%) for callus formation.
The mean number of the obtained calli on M1 and M2 medium (6.5 and, 6.6, respectively) was higher than that of M3 and M4 medium (5.6 and 3.1, respectively).
2. Plant regeneration: -
2.a-Indirect regeneration from calli:
In the present experiment the mean numbers of regenerated shoots showed highly significant differences between genotypes. Amcostar exhibited large number of shoots under all factors studied as compared to other genotypes. The mean numbers of shoots developed from Amcostar on all media (6.36 was higher than that developed from other genotypes Peto-86 (4.95), Castle-Rock (3.67), Super Strain B (4.50), Heins 2710 (2.34), Super Marmand (2.74) and 12-M (1.92) respectively. MS medium containing 2 mg/L BAP + 0.6 mg/L IAA (M4) as well as 2 mg/L BAP and 0.2 mg/L NAA (M2) were the best medium for shoot regeneration frequency 42.88% and 34.84% respectively.
The effect of genotypes and media interaction on plant regeneration from callus was highly significant where the highest percentages of regeneration were achieved from Amcostar and Peto-86 genotypes (53.13% and 35.94%, respectively), with mean numbers of shoots 6.36 and 4.95, respectively while the lowest regeneration percentage was achieved from 12M and Heins genotypes (7.81% and 9.38%, respectively), with a mean 1.92 and 2.34, respectively. The best combination for indirect regeneration was Amcostar genotype on M4 medium (93.8% with a mean 9.7) while all genotypes showed no response on M1 medium
2.b- Direct plant regeneration: -
The highest shoot regeneration value (70.83) was achieved from Super Strain-B, and the lowest one was (10.42 %) from Heins 2710 genotype. Regarding the medium type the R1 medium was the best one for all genotypes and the shoot regeneration percentages was 66.96 while the R2 medium was the lowest one for shoot regeneration. The regeneration percentage was (24.11 %). There are no shoots regenerated from Heins 2710 on R2 medium. The genotypes Peto- 86, Super Strain B, Castle Rock, and Amcostar able to regenerate shoot on all the medium types by successive percentages. The best combination was Super Strain B, Amcoster, Castle Rock, and Peto –86 on R1 medium (100, 93.75, 93.75, 93.75) respectively.
3. Callus growth and Plant regeneration under stress: -
3.a - Callus growth dynamic:
The growth dynamic of callus tissue was evaluated during the growing period of 30 days on the basic medium MS supplemented with different concentration of PEG. The fresh weights of callus at the end of 30 days growth period for five genotypes Peto-86, Castle Rock, Super Strain-B, Amcostar and Super Marmand were 2.44, 2.63, 1.64, 1.75 and 1.48 g / culture, respectively. The concentration of PEG in the medium have a great effect on callus fresh weight and growth value, the fresh weights of callus after 30 day of growth of the five genotypes on the PEG concentration (0.0, 25.0, 50.0, 75.0, and 100.0 g / Liter were 2.65, 2.43, 1.99, 1.63, and 1.20 gram / culture respectively. The concentrations of PEG in the medium showed a great effect on the growth value at the end of 30 days growing period. The growth values for callus growing on MS medium relative to the control were 52.26, 32.20, 53.04, 37.72 and 63.84 % for the Peto-86, Castle Rock, Super Strain-B, Amcostar and Super Marmand, respectively
3.b- Callus growth and mass of shoots:
The mass of cullus and shoots regenerated directly from explants were evaluated after 60 days of growing on regeneration MS medium supplemented with different concentration of PEG. The fresh weight of the explants and its callus and shoot formed after 60 days was measured in gram. The means of callus and shoots of different genotypes of tomato after 60 day growing period were 3.481, 5.138, 5.834, 3.806, and 4.510 gram / explants for the 0.00, 25.00, 50.00, 75.00, and 100.00 gram / Liter. The highest dry weight was achieved when the Peto-86 explants were cultivated on MS supplemented with 75.00 gram PEG. The means of dry weight of callus for the different genotypes ranged between 34.1 to 44.35 mg / culture. These means were 42.8, 36.7, 44.35, 39.3, and 34.1 mg / culture for Peto-86, Castle Rock, Super Strain-B, Amcostar, and Super Marmand, respectively, while the means of dry weight for different concentration 00.0, 25.00, 50.00, 75.00, and 100.00 gram/liter PEG were 1, 38.65, 45.05, 38.05, and 46.4 mg / culture
3.c- Plant regeneration under PEG treatments:-
The averages of shoots regenerated from cotyledon segment explants of different genotypes ( Direct regeneration were ( 8.00, 25.00, 43.00, 51.00 and 11.00 % ) for Peto 86 , Castle Rock , Super Strain-B , Amcostar and Super Marmand , respectively .In the same time this averages for shoots regenerated from callus culture (indirect regeneration) were (64.0, 82.0, 91.0, 95.0 and 64.0 %) for Peto- 86 , Castle Rock , Super Strain , Amcostar and Super Marmand genotypes , respectively .
3.d- Plant regeneration under mannitol treatments:-
Shoots regeneration frequency of cotyledon segment of tomato genotype ranged from 12.00 to 82.40 %. The mean numbers of regenerated shoot per explants were 0.99, 1.53, 2.14, 2.06 and 0.952 for Peto 86, Castle Rock, Super Strain-B, Amcostar and Super Marmand genotype, respectively. The highest shoot regeneration was achieved on the explants of Super Strain B it was 2.14 shoot per explants. The mean number of shoots per explants was decreased by increasing the concentration of mannitol in the medium the mean numbers of shoots per explants were 1.811, 1.59, 1.50, 1.34 and 1.43 for the Mannitol concentration 0.0, 25.0, 50.0, 75.0 and 100.0 mM/L, respectively. The regeneration frequencies were 59.2, 47.2, 41.6, 19.2 and 38.4 % respectively for control 0.0, 25.0, 50.0, 75.0 and 100.0 mM/L. The regeneration capability it’s self affected strongly by the increasing of mannitol in the medium.
4- Evaluation of R1 somaclones:
4 -1- Hydroponics experiment:
For evaluation the parents and the regenerated somaclones plants under drought stress the plants were cultivated in a hydroponics experiment. Plants of 11 tomato ( Lycopersicon esculentum Mill. ) somaclones were derived from 3 tomato genotypes; seeds of 7 somaclones (As-1, As-2, As-3, As-4, As-5, As-6 and As-7) were derived from Amcostar (As), 3 somaclones (SS-1,SS-2 and SS-3) were derived from Super Strain B (SS) and one somaclone (SM-1) was derived Super Marmand (SM) were germinated in medium of vermiculite peatmos (3:1) v/v) in seedling tray. Fourteen days after sowing, seedlings with tray were transferred to half strength Hoagland nutrient solution in basin, which was continuously aerated. Distilled water was added regularly to replace the water lost by transpiration. After three additional weeks the plants were divided into two groups and subjected for 4 weeks growth under to the following treatments:
(1) Lowering the osmotic potential of the nutrient solution with two consecutive additions of 5% polyethylene glycol (PEG) 6000 to a final concentration of 10% (w/v) , (equivalent to –0.112 Mpa , Money 1989) (2) Standard nutrient solution (control).
were germinated in medium of vermiculite peatmos (3:1) v/v) in seedling tray. Fourteen days after sowing, seedlings with tray were transferred to half strength Hoagland nutrient solution in basin, which was continuously aerated. Distilled water was added regularly to replace the water lost by transpiration. After three additional weeks the plants were divided into two groups and subjected for 4 weeks growth under to the following treatments:
(1) Lowering the osmotic potential of the nutrient solution with two consecutive additions of 5% polyethylene glycol (PEG) 6000 to a final concentration of 10% (w/v) , (equivalent to –0.112 Mpa , Money 1989) (2) Standard nutrient solution (control).
4.1.1. Fresh weight:
Fresh weight showed that all somaclones were heavier in their fresh weight than its parent except As-5, SS-1, SS-2, SS-3, and SM-1 under control conditions. On the other hand, with growing these parents and somaclones under either salinity or drought stress their behavior was completely differed. Whereas, under drought stress (using PEG); the fresh weight of all genotypes (parent + somaclone) was sharply decreased compared to control plants. In meantime, the results show that all somaclones which produced from Amcostar parent were more affected with drought stress than their parent. While, some somaclones of other parent were more tolerant for drought stress and had heavier fresh weight compared with its parent i.e., AS-5, SS-1, SS-2, and SS-3 produced from Super Strain-B and SM-1 produced from Super Marmand. The fresh weight ranged from 5.32 to 28.53 gm with mean 14.85 at control treatment. Meanwhile PEG treatment produced 1.29 to 7.07 gm by mean 3.68.
4-1-2 Dry weight:
The dry weight of all genotypes (parent + somaclones) was sharply decreased compared to control plants. The results showed that all somaclones which produced from Amcostar parent were more affected with drought stress than their parent, while, some somaclone of other parent were more tolerant for drought stress and had heavier dry weight compared with its parent, AS-5, SS-1, SS-2, SS-3, and SM-1. The dry weight ranged from 0.47 to 2.82 gm by mean 1.457 at control treatment, while PEG treatment gave 0.26 to 0.89 gm by mean 0.513.
4.1.3. Sodium and potassium content:
The mean of sodium ion was increased by drought stress and it was ranged from 1.156 to 2.027 with average 1.684 while the mean of potassium ion ranged from 3.144 to 5.116 with average 3.969.
4.1.4. Proline content:-
Proline accumulation in dry matter of different plant organs i.e. young leaves, old leaves, stem and roots were significantly differed from one organ to another. The data showed that the proline content in the young leaves was higher than all other tested organs either under control or stress treatments. It means that the young leaves will be the first organ which affected with either drought or salinity stress by mean 10.97 compared with 0.7 in control. The somaclones AS-2, AS-4, AS-5, AS-6 and AS-7 were higher in response for drought stress than the parent AS.
4..2. Field evaluation:
Seeds obtained from R0 regenerated plants were tested in a field experiment where the tomato (Lycopersicon esculentum Mill.) cv. Amcostar (AS) and their 6 somaclones (AS-1, AS-2, AS-5, AS-6, AS-7 and AS-8) were used.
4.2.1. Stem length:
The differences of stem length were high significant between treatments and genotypes where the stem length was decreased by drought treatments (.67 at control 57.95 at regime-1 and 49.91 at regime –2) where the average length of the parent was 65.11. The general mean values of length of (AS-1, AS-2, AS-5, AS-6, AS-7 and AS-8) were (51.67, 54.00, 50.00 59.00, 58.33 and 58.67).
4-2-2- Leaf area:
The leaf area was differed between the parent and some somaclones, (AS-1, AS-2, AS-5, AS-6, and AS-8) where the means were (85.33, 82.67, 88.56, 95.67,and 82.44 respectively).
4-2-3-Number of branches:
The number of branches was not significant among genotypes.
4-2-4- Early flowering and fruit set:
The early flowering process was measured at three dates 16th, 30th October and 6th November, results show that the As-5 and As-8 were the earliest somaclones in their flowering process compared to other somaclones or donor parent. On the other hand, these plants under regime 2 treatment were the earliest in flowering in comparison to other drought treatments. These results may be as a normal responsibility to the drought process or reducing the quantity of water to induce plant toward early flowering and fruiting. All somaclones were earlier in fruit setting compared with the donor parent in all three times except the third date (6-11-2004), the somaclone As-1 was the lowest one in this respect. The treatment of regime 2 was the highest in early fruit setting at all dates. The somaclones AS –2 and AS –5 had the highest percentage of early fruit setting at all the three dates. The somaclone AS-2 in the treatment of regime 2 was the earliest in fruit setting.
4-2-5- Pigment content:
All genotypes were differed from each other in their contents of both chlorophyll a and b. The highest values of this trait were obtained with both As –2 and As – 7 genotypes. The plants under the second regime treatment showed the highest values of chlorophyll a and b. Also, data show that all somaclones were less than the donor parent in their carotenoids content. Also we can observe that, the carotenoids content decreased by reducing of water.
4-2-6-Number and weight of early fruits:
The results revealed that the number of early fruits / plant after one month from the blooming of first flower was increased under the 2nd regime of irrigation (2.16) while it was (1.73) at 1st regime and 1.27 for control plant. The weight of early fruits was 194.4,154.8 and 114.7 gram / plant for 2nd, 1st and control plants , respectively.
4-2-7- Total number and weight of fruits:
The water deficit reduced the mean number of total fruit yield and subsequently the weight of the total yield for all genotypes. On other hand the water deficit increased the number of early fruit/plant and the weight of early fruit in general. Three somaclones (AS-8, AS-7 and AS-6) were superior than other genotypes even donor parent, these somaclones were produced higher weight of total yield/plant.
4-3-3- The cytological study:
The examination of the behavior of meiotic chromosomes of parental plants and their six R1 somaclones (AS-1, AS-2, AS-5, AS-6, AS-7 and AS-8) may allow detecting the cytological stability. The results revealed that the percentages of abnormal cells ranged from 3.5 to 8.3 %. By examination of 500 cells from the parent, the abnormal cells percentage was 3.5 % while these percentages were ranged from 5.5 to 8.3 % in the somaclones cells. The highest percentages of univalancy 79.6 % from the total abnormal cells were observed in plants of somaclone (As-2) and the lowest percentage 69.2 % in (As-5) was observed. Multivalent ranged from 12.4 to 4.6 % in somaclone As-8, As-7, respectively. The highest percentage of lagging in somaclone As-7 was 17.7 % and the lowest percentage of parental plants 6.1 %. The Bridges percentage was 1.7 % in As and zero in As-2, As-5, As-6, and As-8. Data showed that, the highest percentage of stickiness 4.2 % was observed in somaclone As-6 whereas somaclone As-7 exhibited the lowest percentage 1 % .
In conclusion, this study gives more light on the regeneration system for different tomato genotypes and cultivars grown in middle Egypt region from (Peto-86 , Castle Rock , Super Strain-B , Heins 2710 , Amcostar , Super Marmand and 12M) . All genotypes were able to form calli from different explant types (Cotyledon leaf and hypocotyls). M2 medium (MS medium + 2 mg/LBAP + 0.2 mg/l NAA) could be recommended as appreciate medium for callus induction for all tested tomato genotypes. Also the result showed that Cotyledon leaf explants were better than hypocotyls explants for callus formation. Shoots could be regenerated from callus of hypocotyls and cotyledon on M2, M3 and M4 medium.
The best genotype for ability plant tissue technique is Amcostar on M4 medium. Also the results of this work clarified that the in vitro selection for water stress could be applied using mannitol or PEG. Shoots could be regenerated from all genotypes in the presence of mannitol or PEG in the medium. The obtained results showed that regeneration frequency was depended on the genotype and PEG or mannitol concentration in the regeneration medium. The evaluation of hydroponics experiment revealed that the highest somaclone for proline accumulation was AS-2, AS-3, and AS-7 somaclone. The field experiment evaluation indicated that AS-2, AS-6, and AS-8 were the best somaclones for total yield characters under water deficit. These somaclones could be used for further studies to produce a high tolerant tomato genotypes for drought.