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Abstract SUMMARY This study was carried out in plant tissue culture laboratories, Department of plant biotechnology, Genetic Engineering and Biotechnology Research Institute (GEBRI), Sadat City University and South El-Tahrir research station, Horticulture Research Institute, Agriculture Research Center, Ministry of Agriculture, Egypt, during the period from 2008 to 2013. This work aimed to: I. Production of in vitro scions of Balady mandarin by use different explants (shoot tips, nucellar embryos and cotyledon). II. Genetic examinations of germinated embryos arise on Balady mandarin seeds to determined nucellar embryos and sexual embryo by using PCR methods. III. Factors affecting grafting of in vitro produced Balady mandarin scions on in vivo grown volkamarina rootstocks in greenhouse. 1- Micropropagation of Balady mandarin. a. Explants sterilization Experiment (1): Effect of sodium hypochlorite (NaOCl) concentrations on contamination and survival percentage of shoot tips and seeds of Balady mandarin. Shoots tips and seeds of Balady mandarin explants were sterilized in the laminar air flow chamber using different concentrations of NaOCl (1.5, 2.5, 3.5 and 4.5% v/v) for 15 minutes and 70% ethanol for 5 seconds. Results cleared that: - Shoot tips and seeds which were sterilized with NaOCl (3.5 and 4.5%) and 70% ethanol for 5 seconds, recorded the highest number of sterilized explants (70% and 80% for shoots tips, 72% and 84% for seeds, respectively). -The high concentration of NaOCl (4.5%) the low shoots and seeds survival percent (24 and 36%, respectively). the lowest concentration of NaOCl (1.5 and 2.5%) possessed the highest survival percent for both shoots (100 and 90%, respectively) and seeds (100 and 96%, respectively). …………………………………………………………SUMMARY 81 Experiment (2): Effect of sterilization methods and period after cultured on seeds responses of Balady mandarin in vitro. - Seeds were sterilized by 3.5% NaOCl for 15 min and 70% ethanol for 5 second. The fruit group was burned in the laminar air flow and opened with scalpel and seeds were collected then all seeds were cultured in jars contained 50ml MS medium the following results were observed: - Sterilization methods and period after cultivation revealed that flamed method in all period after cultivation were significantly maximized number of sterilized seeds. - Sterilization methods and period after cultivation revealed that flamed sterilization method after 93 days of cultivation possessed the highest percent germinated seeds (90%). - The significantly highest number of germinated embryos / seed (4.17 embryos/seed) was possessed by flamed sterilization method after 103 days. b. Multiplication stage I. Shoot formation of Balaby mandarin derived from shoot tips (as initial explants) in vitro. Experiment (3): Effect of BAP and NAA concentrations on shoot formation of Balady mandarin derived from shoot tips (as initial explants) in vitro. - Sterilized shoots of Balady mandarin were planted on culture jars (210ml) contained 50 ml MS medium supplemented with different concentrations of BAP (0.0, 0.25, 0.50 and 1.00 mg/l BAP) or in combination with different concentrations of NAA (0.0, 0.50, 0.75and 1.00 mg/l NAA). - Interaction between BAP and NAA concentrations cleared significantly high shoot number (2.4 shoots/explants) at concentration 1.00 mg/l BAP and 0.00 mg/l NAA. Control gave the highest shoot length (1.60cm) followed by 0.50mg/l BAP individual (1.40 cm) and 0.25mg/l NAA individual (1.40 cm) with no significant differences between them. …………………………………………………………SUMMARY 82 II.Shoots derived from nucellar embryos germinated from seeds (as initial explants) in vitro. DNA finger print of germinated embryos of Balady mandarin seeds Balady mandarin seeds were germinated in vitro. Germinated embryos were arranged from the tallest to the shortest and labeled from 1st, 2nd, 3rd, 4th to 5th according to its tall. Determination of sexual embryo of Balady mandarin - Nine primers (OP-A01, OP-A12, OP-A17, OP-A18, OP-B12, OP-N16, OP-S147, OP-S238 and OP-S253) which were used for DNA amplification. DNA (RAPD) primers gave 395 amplified fragments (85 comparison rows) that ranged between 1650 and 150bp, including 56 total polymorphic fragments (PF), 67 total common fragments (TCF) and 18 total specific fragments (TSF). Four fragments were unique, appeared in the RAPD pattern of 5th embryo and did not appear in another embryos, also, there were 14 negative amplified fragments which appeared in the tallest four embryos and disappear in the 5th one Phylogenetic relationships among five samples of Balady mandarin embryos based on RAPD. The closest relationship was scored between 1st, 2nd, 3rd and 4th embryos nearly 100%. While, similarity between the four embryos and the 5th embryo was 84%. These result cleared that the 5th embryo is the sexual embryo in balady mandarin Experiment (4): Effect of BAP and NAA on shoots multiplication of nucellar embryos of Balady mandarin after 1st and 2nd subcultures. Shoot tips explants derived from nucellar embryos were planted on jars (210ml) contained 50 ml MS medium supplemented with different concentrations of BAP (0.0, 0.25, 0.50 and 1.00 mg/l BAP) or in combination with different concentrations of NAA (0.0, 0.50, 0.75and 1.00 mg/l NAA) After 1st subculture - The highest level of shoot number on MS medium containing 0.75 mg/l BAP in combination with 0.00 mg/l NAA or 1.00 mg/l BAP in combination with 0.50 mg/l …………………………………………………………SUMMARY 83 NAA (2.00 and 2.00 shoots/explant, respectively) with no significant differences between them. -The tallest shoots on MS medium supplemented with 0.00 BAP in combination with 0.25 or 0.50 mg/l NAA and MS medium supplemented with 1.00 mg/l BAP in combination with 1.00 mg/l NAA (2.00 cm for each treatment). After 2nd subculture - The highest value of shoots number/explant on MS medium supplemented with 0.75 mg/l BAP in combination with 0.00 mg/l NAA (8.80 shoots/explant). -Interaction between BAP and NAA concentrations revealed that basal MS nutrient medium resulted in the highest shoot length (4.60 cm) compared with all combination of BAP and NAA. -Effect of BAP and explants type on shoots multiplication of Balady mandarin after two subculture. -Shoot multiplication of explants derived from ncellar embryos was superior at the most BAP concentrations (0.50, 0.75 and 1.00 mg/l) (3.4, 8.8, and 5.6, respectively). Experiment (5): Effect of cytokinins types and concentrations on growth parameters of shoot tips derived from nucellar embryos of Balady mandarin after 1st and 2nd subculture. -Shoot tips explants derived from nucellar embryos were planted on jars (210ml) contained 50 ml MS medium supplemented with different cytokinins (BAP, Kin, AS) different concentrations (0.0, 0.25, 0.50 and 1.00 mg/l BAP). After 1st subculture - The highest shoots number was observed on MS medium supplemented with 0.75 mg/l BAP followed by 1.00 mg/l BAP and 1.00 mg/l AS (4.80, 2.80 and 1.80 shoots/ explant). While, the lowest shoots number was observed on free cytokinin MS medium (1.00 shoot/explant), MS medium supplemented with 0.50 and 0.75 mg/l kin gave the highest shoot length (2.40 cm) followed by free MS medium (2.00 cm). - The medium containing 1.00 mg/l BAP gave the lowest shoot length (1.00 cm). After 2nd subculture …………………………………………………………SUMMARY 84 - The highest significant shoot number/explant through second subculture resulted from the explant cultured on MS medium supplemented with 0.75 and 1.00 mg/l BAP (8.80 and 5.60, shoots/explant respectively), Interaction between cytokinin types and concentrations conducted that 0.50 and 0.75 mg/l kin and 0.50 mg/l AS resulted in the highest shoot length (5.40, 5.00 and 4.40cm, respectively) with no significant differences between them. Experiment (6): Effect of different seeds age and period after culture on somatic embryogenesis from cotyledon of Balady mandarin. Balady mandarin fruits were harvested in different dates (1 September, 1 November and 1 January) cotyledons were separated as after swelling in MS medium. Then, it were cultured on MS medium on jars (210 ml containing 50 ml medium). - After 120 days of culture, the cotyledons separated from fruits which were harvested in 1st November resulted in maximum number of differentiated somatic embryos (6.0 differentiated somatic embryos /cotyledon) while, the seeds separated from fruits which were harvested in 1st january minimized number of differentiated somatic embryos (3.4 differentiated somatic embryos/cotyledon). 2- Micrografting of Balady mandarin in vitro produced scions on in vivo volkamarina rootstock growing in greenhouse. Experiment (7): Effect of different cytokinin types and concentrations on micrografting success of Balady mandarin scions derived from tissue culture grafted on volkamariana rootstocks grown in greenhouse after 15, 30 and 45 days. Microshoots of Balady mandarin were cultured on MS media supplemented with various cytokinin types (BAP, Kin and AS) at different concentrations (0.0, 0.50, 0.75 and 1mg/l) for one month before used as scions in greenhouse. - After 30 days from culture in vitro these shoots have been grafted on volkamariana rootstock 0.3cm in diameter in greenhouse by pen tip grafting method. After 15 days …………………………………………………………SUMMARY 85 of grafting, scions derived from MS medium supplemented with adenine sulphate (1.0 mg /l) gave the highest percentage of successful grafts (33%). - After 30 days of grafting, scions derived from MS medium supplemented with 0.75mg/l AS maximized percentage of successful grafts (40%). After 45 days of grafting, scions derived from MS medium supplemented with 1.0 mg/l Kin maximized successful grafts percentage (37 %) compared with control (30%). The highest value of both scion length and leaves number / scion were resulted from scions derived from MS medium supplemented with 1.0 mg/l BAP (8.3cm and 6.3 leaves /scion, respectively). Experiment (8): Effect of different auxin types and concentrations on micrografting success of Balady mandarin scions derived from tissue culture on volkamariana rootstocks grown in greenhouse after 15, 30 and 45 days Produced shoots from multiplication stage are cultured on MS media supplemented with various auxin types (NAA, IAA and IBA) at different concentrations (0.0, 0.5, 1.0 and 2.0 mg/l) for one month before used as scions in greenhouse. - After 30 days from culture in vitro these shoots have been grafted on volkamariana rootstock diameter 0.3c min greenhouse by pen tip grafting method. After 15 days of grafting, Balady mandarin scions derived from MS medium supplemented with 0.50 mg/l NAA maximized successful grafts percentage (57%) compared with control (22%). - After 30days of grafting Balady mandarin scions derived from MS medium supplemented with 0.5 mg/l IAA or 0.5 mg/l NAA gave the highest percentage of successful grafts compared with control (57, 57and 30%). - Balady mandarin scions derived from MS supplemented with 0.5 mg/l NAA significantly maximized leaves number (6.0 leaves/graft). Also, Balady mandarin scions derived from MS supplemented with 2.0 mg/l NAA concentrations gave the highest scions length (5.66 cm) in greenhouse. …………………………………………………………SUMMARY 86 - After 45 days of grafting, the highest value of both scion length and shoots number/scion were resulted from scions derived from MS medium supplemented with 0.5 mg/l IBA (10.00 cm and 3.0 shoots/scion, respectively). Also, scions derived from MS medium supplemented with 1.0 and 2.0 mg/l IBA resulted in significant maximized leaves number (15.00 and 15.00 leaves/scion). Experiment (9): Effect of different concentrations of paclobutrazol (PP333) on micrografting success of scions of Balady mandarin derived from tissue culture on volkamariana rootstocks grown in greenhouse after 15, 30 and 45 days, of grafting. Produced shoots from multiplication stage are cultured on MS medium supplemented with paclobutrazol (PP333) different concentrations (0.2, 0.4, and 0.8 mg/l PP333) for 30 days (in vitro). - After 30 days from culture in vitro, this shoots have been grafted on rootstock diameters 0.3 in greenhouse by pen tip grafting method. - After 15 days of grafting, no response was observed among all treatments of PP333. On the other hand; scions derived from control (MS without PP333) resulted in two successful grafts with 22% successful grafts%. After 30 days the highest successful grafts percentage (88%) was obtained from 0.60 mg/l PP333 compared with control (27%). - After 45 days of grafting, scions derived from MS supplemented with 0.6 mg/l PP333 gave the highest percentage of successful grafts (88%) compared with control (27% successful grafts). - The scions derived from MS supplemented with 0.0, 0.2, 0.4, and 0.6 mg/l PP333 significantly maximized leaves number (5.0, 5.0, 4.5 and 4.3 leaves/graft, respectively) and graft length (5.1, 5.5, 4.5 and 4.7 cm, respectively). - The high concentration of PP333 (0.8, 1.0 and 1.2 mg/l) gave the lowest level of leaves number and scions length. …………………………………………………………SUMMARY 87 Experiment (10): Effect of in vitro PP333 concentrations and in vivo volkamariana rootstock diameter on success of pen tip and side pen grafting method of Balady mandarin scions derived from tissue culture in greenhouse. Volkamariana seeds were cultured in a greenhouse in plastic bags three months before grafting. Produced micro shoots of Balady mandarin from multiplication stage were cultured on MS medium supplemented with paclobutrazol (PP333) at different concentrations (0.0, 0.2, 0.4, and 0.8 mg/l PP333) for 30 days (in vitro). After 30 days from culture in vitro, these shoots have been grafted on different diameters volkamariana rootstock (0.3, 0.4 and 0.5 cm) in greenhouse by pen tip grafting and Sid pen grafting method. 1) Tip pen grafting method The highest number of successful grafts were obtained when diameter of rootstock 0.3 cm in combination with 0.8 mg/l PP333 (7.7 successful grafts) successful grafts percentage (77%). 2) Side pen grafting method The highest successful grafts number and percentage were obtained when diameters of in vivo volkamariana rootstocks were 0.3 or 0.4 cm in combination with 0.8 mg/l PP333 (6.0 and 60%, respectively). 3) Comparison between grafting methods Comparison between the effect of different concentrations of PP333 on grafting successful % of different grafting methods (tip pen method and side pen method) is showed that tip pen method was superior on side pen method in all PP333 concentrations. |