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Development Proposal of

Clonal Propagation for Superior Genotypes of Date Palm

Through Tissue Culture Technique


In Aceh Province Republic Indonesia

Habib Bugak Corporation


2016

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Summary
Date palm is one of the most important fruit crops in arid regions of North Africa and Middle East,
but has not been planted commercially in Indonesia. Date palm is commonly propagated by seeds
and offshoots. Seed propagation is the easiest method but half of the seed progeny are male and half
are female trees, and are not true-to-type trees. Offshoot propagation is not commercially practical
because only a limited numbers of shoots produced and is difficult and laborious.
Tissue culture of date palm enables large-scale mass multiplication of genetically uniform female
plants of superior cultivars. Tissue culture of date palm has been conducted via organogenesis or
somatic embryogenesis (SE) routes. Tissue culture procedure of date palm will be developed using
superior genotypes of known superior cultivars of date palm as explant sources.
The long-term objective of this project is to develop propagation technology for mass production of
clonal planting material of superior genotypes of date palm through organogenesis and/or SE. The
technology developed will be implemented at the future tissue culture laboratory established in
Aceh with production capacity of 1.000.000 date palms annually.

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I.

INTRODUCTION

Date palm (Phoenix dactylifera L.) is a dioecious, perennial monocot plant species of the Arecaceae family. It
is one of the oldest and most important fruit crops cultivated in arid regions of North Africa and Middle East
countries. Date palm has been planted in Indonesia mainly for ornamentals because the plant has rarely produced
flowers and fruits. However, recently date palm has been grown and fruited successfully in the tropical area of
Thailand and now is becoming one of the most-wanted fruits in this country.
As the biggest Moslem country, Indonesia should be the leader in the development of date palm in the
tropics. Since the R & D on date palm has already been conducted extensively for many years in other countries,
date palm R & D in Aceh must be focused on the selection of superior varieties adaptable to agroclimate conditions
of Aceh and on mass clonal propagation of superior varieties to provide rapidly elite planting materials to be
planted in Aceh and the rest of Indonesia.
Date palm is commonly propagated by seeds and offshoots. Seed propagation is the easiest method but
approximately half of the seed progeny are male and half are female trees, and the progeny are not true-to-type
trees. Offshoot propagation is commonly used in North Africa and Middle East countries, but it is not commercially
practical because only a limited numbers of shoots produced and this method is difficult and laborious. Therefore,
plant tissue culture technology is a potential alternative to propagate date palm. Tissue culture of date palm
enables large-scale mass multiplication of genetically uniform female plants of superior cultivars.
Research on tissue culture of date palm has been conducted since 1970s (Tisserat, 1979) using direct or indirect
organogenesis and somatic embryogenesis routes (Bekheet, 2013). Direct route (without the formation of callus)
is genetically more stable (lower chance of flower/fruit abnormality). While this indirect route (through callus
formation) are genetically unstable and cause genetic variation in crop propagation. Therefore, in this study will be
used for direct organogenesis.
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II.

OBJECTIVES

Long-term objective: to develop clonal mass propagation technology of date palm.


First year objective: to produce planlets from direct organogenesis of date palm.

III.

EXPECTED OUTPUTS

First Year : In vitro shoot and Planlets


Second Year : Acclimatization and Transpanting

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IV.

MATERIALS AND METHODS

1. Culture Media
MS + 30 g / l of sugar, 7 g gelatin, 1-2.5 g of active carbon. PH Media 5.7. Then sterilized for 15 minutes in an autoclave at a
temperature of 121 0 C and a pressure of 1 kg / cm 3.

2. Explant
Explants used are off shoots of seedlings aged 2-3 years from seed to cultivate the obvious.
Exsplant washed under running water. Then soaked in fungicide and bactericide 0, 2 g / 100 ml for 30 minutes and then rinsed with
sterile water 3 times then soaked in 30% Clorox for 30 minutes and then soaked in 20% Clorox for 20 minutes and then rinse with
sterile water 3 x soaked in Clorox last 10 % for 10 minutes and rinsed 3 times in sterile water.
Then on a petri dish, go and get rid of all the leaves that cover the buds and shoots cut lengthwise 4 -6 pieces and move them to the
culture medium.
After the incubation room, placed for 3 -6 months and was not given the light to stimulate the formation of buds.
3. Medium Composition for the initiation and propagation
MS (Murashige and Skoog, 1962) medium will be used since most of tissue culture protocols of date palm treatments use this medium
that will be used are the type and concentration of plant growth regulators (auxins and cytokinins) use of activated charcoal and level
of Carbohydrate ,

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4. The formation of shoots and multiplication


Seedlings appear after about 3-6 months of initiation. After the buds are formed to move the culture bottle of the light. The buds are
formed and then transferred to new media every 4 weeks. Total subculture to subculture limited to 6-7 in order to avoid genetic
variation. The concentration of growth hormone used is not too high so that the plants remain true - to - type.
In multiplication, will be trying to use a solid medium with agar 7 g / l and a liquid medium without agar shaken shaker. Liquid
medium shaken shaker so remain mixed media throughout the bottle culture and aeration also going well.
Multiplication in a liquid medium that whipped-cream / stirred culture of palm varieties Maktoom produce seedlings more than a
solid medium. (Khirellah, at all, 2007).
5. Rooting
Rooting the culture of these dates will be using some of the media:
MS is not given any hormones after one month was transferred to MS medium with the addition of coconut milk 100 ml / l
media.
It will also use media MS + NAA 1 ppm, 0.5 ppm BA and kinetin 0.5 ppm.
Then after one month was transferred to MS + NAA 0.1 mg / l. In every treatment there is added PEG (Polythilene glycol) to
strengthen plantlets during the acclimatization.
6. Acclimatization
Plantlets were ready for acclimatization if: shoots 10 cm high, looks sturdy plantlets, have 2 -3 leaves and roots are more open than
three. Plantlets were acclimatized and ready for hardening process for 1 week.
After one week plantlets were washed in running water to remove the gelatin. The roots dipped in fungicides and grown in media
acclimatization.
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Media acclimatization to try are:


1) Husk fuel manure + 1: 1
2) 100% Bamboo Compost
3) husk + sand + manure 1: 1: 1
4) 100% husk
Once on the ground with hermetically sealed transparent plastic for one week. Then open. Preserved for 2 - 4 weeks after the seeds
are then transferred to a strong polybag containing media:

Soil + manure 1: 1
Soil + chaff manure + 1: 1: 1
Soil + sand + manure 1: 1: 1
Soil + sand + compost bamboo 1: 1: 1

After 3 - 4 months dipolybag seeds ready to be moved to the garden.


Procurement The tools of production:
Test tube and place (of aluminum) a total of 100 tubes (diameter ...)
Shaker for 20 culture bottles
Aluminum foil
Materials Chemistry:
NOA
PEG (polyethylene glycol)
Carbon
2ip
Procurement Support Incubation:
Culture rack and lights (30 steps) with light using the new model (but not long fluorescent light bulb threaded with 10 watts) for 1 to
2 rugs bulb threaded. because:
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1. TL Lamp 36 - 40
2. TL Lamp long size is not easy in electrical stores
3. With light bulb threaded for plants relatively stable for the long term and effective.
Schedule of research activity in 2016
Month (2016)
Activity

Indicators
1 2 3 4 5 6 7 8 9 10 11 12 13 14

Preparation

Materials & Equipment are available

Collection of Explant Source


Offshoots
The buds are formed
Initiation
The number of seedlings to be a lot of
Propagation buds
Plantlets ready acclimatized
Rooting
Seedlings are ready transpalanting
Acclimatization
Seedlings are ready for planting field
Transplanting
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V.

Advisors

PERSONNEL

Prof. Dr. Abdul Aziz Darwis, MSc.


Dr. Habib Shechan Shahab, SH.
Dr. Amirudin Idris, M.Si.
Dr. Said Haran, M.Sc.
Dr. Agus Sabti M.Sc.
Dr. Hilmy Bakar, MA.
Dr. Ermawita, M.Sc.
H. Nursyamsu, M.Si.

Resaerachers :

Dr. Halus Satriawan M.Si.


Can. Dr. Jaswar, M.Sc.
Agusni, SP. MP.
Ety Sulastianti, SS
Muhammad Adil, SS

Technicians & Laborans

Chairul Miswar, Said Agus Saputra, Fitria, AMd. Ari,


Dodi, Ibrahim, Fatma Welda, SSy. Eka Sri Astuti,
Erry Tri Rahayu, Mayasari, Netty, Santy, Eem Emelia
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VI. BUDGET

& CASH FLOW

FINANCE:

YEAR 0

Investment costs

-US$2,684,211

Pre-Operation costs

-US$107,368

Working capital

-US$315,789

Total Investment Costs

-US$3,107,368

YEAR 1

YEAR 2

US$0

US$0

Operating benefits

US$25,263,158

Total Operating Benefits

US$0

US$25,263,158

Operating costs

-US$2,656,632

-US$2,656,632

Total Operating Costs

-US$2,656,632

-US$2,656,632
US$22,606,526

Net Cash Flows

-US$3,107,368

-US$2,656,632

Debt financing

US$2,175,158

US$1,859,642

Net Financing Flows


Net Cash Flows (Equity)

-US$932,211

-US$913,358

-US$1,694,229

-US$1,710,347

US$20,912,298

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VI.

REFERENCES

Abd El Bar O.H. & M.M. El Dawayati (2014). Histological changes on regeneration in vitro culture of date palm
(Phoenix dactylifera) leaf explants. Australian J. Crop Sci. 8(6): 848-855.
Abul-Soad A.A. & S.M. Mahdi (2010). Commercial production of tissue culture date palm (Phoenix dactylifera L.) by
inflorescence technique. J. Gen. Eng. Biotechnol. 8(2): 39-44.
Alkhateeb A.A. (2006). Somatic embryogenesis in date palm (Phoenix dactylifera L.) cv. Sukary in response to
sucrose and polyethylene glycol. Biotechnol. 5(4): 466-470.
Aslam J., S.A. Khan, A.J. Cheruth, A. Mujib, M.P. Sharma & P.S. Srivastava (2011). Somatic embryogenesis, scanning
electron microscopy, histology and biochemical analysis at different developing stages of embryogenesis in six
date palm (Phoenix dactylifera L.) cultivars. Saudi J. Biol. Sci. 18: 369-380.
Bekheet S. (2013). Date palm biotechnology in Egypt (review article). App. Sci. Rep. 3(3): 144-152.
Boufis N., M. Khelifi-Slaoui, Z. Djillali, D. Zaoui, A. Morsli, M.A. Bernards, A. Makhzum & L. Khelifi (2014). Effects of
growth regulators and types of culture media on somatic embryogenesis in date palm (Phoenix dactylifera L.
cv. Degla Beida). Sci. Hort. 172: 135142.
Eshraghi P., R. Zarghami & M. Mirabdulbaghi (2005). Somatic embryogenesis in two Iranian date palm cultivars.
African J. Biotechnol. 4(11): 1309-1312.
Fki L., N. Bouaziz, W. Kriaa, R. Benjemaa-Masmoudi, R. Gargouri-Bouzid, A. Rival & N. Drira (2011). Multiple bud
cultures of Barhee date palm (Phoenix dactylifera) and physiological status of regenerated plants. J. Plant
Physiol. 168: 1694-1700.
Murashige T. & F. Skoog (1962). A revised medium for rapid growth and bio assays with tobacco tissue culture.
Physiol. Plant. 15: 473-497.
Tisserat B. (1979). Propagation of date palm (Phoenix dactylifera L.) in vitro. J. Exp. Bot. 30: 1275-1283.
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Annex 1. List of major laboratory equipment


Equipment
Autoclave, 400 & 1.200 L capacity
Balances, normal and sensitive with accessories
Burners
Freezer
Dish washing machine
Electric power generator 80 Kva
Laminar air flow cabinet
Media dispenser 10-500 mL
Microscope, light and binocular
Oven
pH meter
Refrigerator
Stirrer, magnetic hot plate
Thermohygrometer
Vacuum pump
Water bath, 5-100C
Water purification system
Rotary shaker
Electric timer for photoperiodic

Quantity
2
4
10
2
2
1
6
2
2
2
2
2
4
4
1
1
1
2
10

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Annex 2. List of culture vessels for the annual production of 100.000 plants
(Zaid et al., 2011)
Item
Test tube, 24 x 200 x 1.2 mm, rimless
Test tube plastic closures, autoclavable
Racks, stainless steel, hold 48 tubes
Pickle jars 370 mL
Twist-off cup, diameter 63 mm, polypropylene
Media bottle, 500 mL capacity, with polypropylene cap

Quantity
150.000
150.000
4.000
50.000
50.000
30.000

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Annex 3. List of basic nutrients and chemical reagents used in the first year
Item

Quantity

Macro nutrients : NH4NO3


KNO3
CaCI2.2H2O
MgSO4.7H2O
KH2PO4
NaH2PO4
FeSO4.7H2O
Na2EDTA

10 kg
10 kg
7 kg
5 kg
5 kg
5 kg
3 kg
3 kg

Micro nutrients : MnSO4.H2O


ZnSO4.7H2O
H3BO3
KI
Na2MoO4.2H2O
CuSO4.5H2O
CoCl2.7H2O
Agar powder
Auxins : 2,4-D
IAA
NAA
IBA
Cytokinins: Kinetin
BAP
2-Ip

3 kg
1 kg
1 kg
0.5 kg
0.1 kg
0.1 kg
0.1 kg
100 kg
50 gr
20 gr
20 gr
30 gr
20 gr
30 gr
20 gr

Other Plant Growth Regulator : GA3


TDZ

10 gr
10 gr

Vitamins : Nicotinic Acid

50 gr

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Thiamine
Pyridoxin
Myo- Inositol
Ascorbic Acid
Amino acids : Glycine
Carbohydrates : Sucrose
Activated charcoal
Other chemicals : PVP
HCl 10 M
NaOH powder
Universal pH indicator
Spiritus
Ethanol 95%

100 gr
50 gr
2kg
100 gr
200 gr
100 kg
10 kg
500 gr
2 ltr
2 kg
10 box
500 ltr
1000 ltr

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Daar Al-Fallah Tissue Culture Laboratory


http://dafataman.com/
S
S

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