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Contents
PART I
GERMPLASM IN VITRO CONSERVATION
1 Storage and Viability Assessment of Date Palm Pollen . . . . . . . . . . . . . . . . . . . . . . .
3
2 In Vitro Conservation of Date Palm Tissue Cultures . . . . . . . . . . . . . . . . . . . . . . . .
15
3 Cryopreservation of Date Palm Pro-Embryonic Masses
Using the D Cryo-plate Technique. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25
4 In Vitro Cryopreservation of Date Palm Caulogenic Meristems. . . . . . . . . . . . . . .
39
5 In Vitro Conservation of Date Palm Shoot-Tip Explants
and Callus Cultures Under Minimal Growth Conditions. . . . . . . . . . . . . . . . . . . . .
49
6 In Vitro Conservation of Date Palm Somatic Embryos
Using Growth-Retardant Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
61
7 Encapsulation of Date Palm Somatic Embryos: Synthetic Seeds. . . . . . . . . . . . . . .
71
PART II
MOLECULAR ANALYSIS OF IN VITRO CULTURES
8 Evaluation of Clonal Fidelity of Micropropagated Date Palm
by Random Amplified Polymorphic DNA (RAPD). . . . . . . . . . . . . . . . . . . . . . . . . .
81
9 Molecular Identification of Fungal Contamination
in Date Palm Tissue Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
91
PART III
GENETIC DIVERSITY AND CULTIVAR IDENTITY
10 Genetic Diversity Analysis of Date Palm Using Random
Amplified Polymorphic DNA (RAPD) and Inter-Simple
Sequence Repeat (ISSR) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 105
vii
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viii
Contents
11 Date Palm Genetic Diversity Analysis Using
Microsatellite Polymorphism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113
12 Assessing Date Palm Genetic Diversity Using Different
Molecular Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 125
13 Molecular Analysis of Date Palm Genetic Diversity Using
Random Amplified Polymorphic DNA (RAPD)
and Inter-Simple Sequence Repeats (ISSRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143
14 Determining Phylogenetic Relationships Among Date Palm
Cultivars Using Random Amplified Polymorphic DNA
(RAPD) and Inter-Simple Sequence Repeat (ISSR) Markers . . . . . . . . . . . . . . . . . 153
15 Genotyping and Molecular Identification of Date Palm
Cultivars Using Inter-Simple Sequence Repeat (ISSR) Markers . . . . . . . . . . . . . . .
173
16 Molecular Identification of Date Palm Cultivars Using Random
Amplified Polymorphic DNA (RAPD) Markers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 185
PART IV
GENDER IDENTIFICATION
17 Early Sex Identification in Date Palm by Male-Specific
Sequence-Characterized Amplified Region (SCAR) Markers . . . . . . . . . . . . . . . . . 199
18 Gender Identification in Date Palm Using Molecular Markers . . . . . . . . . . . . . . . . 209
19 Development of Sex-Specific PCR-Based Markers in Date Palm . . . . . . . . . . . . . . 227
20 Date Palm Sex Differentiation Based on Fluorescence
In Situ Hybridization (FISH) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245
PART V
GENOMICS
21 Characterization and Amplification of Gene-Based
Simple Sequence Repeat (SSR) Markers in Date Palm . . . . . . . . . . . . . . . . . . . . . . . 259
22
Mitochondrial Molecular Markers for Resistance to Bayoud
Disease in Date Palm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 273
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Contents
ix
23 Analysis of Expressed Sequence Tags (EST) in Date Palm. . . . . . . . . . . . . . . . . . . . 283
24 Development of Genomic Simple Sequence Repeats (SSR)
by Enrichment Libraries in Date Palm . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 315
25 MicroRNA Expression in Multistage Date Fruit Development . . . . . . . . . . . . . . . 339
PART VI
PROTEOMICS
26 Proteome of Abiotic Stress Tolerance in Date Palm . . . . . . . . . . . . . . . . . . . . . . . . . 355
27
Electrophoresis-Based Proteomics to Study Development
and Germination of Date Palm Zygotic Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . 365
28 Date Fruit Proteomics During Development and Ripening Stages . . . . . . . . . . . . 381
Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
399
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Chapter 1
Storage and Viability Assessment of Date Palm Pollen
Abstract
Pollen storage and viability are very important for pollination, breeding, biodiversity, biotechnology,
conservation, and other biological and non-biological studies of the date palm. Optimizing procedures
and duration of storage are important for effective and long-term date palm pollen storage and viability.
Here we describe pollen storage methods, such as room temperature (25–30 C), refrigeration (4 C),
storage at 4 C in desiccators, deep freezer (20 C), and cryopreservation (196 C). Based on pollen
viability by staining and in vitro germination methods, cryopreservation is the best method for long-term
storage without any significant effect on pollen viability (75–84%); however, the percentage of pollen
viability depends on the storage period.
Key words Acetocarmine, In vitro germination, Pollen storage, Pollen viability
1
Introduction
Pollen viability affects pollen germination and delivers the nuclei to
the embryo sac for compatible fertilization [1]. Date palm is a
dioecious plant, and cross-pollination is the general rule.
Hence, date palm pollen storage is an expedient practice; however,
storage methods and pollen viability are subjects worthy of
research. Pollen can retain its viability much longer under dry
conditions. Pollen is collected at anthesis and stored in stoppered
bottles [2], refrigerated or at room temperature for 4 weeks without losing viability and performance [3]. Various pollen storage
methods indicate that short-term storage (230 days) is the best
in desiccators, whereas freeze-drying is effective for long-term
storage [4]. Previously, viability of pollen grains stored at various
conditions has also been examined at room temperature
(25–30 C), in a refrigerator (3–4 C) [5], at 4 C in desiccators
(anhydrous calcium chloride) [4], deep freezer (20 C) [6], and
cryopreservation at 196 C [7].
Furthermore, pollen viability can be determined with Alexander’s stain, fluorescein diacetate (FDA), acetocarmine or in vitro
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4
Maryam et al.
pollen germination [8]. Acetocarmine is widely used to test the
germination rate of stored pollen grains on germination [9];
however, viability of fresh and stored pollens (1–12 months) can
also be evaluated by applying different storage methods and germination tests, as different storage conditions had no apparent effect
on pollen viability as tested with acetocarmine. Different media are
used to compare stored and fresh pollen germination rates and
tube elongation indicating significant decrease in pollen tube
length in stored pollen grains [5]. The pollen germination rate
was poor (8.5%) when stored at 0 C for 6 weeks; however, the best
temperature for pollen tube elongation was 30 C [10]. Phoenix
pollen has been stored cryogenically by immersing pollen, wrapped
in aluminum foil, in liquid nitrogen (196 C) for long-term
storage [7, 11]. Pollen viability gradually decreases although its
shelf life is enhanced through low-temperature storage.
Germination testing is highly reliable to determine pollen viability [12, 13]. On the other hand, pollen capacity to fertilize the
ovule and set fruit is considered a measure of date fruit production
[14]. In this chapter, we describe pollen storage and viability testing
procedures, which are important for date palm pollination and
production, due to asynchronized inflorescence emergence and
anthesis between male and female date palms.
2
Materials
2.1
Plant Material
2.2 Reagents
and Supplies
Pollen grains collected from mature spathes of date palm Halawy cv.
(see Note 1).
1. Pollen waxing: 200 g paraffin wax, paraffin film (see Note 2).
2. Pollen germination medium: 0.42 g/L Ca(NO3)2.2H2O
(see Note 3), 0.2 g/L H3BO3, 0.1 g/L KNO3, 0.22 g/L
MgSO4.7H2O, 200 g/L sucrose, and 10 g/L agar in distilled
water.
3. Acetocarmine staining solution (1%): 100 mL of 45% glacial
acetic acid, 1 g carmine powder.
4. Cryopreservation: Liquid nitrogen (196 C).
2.3
Equipment
1. Instruments: Hot air oven, magnetic stirrer, weighing balance,
pH meter, autoclave, refrigerator, deep freezer, microscope,
desiccator, and thermometer.
2. Glassware: Cryotubes (1 mL), glass test tubes (10 mL), beakers
(50, 500 and 1000 mL), Petri plates, and measuring cylinders
(50, 100, 500 and 1000 mL).
3. Supplies: Parafilm, spatula, pharmaceutical capsules (1 mL),
and wax.
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Storage and Viability of Pollen
3
Methods
3.1
Pollen Collection
5
1. Select mature spathes of male date palm, which are either ready
to open or already opened, and cut at the base (see Note 4).
2. Place cut spathes for 2–3 days in the dark at 25–30 C and
30–40% relative humidity for anther dehiscence.
3. Separate pollens by shaking the inflorescence strands on a paper
sheet (Fig. 1).
4. Dry pollen at room temperature and sieve (40 mesh ¼0.42 mm
pore size) to separate petals and other inert material.
3.2
Pollen Storage
1. Encapsulate pollen for long-term storage in pharmaceutical
capsules as follows:
(a) Take 200 g paraffin wax in 250-mL beaker and heat in
oven at 45 C for 2 min.
(b) Dip pollen-filled capsules in melted wax (35–40 C).
(c) After 8–10 min, transfer each waxed capsule in a Petri
plate, and wrap in paraffin film.
Fig. 1 Collecting (a) and drying (b) of date palm pollen grains after dehiscence
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Fig. 2 Storage of pollen grains at different storage temperature after waxing
2. Place capsules in labeled zipped polythene bags. Store bags
under following storage conditions for up to 12 months
(Fig. 2):
(a) Room temperature (25–30 C).
(b) Refrigerator (4 C).
(c) Storage at 4 C in the desiccators (see Notes 5 and 6).
(d) Deep freezer (20 C).
(e) Cryopreservation (196 C) (see Note 7).
3. For cryopreservation, transfer the dehydrated pollen into cryotubes (0.56 g/mL), close the lid, and immerse in liquid nitrogen for 15 min.
4. Store the cryotubes in a freezer at 196 C.
3.3 Pollen Viability
Tests
3.3.1 Pollen Germination
Test
1. Soak glassware in liquid detergent for 1 h and thoroughly wash
with hot water. Rinse the glassware with double-distilled water,
and air-dry before use (see Note 8).
2. Prepare the germination medium as follows:
(a) Weigh the chemicals for germination medium individually
(for 1 L medium), and put in a 500-mL glass beaker. Add
300 mL distilled water to the beaker, and place on magnetic stirrer for dissolution of chemicals (see Note 9).
(b) Adjust the pH of medium to 5.7 prior to the addition of
agar.
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Storage and Viability of Pollen
7
Fig. 3 Thawing of pollen grains after storage at 20 C
(c) Weigh 10 g agar, put it in 100 mL glass beaker, add
100 mL distilled water, and boil.
(d) Pour 100 mL boiled agar and 300 mL solution of salts and
sucrose into a 1000 mL conical flask. Mix it vigorously;
close the flask with aluminum foil, and autoclave at 121 C
for 20 min.
(e) In a laminar hood cabinet, pour the medium in sterile
50 mm diameter Petri plates and allow to solidify.
3. Sterilize camel hair brush for spreading pollen on the germination medium.
4. Thaw the frozen and cryopreserved stored pollen grains by
swirling tubes in water bath, at 45 C, until the ice melts
(Fig. 3, see Note 10).
5. After thawing, dust the stored pollens with a camel hair brush
in Petri plates containing pollen germination medium under a
laminar airflow cabinet to minimize the risk of contamination
(Fig. 4).
6. Seal the plates with paraffin film, and incubate in a growth
room at 25 2 C and 16-h photoperiod of 100 μmol/m2/s.
7. Count the number of germinated and total number of pollen
grains (see Note 11) in each visible field of the microscope
using 200 magnification within 1–24 h (Fig. 5, see Notes
12–14).
8. Calculate pollen germination percentage by using the following formula (see Note 15).
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Fig. 4 Culture of pollen grains on the germination medium
Fig. 5 Viability of 12-month stored pollen grains tested by acetocarmine staining
(magnification 200) [NV Non-viable, V Viable]
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Storage and Viability of Pollen
Germination ð%Þ ¼
9
Number of germinated pollen grains
100
Total number of pollen grains
9. Measure pollen tube length (see Note 16) at successive intervals under a microscope equipped with an eyepiece and stage
micrometer using 200 magnification (see Note 17).
3.3.2 Pollen Staining
Test
1. Prepare the acetocarmine solution for pollen staining as
follows:
(a) Boil 100 mL 45% glacial acetic acid in a glass beaker.
(b) Weigh 1 g carmine powder, transfer to boiling glacial
acetic acid, and boil gently for 5 min in a reflux condenser.
Shake well and filter when cool.
(c) Store the solution in a tightly closed screw-capped
dark glass bottle under cool (4 C) and dry conditions
(see Note 18).
2. Place one drop of 1% acetocarmine solution on a glass slide.
3. Place a drop of pollen suspension on glass slide, put a cover slip
on top of it, and gently press the cover slip to remove extra stain
with tissue paper/filter paper (see Note 19).
4. Examine stained and unstained pollens under the microscope
at 200 magnification (Fig. 6, see Note 20).
Fig. 6 In vitro pollen germination after 12-month storage (20 C) incubated at
28 C (magnification 200)
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Maryam et al.
5. Calculate stained pollen percentage by using the following
formula.
Staining ð%Þ ¼
4
Number of stained pollen grains
100
Total number of pollen grains
Notes
1. The age of the date palms in this study was about 30 years.
2. Wax the capsules, and wrap with paraffin film as a precaution to
avoid possible capsule cracking that may occur due to dryness.
3. Calcium nitrate is also important for date palm pollen in vitro
germination. Application of certain concentrations of calcium
nitrate in culture media increased the pollen germination rates,
but its higher concentration could decrease it. Pollen germination and pollen tube growth are regulated by transferring
inorganic Ca2+ and K+ ions across the plasma membrane.
Calcium also plays a role in determining the direction of pollen
tube growth. For in vitro pollen germination and pollen tube
growth, boron ion (B+) is one of the essential factors.
4. Select and cut a male spathe, either already split open or about
to split open, and has brown color and soft texture. To prevent
wind or bees from causing loss of pollen, it is recommended
that the freshly opened spathe be cut early in the morning.
5. Before placing pollen in desiccators, fill it with new silica gel for
effective moisture control.
6. Desiccators are used to store dried samples in dry atmosphere
by using desiccants like silica gel, whereas pollen storage in
a refrigerator at low temperature slows down metabolic
processes and does not lead to pollen drying.
7. Ultra-low temperature (196 C) is the best for long-term
storage of date pollen without any deteriorating effect on
viability.
8. Prepare all solutions using distilled water. Chemicals and
reagents should be of analytical grade.
9. Gradually increase the speed of stirrer for uniform movement
of magnet, otherwise it will move abruptly and the beaker may
break. If chemicals do not dissolve, increase the temperature up
to 40 C.
10. Do not expose the frozen pollen capsules directly to hot water,
but thaw them using glass test tubes in a beaker filled with
hot water. Swirl these tubes in hot water at 65 C for 10 min.
Then place the capsules on Petri plates, remove the paraffin and
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Storage and Viability of Pollen
11
wax using tissue paper, and collect the pollen on labeled Petri
plates.
11. Pollen germination percentage during incubation period at 3
and 24 h ranges from 15 to 85%, respectively.
12. Pollen incubation on culture media up to 24 h ensures maximum germination.
13. A pollen grain is considered germinated when the pollen tube
length is equal to or greater than the diameter of the grain.
14. Pollen culture medium can become contaminated if incubated
for duration longer than 24 h. Although sterilized culture
media and sterile Petri plates are used, the culture media can
become contaminated when Petri plates are opened during
microscopic studies.
15. Count germinated and total pollen in at least five microscopic
fields, add accordingly, and then calculate the pollen germination percentage according to the given formula. For example,
in the visible field of microscope, the total number of pollen are
90, out of which 75 pollen germinated, then the percent pollen
germination is 83%.
16. Pollen tube length varies from 44 to 196 μm at 3 and 24 h,
respectively.
17. Place the ocular micrometer inside the body tube of the microscope by unscrewing the eyepiece of the microscope. Then
place the stage micrometer on the stage of the microscope,
and focus the scale for calibration. The stage and ocular
micrometer scales must be in the same direction. Compare
and count the number of divisions of the point where both
scales coincide with each other under the microscope with 40
object and 10 eyepiece magnification. Each of the 100 parts
of the stage micrometer scale represents 0.01 mm, i.e., 10 μm
(Fig. 7). For example, if 50 ocular micrometer scales are equal
to 65 scales of stage micrometer, then
50 scales of ocular micrometer ¼ 0.65 mm
1 scale of ocular micrometer ¼ 0.65/50 ¼ 0.013 mm ¼ 13 μm
After calibration, place the sample slide on the stage under the
microscopic object for pollen tube length measurement. It
is already determined that each scale of ocular micrometer is
equal to 13 μm at 40 object and 10 eyepiece magnification
of the microscope. The length of the pollen tube will be the
number of scales multiplied by 13 μm.
18. Since the solution contains 45% glacial acetic acid, it is recommended to store the solution in a tightly closed dark screw cap
glass bottle, in cool, dry, well-ventilated place away from
inflammable material.
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Maryam et al.
Fig. 7 Illustration of ocular and stage micrometer used for pollen tube
measurement
19. Place cover slip gently and press it slightly with care to avoid air
bubbles.
20. The pollen grains appearing normal and stained red are considered viable, whereas weakly stained or colorless are recorded
as non-viable. The viability of stained pollen varies from 65 to
87% at storage periods of 1 and 12 months, respectively.
Acknowledgment
This work was supported by the Higher Education Commission,
Pakistan and Plant Tissue Culture Cell, University of Agriculture,
Faisalabad 38040, Pakistan.
References
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(1988) Effect of storage and temperature on
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48:119–122
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Storage and Viability of Pollen
11. Tisserat B, Ulrich JM, Finkle BJ (1981) Survival of Phoenix pollen grains under cryogenic
conditions. J Crop Sci 23:461–470
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in some stone fruits. J Agric Forest 23:383–389
13
13. Rodriguez-Riano T, Dafni A (2000) A new
procedure to assess pollen viability. Sex Plant
Reprod 12:241–244
14. Boughediri L, Bounaga N (1987) In vitro germination of date pollen and its relation to fruit
set. Date Palm J 5:120–127
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