الفهرس 
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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).
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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.
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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
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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
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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
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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.
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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.
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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.