shoot-tip grafting in vitro · 2021. 2. 8. · shoot-tip grafting in vitro to obtain citrus...
TRANSCRIPT
Project TCP-BZE-3402
Technical Manual
SHOOT-TIP GRAFTING IN VITRO
TO OBTAIN CITRUS PLANTING MATERIAL FREE
OF GRAFT-TRANSMISSIBLE PATHOGENS AND
FOR THE SAFE MOVEMENT OF CITRUS
BUDWOOD
SHOOT-TIP GRAFTING IN VITRO
TO OBTAIN CITRUS PLANTING MATERIAL FREE
OF GRAFT-TRANSMISSIBLE PATHOGENS AND
FOR THE SAFE MOVEMENT OF CITRUS
BUDWOOD
Olga Mas and Romualdo Pérez (retired Senior Researchers of the Tropical Fruit Crops Research Institute, IIFT, Havana,
Cuba) E-mail: [email protected]
Prepared under the Project TCP-BZE-3402 - Assistance to Manage Huanglongbing in Belize
© FAO, 2014
Food and Agriculture Organization of the United Nations
Belize, 2014
Table of Contents
INTRODUCTION ............................................................................................................................................... 2
1.1 MANDATORY CITRUS CERTIFICATION PROGRAMS ................................................................................................ 3 1.1.1 Quarantine Program ....................................................................................................................... 3 1.1.2 Clean Stock Program ....................................................................................................................... 3 1.1.3 Certification Program...................................................................................................................... 4
II. WAYS OF OBTAINING PATHOGEN-FREE BUDWOOD ............................................................................... 8
2.1 SELECTION OF TREES ON THE FIELD (CLONAL SELECTION) ........................................................................................ 8 2.2 PLANTS OF NUCELLAR ORIGIN ........................................................................................................................... 8 2.3 THERMOTHERAPY .......................................................................................................................................... 9 2.4 SHOOT-TIP GRAFTING IN VITRO....................................................................................................................... 10
III. SHOOT-TIP GRAFTING IN VITRO (STG) .................................................................................................. 12
3.1 SELECTION OF MOTHER TREES FROM THE LOCAL CULTIVARS (CLEAN STOCK PROGRAM) ............................................. 12 3.2 OBTAINING IN VITRO ROOTSTOCKS .................................................................................................................. 12 3.3 SOURCES OF FLUSHES FOR SHOOT TIPS ............................................................................................................. 13 3.4 ROOTSTOCK PREPARATION ............................................................................................................................ 15 3.5 ISOLATING THE SCION AND PERFORMING THE GRAFT ........................................................................................... 15 3.6 CARE OF IN VITRO STG PLANTS ...................................................................................................................... 16 3.7 TRANSFER OF SHOOT-TIP GRAFTED PLANTS TO EXTERNAL ENVIRONMENTAL CONDITIONS ............................................ 17
3.7.1 Transfer to pots ............................................................................................................................. 17 3.7.2 Re-grafting .................................................................................................................................... 18
3.8 PLANT MAINTENANCE .................................................................................................................................. 20 3.9 FACTORS INFLUENCING SHOOT-TIP GRAFTING RESULTS ........................................................................................ 22
3.9.1 Rootstock chosen and variety used as scion ................................................................................. 22 3.9.2 Shoot tip size ................................................................................................................................. 22 3.9.3 Pathogen ....................................................................................................................................... 23 3.9.4 Skill of the person performing STG ................................................................................................ 23
IV. DIAGNOSTIC TESTS FOR PATHOGENS ................................................................................................... 24
V. APPLICATIONS OF SHOOT-TIP GRAFTING IN VITRO ............................................................................... 27
5.1 RECOVERY OF PATHOGEN-FREE CITRUS PLANT MATERIAL ..................................................................................... 27 5.2 RECOVERY OF PATHOGEN-FREE PLANTS OF OTHER WOODY SPECIES ........................................................................ 27 5.3 OTHER APPLICATIONS OF STG IN PLANT SPECIES ................................................................................................ 27 5.4 SEPARATION OF PATHOGENS IN MIXED INFECTIONS ............................................................................................ 28 5.5 STUDIES ON GRAFT INCOMPATIBILITY AND ON HISTOLOGICAL AND PHYSIOLOGICAL ASPECTS OF GRAFTING ..................... 28 5.6 APPLICATIONS OF STG IN OTHER CITRUS RESEARCH ............................................................................................ 28 5.7 IMPORTATION OF CITRUS GERMPLASM: QUARANTINE PROGRAMS ........................................................................ 29
VI. SAFE MOVEMENT OF CITRUS BUDWOOD ............................................................................................. 30
VII. REQUIREMENTS, CULTURE MEDIA AND PROTOCOLS ....................................................................... 32
7.1 REQUIREMENTS .......................................................................................................................................... 32 7.1.1 Facilities ........................................................................................................................................ 32 7.1.2 Equipment ..................................................................................................................................... 32 7.1.3 Reagents ....................................................................................................................................... 33 7.1.4 Glassware, instruments and other ................................................................................................ 34
7.2 STOCK SOLUTIONS AND CULTURE MEDIA ......................................................................................................... 36 7.2.1 Stock Solutions .............................................................................................................................. 36 7.2.2 Culture Media ............................................................................................................................... 37
7.3 PROTOCOLS ................................................................................................................................................ 41 7.3.1 In vitro sowing of seeds from the selected rootstock .................................................................... 42 7.3.2 Collecting and surface sterilizing flushes ...................................................................................... 46 7.3.3 Rootstock preparation .................................................................................................................. 48 7.3.4 Isolating the scion and performing the graft ................................................................................ 51 7.3.5 Removing rootstock sprouts ......................................................................................................... 54 7.3.6 Budstick culture in vitro ................................................................................................................ 56
VIII. BIBLIOGRAPHY ................................................................................................................................. 66
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IX. ACKNOWLEDGEMENTS ......................................................................................................................... 73
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INTRODUCTION
The diseases caused by viruses, viroids, bacteria and phytoplasms resulting in
important economic losses are widely disseminated globally as a result of their
propagation by grafting without sanitary control on the trees taken as source of the
buds. Top-working is also a major form in which all graft-transmissible pathogens
are spread. Although insect vectors exist for some of the pathogens, man has been
undoubtedly their main transmitter.
These pathogens present in the citrus plants result in significant, negative effects on
productivity, longevity and vigour, as well as on the quality of fruits and furthermore,
introduce limitations in the use of several rootstocks.
The use of disease-free, genetically certified planting material is indispensable to
guarantee the use of potentially high-yielding material and to take the necessary
measures to reduce, as much as possible, the damage caused by pathogens that
are transmitted by vectors. Only then will orchards with high productivity and good
quality fruits will be obtained. It is essential to make certification of planting material
in citrus-growing countries mandatory, in order to avoid the propagation of disease-
causing pathogens and to reduce the probability of introducing symptomless plant
material and plants that contain pathogens destructive to the crop. The existence of
procedures to detect and eliminate such pathogens supports the possibility of
establishing mandatory certification programs for citrus planting material.
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1.1 Mandatory Citrus Certification Programs
For certification of planting material, it is necessary to establish three closely
inter-related programs, which are briefly described.
1.1.1 Quarantine Program
In every country, there is a constant demand for new citrus cultivars developed in
other citrus-growing countries. Uncontrolled importation of exotic germplasm can
result in the importation of new pests and pathogens, some of which could cause
serious economic losses to the entire industry.
One of the functions of a Quarantine Program is to ensure safe importation
of exotic germplasm without introducing new pests or diseases. Such a
program is generally the responsibility of the Plant Protection Services in the Ministry
of Agriculture.
Under the Quarantine Program, imported germplasm is placed in quarantine either
at an isolated location away from citrus production areas or separated by rigid
physical barriers, or via the use of in vitro approaches. The imported germplasm is
then indexed for the presence of graft-transmissible pathogens, recovered free of
specific pathogen(s) by shoot-tip grafting and / or thermotherapy, then re-indexed to
ensure freedom from known graft-transmissible pathogens.
1.1.2 Clean Stock Program
In areas where there is a long history of citrus production, local varieties that perform
well under local conditions are often selected. It is necessary to recover healthy
budwood from these unique locally grown selections in order to introduce them into
the industry through the citrus certification program. The purpose of a Clean Stock
Program is to produce pathogen-free germplasm from locally-selected clones often
best adapted to local climate and soils.
Several steps are involved in the establishment of a Clean Stock Program:
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1) selection of mother trees from local cultivars
2) indexing of the selected mother trees to detect possible graft-transmissible
pathogens
3) recovery of pathogen-free plants by shoot-tip grafting in vitro and / or
thermotherapy
4) indexing of the plants that are recovered
5) horticultural evaluation of the healthy plants, and
6) maintenance of healthy plants under protected conditions.
Since infections by graft-transmissible pathogens are often latent, indexing must be
done on the selected mother trees in order to determine which pathogen(s) need to
be eliminated. Such a program is usually implemented in research institutions and
requires the involvement of experts in horticulture, virology and in vitro culture.
Horticultural evaluation of the recovered plants is absolutely essential. While there
has never been any report of shoot-tip grafted plants expressing abnormal traits,
there is always a chance that a spontaneous bud sprout can occur or the human-
induced risk that labels have been misplaced. Additionally, if the horticultural
evaluation is carried out in a way that includes yield records, this information allows
growers to select the more productive clones over a period of time.
Maintenance of the pathogen-tested recovered germplasm to avoid
re-infection by graft-transmissible pathogens is essential. In the case of graft-
transmissible pathogens vectored by insects or mites, it is essential that the clean
stock plants be maintained either in an insect-free screenhouse or a greenhouse.
The plants in the Clean Stock Program need to be re-indexed on a regular,
recurring basis for the graft-transmissible pathogens which are present in the
country, to ensure that their disease-free status is maintained.
1.1.3 Certification Program
This Program guarantees the sanitary and true-to-type status of the nursery material
during the process of commercial propagation through nurseries. The Quarantine
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Program and Clean Stock Program provide the source of pathogen-tested material
which is distributed to nurseries and growers by the Certification Program.
This program consists of legal regulations for the different components and is
usually entrusted to institutions with legal authority to impose restrictions and carry
out inspections of these components.
The components of Certification Programs are as follows:
1. Protected Primary Foundation Block / Protected Germplasm Block. This
comprises pathogen-tested plants recovered through the Clean Stock and
Quarantine Programs. The plants have been verified to be of the highest
horticultural quality and to have undergone recurring indexing to verify their
pathogen-tested status over time. They are grown under protected conditions
(screenhouses / greenhouses). These trees are the primary source of
budwood for the establishment of a Protected Foundation Block.
2. Protected Foundation Block. Trees must be propagated using budwood
from the Protected Primary Foundation Block. These trees are grown in
containers large enough to allow them to fruit so that trueness-to-type of the
fruit can be monitored. They are indexed on a regular recurring basis
3. Protected Budwood Increase or Multiplication Block. This block provides
for a catalytic increase of budwood from the Protected Foundation Block, for
the propagation of certified nursery plants that will be planted in the field. A
time limit is set for plants in this Block to prevent the possible propagation of
undetected mutations.
4. Certified Nurseries (Multiplying and Commercial Nurseries). Buds used
to produce these plants come from the Protected Budwood Increase Block
and seeds for rootstock propagation from the Seed Source Trees.
Certified Nurseries (as well as the three previous components mentioned
earlier) should be located at a certain distance from established citrus
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orchards. Nurseries must keep records to demonstrate that they have
complied with regulations established for the components of the Certification
Programs.
5. Seed Source Trees. Citrus certification programs allow for the propagation
and certification of true-to-type rootstock Seed Source Trees that have been
tested to be free from known graft-transmissible pathogens. While in the past
only scarce reports on seed transmission of psorosis virus and psorosis-like
pathogens – in seeds of Poncirus trifoliata (L.) Raf. and in hybrids having
P. trifoliata as one of the parents – had been published, recent reports have
shown seed transmission of several citrus pathogens such as citrus
variegated chlorosis (CVC), witches’ broom of lime and citrus leaf blotch virus
(CLBV). Therefore, Seed Source Trees have to be tested on a recurring
basis for seed-borne pathogens that may be present in the region.
6. Variety Blocks or Lots for Horticultural Evaluation. The purpose of these
blocks is to monitor the trueness-to-type and horticultural quality of the
material propagated (cultivars and rootstocks).
In Certification Programs, plants should be grown with the best available cultural
practices. Special precautions should be taken to control pests and fungal diseases.
All pruning and grafting tools should be adequately disinfested prior to their use in
any operation. Careful labelling of plants during the whole process of propagation is
very important.
A Certification Program requires, as an essential step, to have an initial
selected material that comes from a sanitary and genetic improvement program.
The Certification Program does not increase the initial quality of the plant material
but is geared to prevent its deterioration during the multiplication process.
Once established, Certification Programs tend to perpetuate themselves on
account of the multiple advantages they offer to the grower. They are designed to
not only prevent the introduction of highly-destructive diseases, but to also prevent
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their dissemination from infected material within a country. The support, cooperation
and active participation of all stakeholders are vital for its successful implementation.
The progress of Citrus Certification Programs was accelerated by the development
of the shoot-tip grafting technique (Navarro et al., 1975; Diagram 1).
DIAGRAM 1. SYSTEM FOR THE PRODUCTION OF CITRUS CERTIFIED PLANTING MATERIAL
QuarantineQuarantine ProgramProgramIImportationmportation of varietiesof varieties
CleanClean StockStock ProgramProgramLocal Local selectionsselections
CertificationCertification ProgramProgram
Shoot-tip grafting
o Protected Germplasm Block
o Protected Foundation Block
o Protected Multiplication Block
o Certified Nurseries
o Lots for Horticultural Evaluation
o Seed Source Trees
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II. WAYS OF OBTAINING PATHOGEN-FREE BUDWOOD
When graft-transmissible pathogens have been diagnosed and no source of healthy
planting material is available, the only solution is to eliminate the infection from the
diseased material, thus enabling the reutilization of valuable, pathogen-free
resources.
Several ways to obtain healthy citrus planting material have been considered.
2.1 Selection of trees on the field (clonal selection)
Healthy trees may be found in the existing orchards using appropriate diagnostic
tests. In this case, the feasibility of using them as the source of propagation material
of certain clones should be considered. Success is, however, difficult in this search,
and in view of the existence of highly-reliable techniques for the elimination of
pathogens (as explained later), this method of obtaining healthy citrus material is not
recommended.
2.2 Plants of nucellar origin
In the past, the most widely-used method to recover pathogen-free citrus plants was
the selection of nucellar seedlings. The characteristic polyembryony of most citrus
species allows obtaining nucellar progeny identical to mother plants, through the
conventional germination of seeds. Ovule culture in vitro (in the case of seedless
polyembryonic varieties) and nucellus culture in vitro (in the case of monoembryonic
varieties) were developed for the purpose of obtaining nucellar progeny identical to
the mother plant. On this basis, regardless of the undesirable juvenile characteristics
of the progeny (excessive vigour, thorniness, late bearing), plants of nucellar origin
have been considered for years as an alternative to obtain citrus plants free of
pathogens, taking into account that most pathogens are not transmitted into the
seeds of the fruits on an infected tree, so that plants obtained from these seeds are
free of pathogens.
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Until not long ago, only the transmission of psorosis and psorosis-like pathogens
through seeds of Poncirus trifoliata (L.) Raf. and hybrids having P. trifoliata as one of
the parents had been reported, but recent reports have clearly demonstrated that
several pathogens can be transmitted through seeds to citrus seedlings:
Xylella fastidiosa Wells, a bacterium causing citrus variegated chlorosis
(CVC), can infect and colonize sweet orange fruit tissues including the
seed and can be transmitted via seeds to seedlings
transmission of citrus leaf blotch virus (CLBV) through the seeds of
‘Troyer’ citrange (Poncirus trifoliata (L.) Raf. x Citrus sinensis (L.) Osb.),
‘Nagami’ kumquat (Fortunella margarita (Lour.) Swingle) and sour orange
(Citrus aurantium L.)
transmission of the causal agent of witches’ broom disease of lime
(WBDL) through seeds to seedlings.
These results indicate that citrus nucellar progeny do not constitute a safe method of
obtaining healthy plants, and that the regulations of citrus certification programs may
need to be changed to include increased control of Seed Source Trees. In addition,
international regulations for citrus seed movement should include appropriate
phytosanitary certification.
2.3 Thermotherapy
Thermotherapy is the classical method to obtain plants free of pathogens. This
requires subjecting infected plants to high temperatures (38-40ºC) for extended
periods (weeks or months), which deactivates thermo-sensitive pathogens. This
method is effective against most citrus viruses and other pathogens, although it has
some drawbacks in that some citrus cultivars are sensitive to high temperatures and
thermotherapy is not effective in eliminating certain pathogens such as yellow vein
and dweet mottle viruses, Spiroplasma citri – the cause of stubborn disease –
exocortis and cachexia, of which these two viroids are widely spread.
In spite of this, the combination of thermotherapy with shoot-tip grafting in vitro is
currently recommended to eliminate thermo-sensitive pathogens, taking into account
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the fact that subjecting plant material to appropriate thermo-therapeutic treatments,
leads to successfully obtaining healthy material by combining both methods.
2.4 Shoot-tip grafting in vitro
Shoot tip culture is successfully used to produce pathogen-free plants of numerous
species. The procedure is based on the ability of shoot tips to regenerate whole
plants and also, that this part of the plant is usually free of microorganisms, even
though the rest of the plant may be infected. There are some hypotheses that
explain this effect. One of them is the fact that pathogens move through the vascular
system that is not present in the meristematic cells of the shoot apex. The intense
activity of meristematic cells has also been considered as a limiting factor of virus
replication by competing with them for the necessary molecules.
Failed attempts to produce plants from citrus shoot-tips cultured in vitro led to the
advent of shoot-tip grafting in vitro to recover pathogen-free citrus plants.
Researchers of the University of California pioneered the concept of putting the very
small apex of the shoot on the cut end of a small seedling, growing in a test tube
(Murashige et al., 1972). Navarro et al. (1975) defined the parameters for shoot-tip
grafting in vitro and perfected the technique: the publication “Improvement of
Shoot-tip Grafting in Vitro for Production of Virus-free Citrus” became the
standard for all future work on shoot-tip grafting.
Shoot-tip grafting in vitro is the most effective and currently the most widely used
method for obtaining healthy citrus plants. It works very similar to a traditional graft,
but offers a great efficiency in cleaning citrus, thus avoiding the constraints of using
nucellar plants. Similarly, this technique is the basis of the methodology that has
been recommended for the international exchange of citrus germplasm.
The technique is performed under aseptic conditions, using a stereomicroscope and
appropriate instruments. It consists of grafting a small shoot tip (0.1-0.2 mm from
top to bottom), excised from a new-flush on a plant with some graft-transmissible
pathogen, onto a citrus rootstock obtained by in vitro germination of clean, certified
seeds. The shoot-tip grafting in vitro technique works like any other common grafting
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procedure in preserving the characteristics of the original tree and its quick coming
into bearing.
Shoot-tip grafting has been successful in obtaining bud-lines free of all tested citrus
pathogens, including those that cannot be removed by thermotherapy. At the same
time, the healthy plant produced by shoot-tip grafting shows identical features as the
mother plant, and has no juvenile characteristics so it comes into bearing rapidly.
It is essential to check that the resulting plant is free of pathogens by performing all
the necessary indexing, and once indexing is complete, the reference to a healthy
plant indicates that such a plant is free of pathogens, for which diagnostic test
results were negative and, also taking into account the reliability of the diagnostic
tests used.
Shoot-tip grafting is the basis of Clean Stock and Quarantine Programs currently
developed as an essential part of citrus certification in most citrus-growing countries,
and is also important for establishing a properly-preserved citrus germplasm
collection.
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III. SHOOT-TIP GRAFTING IN VITRO (STG)
3.1 Selection of mother trees from the local cultivars (Clean Stock Program)
Depending on the importance that local selections and cultivars in a country have for
citriculture, mother trees are selected based on their physical features, size, yield,
fruit characteristics and other features. Diagnostic tests are performed to detect the
presence of graft-transmissible pathogens, in order to determine the sanitary status
of the selected plant, before carrying out shoot-tip grafting. This way, at the end of
the cleaning process, the effectiveness of the activity in removing pathogens through
STG can be confirmed.
3.2 Obtaining in vitro rootstocks
Rootstocks for shoot-tip grafting are obtained through in vitro seed germination
(Figure 1). The use of certified rootstock seeds must be ensured, since some
pathogens can be transmitted via seeds.
Figure 1. ‘Troyer’ citrange seedlings ready to be used as rootstocks in STG. In theory, any compatible scion / rootstock combination can be used for STG.
Although the most used rootstock is ‘Troyer’ citrange (Poncirus trifoliata (L.) Raf. x
Citrus sinensis (L.) Osb.), there are others such as ‘Carrizo’ citrange, Poncirus
13
trifoliata, ‘Rough’ lemon (Citrus jambhiri Lush.), ‘Etrog’ citron (Citrus medica L.),
Citrus macrophylla Wester, ‘Rangpur’ lime (Citrus limonia Osb.), sour orange (Citrus
aurantium L.), sweet orange (Citrus sinensis (L.) Osb.), ‘Swingle’ citrumelo (Citrus
paradisi Macf. x P. trifoliata (L.) Raf.), Citrus volkameriana Ten. & Pasq. and
‘Cleopatra’ mandarin (Citrus reshni Hort. ex Tan.) that have been used. The use of
these rootstocks is for different reasons, such as compatibility, faster growth of the
scion when using vigorous rootstocks, and the ease provided by trifoliate stocks to
identify rootstock shoots – due to the trifoliate leaves – for their removal to avoid
impaired development of the grafted shoot-tip.
The protocol for sowing seeds is provided in Chapter VII 7.3.1 of this manual.
3.3 Sources of flushes for shoot tips
The scion (shoot tip) for shoot-tip grafting can be obtained from various sources of
flushes from selected infected trees:
directly from field trees that are naturally in flush; however, flushing is
season-associated and suitable plant material from which obtaining the shoot
tips is not always available
defoliated branches of field trees
nodal sections cultured in vitro
budsticks cultured in vitro, such as those recommended for the safe
movement of citrus budwood (see Chapter VI in this manual)
grafted plants of the desired selections kept in bags or pots and in which
flushing can be induced by total defoliation around two weeks before
performing the STG.
Growth depends on time of the year, environmental conditions and citrus variety.
Grafted plants in bags or pots are recommended considering the following
advantages:
the plants can be placed in a screenhouse or greenhouse near the laboratory
in which STG is performed, thus facilitating the work
the plants can be stripped at a convenient time to induce flushing.
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Grafted plants used as flush sources should be provided with:
an appropriate substrate
favourable environmental conditions as to temperature and humidity
appropriate cultural practices such as irrigation and fertilization.
Once scions on propagating rootstocks have reached a size that can tolerate being
stripped of their leaves (3-4 months after grafting), flushing may be induced by
removing all leaves from the plant and cutting back young, soft growth, by hand.
It is recommended that the defoliated potted plants be placed in a controlled-
temperature room or chamber at 32°C, or placing bud-sticks cultured in vitro as
source of flushes in an incubator at the same temperature. This procedure helps
increase the percentage of plants that are free of thermo-sensitive pathogens.
In approximately two weeks – the period needed for rootstock seedlings to be ready
for STG – any of these variants can produce the flushes necessary for STG (Figure
2).
Vegetative flushes, 1-3 cm long, are used (Figure 3). It is advisable not to collect
larger flushes to avoid shoot-tips that are abscising or otherwise degenerating.
The protocol for collecting flushes and preparing the flush terminal for STG is
provided in Chapter VII 7.3.2 of this manual.
Figure 2. Stripped plant producing flushes. Figure 3. Flushes from a budstick. cultured in vitro.
(Photo: F. Arámburo) (Photo: L. Navarro, ECOPORT)
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3.4 Rootstock preparation
Etiolated rootstocks obtained in vitro nearly two weeks after sowing certified seeds
are ready for use in STG.
Working under aseptic conditions (air laminar flow box) and using sterile dissecting
instruments, the rootstock is prepared for STG: the cotyledons and axillary buds are
excised; the seedling is decapitated, leaving about 1.5 cm of the epicotyl; the root is
cut to a length of 4-6 cm. The removal of the bottom part of the root permits an
easier access of the grafted rootstock into the hole at the supportive paper platform.
As with the other steps of plant material handling, the skill of the person performing
STG is very important in the preparation of the rootstock.
The protocol for preparing the rootstock for STG by making an inverted-T incision is
provided in Chapter VII 7.3.3 of this manual.
3.5 Isolating the scion and performing the graft
The graft type most widely used in STG is the inverted-T incision at the end of the
decapitated epicotyl, although other methods are also used, such as the wedge cut
and the triangular shaped cut (Figures 4, 5 and 6).
Figure 4. Inverted-T Figure 5. Wedge cut. Figure 6. Shoot tip growing from incision. (Photo: John Da Graça, a triangular shaped cut.
ECOPORT) (Photo: F. Arámburo) Particularly for this step, the skill of the person performing the STG is crucial (Figure 7).
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The protocol for isolating the scion and performing the graft is provided in
Chapter VII 7.3.4 of this manual.
Figure 7. STG plant in vitro.
3.6 Care of in vitro STG plants
STG plants are kept at 27°C and exposed to 16 hours daily to illumination of 40-50
µEm-2s-1 (Figure 8) and 8 hours of darkness, or natural lighting. Histological studies
show that three days after placing the shoot tip on the rootstock seedling, there is
some callus development; initiation of vascular differentiation has been observed
seven days after grafting; and there is a complete vascular connection between both
parts eleven days after grafting.
Figure 8. STG plants growing in a culture room. (Photo: F. Arámburo)
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After 4-6 weeks, successful shoot-tip grafted plants are ready for their adaptation to
external environmental conditions.
At this stage, adventitious shoots usually emerge from the rootstock. Such shoots, in
addition to not serving the purpose of the STG, are an obstacle for the development
of the growing graft. Hence, periodic observation of the cultures is necessary, so that
the undesired shoots can be identified as soon as possible and removed with sterile
curved pointed-tip scissors (Metzenbaum scissors) in the laminar flow box. This is the
step at which trifoliate leaves of some of the most-used rootstocks (e.g. citrange) offer
the advantage of an immediate identification.
The protocol for removing rootstock sprouts is provided in Chapter VII 7.3.5 of this
manual.
3.7 Transfer of shoot-tip grafted plants to external environmental conditions
Scions of successful grafts should have at least two expanded leaves before being
transferred to external environmental conditions. This stage is usually attained by
4-6 weeks after the STG is done.
3.7.1 Transfer to pots
Shoot-tip grafted plants can be taken to external environmental conditions by
transferring them directly to pots with appropriate substrate or mixture of
substrates (Figure 9). In this case, STG plants are transferred to pots
containing artificial soil mix suitable for growing citrus. Taking into account that
STG plants have a poor root system, a well-sterilized and light substrate is
required. In this critical period, in order to reduce moisture loss, pots are
enclosed in polyethylene bags (secured with rubber bands) and placed in a
shaded area of a temperature-controlled greenhouse at 18-25°C. After 8-10
days, the bags are opened, and after a further 8-10 days, the bags are
removed and the plants are allowed to grow under standard greenhouse
conditions.
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Figure 9. STG plants transferred to pots. (Photo: C. N. Roistacher, ECOPORT)
In some laboratories there have been many losses in transplanting STG
plants, mostly due to poor growth. This problem can be overcome by re-
grafting.
3.7.2 Re-grafting
The development and growth of the STG plant is accelerated when a re-graft is
done on a vigorous rootstock (Figure 10).
For re-grafting, Citrus volkameriana Ten. & Pasq., Citrus macrophylla Wester
and ‘Rough’ lemon (Citrus jambhiri Lush.) are mostly used. These rootstocks
should be obtained from certified seeds (from the same source and for the
same reason explained for seeds used as rootstocks for STG) sown in pots or
bags and kept inside well-protected screenhouses and should have an
adequate diameter to perform re-grafting on them.
An alternative is to perform a normal T or patch cut on the potted rootstock, at
a height of 20-25 cm. The STG plant is taken out of the test tube and a patch-
shaped cut is done with a scalpel on the rootstock of the STG plant, which is
inserted into the T cut. The graft is covered with a parafilm or polyethylene
strip taking care not to affect the growth area of the STG (Figure 11). The area
of the re-graft is covered with transparent polyethylene to protect it from
19
dehydration. In some laboratories, the rootstock is bent to force the
development of the re-graft. About 20 days later, the polyethylene cover is
taken off, the parafilm strip is removed and the rootstock is decapitated.
Figure 10. Re-graft on vigorous rootstock. Figure 11. Re-graft covered with transparent polyethylene.
(Photo: C. N. Roistacher, ECOPORT) (Photo: F. Arámburo)
Good results have been obtained when re-grafting is done by performing a
side graft on a decapitated vigorous rootstock. This procedure is
described in Chapter VII 7.3.7 of this manual.
Re-grafting is useful because:
a high percentage of graft establishment is achieved
the poor root system of the STG is discarded
a very quick growing rate is achieved, and
the period between STG and the start of diagnostic tests is reduced.
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3.8 Plant maintenance
Each plant must be duly labelled and registered, with the corresponding data
regarding its origin, as well as information on the cultivar or accession and
rootstock, and the date of the operations carried out.
Shoot-tip grafted plants moved to external environmental conditions should be
maintained in screenhouses to ensure that they are protected from possible
disease vectors. Access to the facilities should be controlled and measures for
screenhouses that are part of a certified planting material production system
should be enforced.
While in the screenhouse, the plants should be checked frequently and given the
necessary agronomic treatments. The time required to obtain planting material to
start the essential diagnostic tests depends on the shoot-tip grafted cultivar and
the rootstock on which the shoot-tip grafting and re-grafting were done. When re-
grafting is done on vigorous rootstocks, it is possible to start indexing 3-4 months
after re-grafting.
Horticultural evaluation of the recovered plants is absolutely essential. While
there are no known reports of shoot-tip grafted plants expressing abnormal traits,
there is always a chance that a bud sport (=mutation) can occur or that a human
mistake has been made (e.g. in labelling).
The success rate for STG plants (= graft establishment) is 30-50%. It should be
noted that a single STG plant is enough to obtain healthy plant material from the
cultivar or accession, once diagnostic tests show that this plant is free of the
pathogens for which tests were performed. This plant would then be the point of
departure to obtain the necessary budwood replicating this material – grafting
always on rootstocks from certified seeds in each further graft – from which
enough planting material (buds) will be produced as grafted plants develop. It
does not mean that the objective of STG is to produce a single shoot-tip grafted
plant free of pathogens from a cultivar or accession. Successful establishment of
several healthy STG plants makes available a larger number of healthy
budwood. It is also useful to note that it is not necessary to obtain a large
21
number of shoot-tip grafted plants from the same source (infected cultivar or
accession).
Another point to note is the possible re-infection of the material: since the shoot-
tip grafting in vitro technique planting material free of pathogens is obtained from
an infected source, and the sanitary status of the material – both the original
STG plant as well as the budwood multiplied from it – can only be ensured if and
when it is maintained in aphid-proof screenhouses. In addition, strict measures
should be put in place to prevent the entrance of vectors and correct
management of the plants. Once certified plants from protected nurseries are
taken to the field, re-infection is possible due to vectors of pathogens: both man
and insects are main vectors of citrus diseases if phytosanitary and cultural
practices – particularly during pruning – are not observed.
Plants recovered by STG do not have juvenile characters (Figure 12), as long as
the shoot tips are excised from adult plants. Several thousand plants have been
obtained by STG in different laboratories, and all available data indicate that they
are true-to-type.
Figure 12. ‘Valencia 121’ orange obtained by STG, flowering one year after performing the graft in vitro.
22
3.9 Factors influencing shoot-tip grafting results
There are several factors that influence the percentage of STG plants obtained
and the recovery of healthy plants by STG.
3.9.1 Rootstock chosen and variety used as scion
As in all citrus grafts, the scion / rootstock compatibility is important for
bud-taking. Theoretically, any rootstock which is graft-compatible with the
shoot tip scion variety can be used for STG. The success of grafting is
partially influenced by the degree of compatibility. Though the rootstock
routinely used for STG is 'Troyer' citrange, others are also used, based on
compatibility, that is, 'Rough' lemon is recommended as rootstock when
the objective is to clean lemon varieties.
On the other hand, there is evidence that higher percentage of successful
STG plants are produced when grafted on etiolated rootstocks (seeds
germinated in darkness) than when grafted on rootstocks are obtained
under light, so in the standard procedure, etiolated rootstocks are used.
The age of the rootstock also influences grafting success: 12-16 days
after sowing in vitro is the optimal age for ‘Troyer’ and ‘Carrizo’ citrange
seedlings, which attain a height of 3-5 cm with a diameter of 1.6-1.8 mm
at the point of grafting. Stem height and diameter are more appropriate
parameters than age to determine the optimal stage of seedling
development for grafting.
3.9.2 Shoot tip size
There is evidence that increasing shoot tip size results in higher success rate
of grafts, but there is an inverse proportion between the shoot tip size and the
percentage of healthy plants obtained. The use of a shoot tip composed of
the apical meristem and subjacent tissue plus 2-3 primordial leaves and
measuring 0.1-0.2 mm from top to bottom, is recommended for routine STG
application. This size gives a realistic frequency of successful grafts and
healthy plants.
23
3.9.3 Pathogen
Most pathogens are easy to eliminate while others such as psorosis, concave
gum, impietratura, cristacortis and tatter leaf are more difficult. The recovery
rate of plants, free from the pathogens difficult to eliminate by STG, can be
increased by growing the shoot-tip source plants under relatively warm
conditions: placing the defoliated grafted plants – which are the source of
flushes for STG – in pots or bags in a growth chamber at constant 32°C, or
35°C during the day and 30°C during the night, >90% of pathogen-free STG
plants (including the difficult-to-eliminate pathogens) can be obtained.
3.9.4 Skill of the person performing STG
STG requires considerable dexterity and specific skills. As stated earlier, the
cuts made to the rootstock to perform the incision, the excision of the shoot
tip and its placement in the incision, must be done as quickly and as cleanly
as possible to avoid dehydration of the tissues and possible damage. On the
other hand, it is essential that the person performing the STG follows the
recommendations at each step of the procedure to ensure the appropriate
handling of instruments and plant materials used in STG.
24
IV. DIAGNOSTIC TESTS FOR PATHOGENS
Although diagnostic tests are not part of the shoot-tip grafting technique, it is
important to emphasize they are an essential complement of the work done. It
should never be assumed that a plant is healthy because it has been subjected
to a sanitation treatment. It is vital that every STG plant obtained be subjected
to diagnostic tests for pathogens to permit the sanitary certification of the planting
material produced. That is why it is impossible to bypass this step as an essential
complementary part of the work. In fact – as has been stated earlier – when
reference is made to a healthy plant, it means that such plant is free of pathogens for
which diagnostic test results were negative, also taking into account the reliability of
the diagnostic tests used.
At present, laboratory tests (microscopy; enzyme linked immunosorbent assay,
ELISA; sequential polyacrylamide gel electrophoresis, sPAGE; polymerase chain
reaction, PCR) are used to diagnose viruses, viroids, bacteria and phytoplasmas
causing severe diseases in citrus. However, there is an important group of diseases
that is little characterized and the diagnosis of which is limited to the use of indicator
plants, so bio-indexing to detect graft-transmissible diseases is a must, regardless of
the fact that it takes more time than laboratory tests. In relation to this topic, more
details can be found in the manual “Biological Indexing Procedures of Citrus Graft-
transmissible Pathogens (CGTPs)” (prepared under TCP-JAM-3302).
The selection of the diagnostic procedure for certain pathogen depends on the need
for quick and accurate results, the sensitivity and cost of the procedure as well as the
availability of specific reagents and the requirement of facilities and skilled
personnel.
Once the favourable sanitary status of the budwood obtained is confirmed, the
sanitary certification of the planting material can be provided and the healthy cultivar
or accession incorporated to the Protected Germplasm Block (Figure 13).
25
Figure 13. Protected Germplasm Block of citrus at Bodles Research Station in Jamaica.
In general, from the time of sowing the rootstock seeds in vitro until the essential
diagnostic tests are completed for those pathogens considered necessary, a
period of 16 to 24 months is required to obtain certified budwood (Figure 14). On
the other hand, it is a must to have the results of horticultural evaluations so the
certified budwood can be safely distributed to citrus growers.
26
Shoot-tip grafting: 6-8 weeks in vitro
Re-grafting: 3-4 months
Indexing procedures: 12-18 months
Figure 14. Time required to obtain certified budwood.
27
V. APPLICATIONS OF SHOOT-TIP GRAFTING IN VITRO
5.1 Recovery of pathogen-free citrus plant material
Shoot-tip grafting in vitro has proved to be the most effective method to obtain
pathogen-free citrus budwood and bud-lines free of all tested citrus pathogens.
STG is the basis for Clean Stock Programs in the production systems of
certified propagation material.
Its use guarantees disease-free status of a citrus germplasm collection,
subjecting the accessions to STG prior to establishment of the collection.
Thus, adequate conservation of these valuable plant genetic resources is
attained, making them more useful in research related to plant breeding and for
international germplasm exchange.
5.2 Recovery of pathogen-free plants of other woody species It is noteworthy that STG developed for citrus has also been applied to recover
pathogen-free germplasm in other tree species e.g. avocado (Persea americana
Mill.), grapevine (Vitis vinifera L.), peach (Prunus persica Batschi), cherry
(Prunus avium L.), apricot (Prunus armeniaea L.), almond (P. amygdalus L.),
apple (Malus plumila Mill.), camellia (Camellia japonica L.), pistachio (Pistacia
vera L.) and sequoia (Sequoia dendrongiganteum Buchholz).
5.3 Other applications of STG in plant species
STG has been applied to research on several species for various purposes –
recovery of transgenic plants, propagation, rejuvenation, study of graft union,
germplasm exchange – among these species, cotton (Gossypium hirsutum L.),
Protea cynaroides L., pear (Pyrus communis L. / Pyrus elaeagrifolia Pallas),
passion fruit (Passiflora edulis Sims), cashew (Anacardium occidentale L.),
prickly pear cactus (Estrada-Luna et al. 2002), olive (Olea europaea L.), carob
tree (Ceratonia siliqua L.), apricot, almond and others.
28
5.4 Separation of pathogens in mixed infections Citrus trees are often infected by several pathogens. As STG is most efficient to
eliminate some pathogens, it is possible to recover plants infected with one
pathogen from original trees infected with several pathogens, which is of interest
to Plant Pathology related research.
5.5 Studies on graft incompatibility and on histological and physiological aspects of grafting
Shoot-tip grafting has been useful for studying some incompatible grafts and has
contributed to better know or to an approach to diverse aspects of grafting.
5.6 Applications of STG in other citrus research
Shoot-tip grafting is turning into a very useful technique for propagation and
regeneration of elite genotypes in different research areas. As a matter of fact,
larger tips (up to 1 cm long) are used and different types of incisions are made to
attain close to 100% success rate for STG plants.
Within the research in which STG in citrus has found application are:
the regeneration of somatic hybrids in protoplast fusion which very
frequently produce embryos that would be lost as they do not produce
plants that could be transplanted to the soil
the regeneration of plants from irradiated tips in breeding research aimed
at reducing the number of seeds in fruits
the regeneration of haploid plants of particular value in citrus genetics and
genomics
the production of tetraploid stable plants of monoembryonic genotypes of
significant usefulness in breeding programmes
the regeneration of plants in experiments of somaclonal variation of adult
material as an interesting way to improve citrus
the regeneration of transgenic plants that most of the time show low
rooting efficiency, but can be achieved through STG, increasing the
efficiency of the genetic transformation protocols.
29
5.7 Importation of citrus germplasm: Quarantine Programs
Based on the use of shoot-tip grafting, a successful method through in vitro
cultures has been developed for citrus budwood introduction with a minimum risk
of importing diseases or pests and the safe movement of budwood within a
country. The only materials actually introduced into the country by this method
are the small apices used as scions in STG, on which this method is based. The
procedure is explained later.
30
VI. SAFE MOVEMENT OF CITRUS BUDWOOD
Navarro et al. (1984) proposed a method for the international exchange of citrus
planting material, based on in vitro cultures. The method has been used with
satisfactory results in several countries. It can be used to introduce new citrus
cultivars into the country and safely move citrus budsticks from one place to
another within the same country.
Budsticks are received in sealed transparent plastic bags that can be easily
inspected visually before opening the packages. If the results of the inspection
(no visible pests or any other disorder) permit to open the bags, then steps for
budstick culture in vitro detailed in Chapter VII 7.3.6 of this manual are followed.
The flushes obtained are used as the source of scions for STG. The remaining
material:
the contents of the package (any budstick discarded, fallen petioles and
the plastic bag)
the ends of the budsticks removed before placing the budsticks in vitro
the budsticks after removal of flushes
the remains of the flushes after taking the tips
wash water
or the complete package without opening, if so decided – taking into account the
results of the visual inspection – is destroyed in an autoclave, so the only part of
the introduced material that remains is the tip of a successful STG plant.
STG plants are maintained under supervision and control at the Post-entry
Station, until the diagnostic tests are complete and a decision is taken to
propagate the material.
This method based on in vitro cultures has several advantages over the
traditional quarantine method:
pests and diseases that may be present in the original material are
eliminated in the early stages of introduction, and this shortens the
quarantine period
31
test tubes or glass jars substitute the high cost special facilities of the
traditional quarantine method, and
the quarantine stations that adopt this procedure may be located at citrus
research stations instead of being located in isolated areas.
The procedure explained in Chapter VII 7.3.6 includes an alternative in which
thick test tubes are replaced by glass jars (lower cost, easy acquisition) and the
agar is replaced by river sand or zeolite as support (lower costs). Additionally,
the cultured budsticks are maintained in a room with natural light without artificial
illumination, with consequent energy-saving.
Considering all the advantages of the shoot-tip grafting in vitro over other
ways of obtaining pathogen-free budwood, and taking into account the
excellent results, both in cleaning and in the introduction of citrus
varieties, that have been obtained in many citrus growing countries, the
application of this technique is recommended as the basis for Clean Stock
and Quarantine Programs within the production systems of certified
propagation material that urgently need to be developed in countries
where current propagation methods do not provide the necessary sanitary
guarantees.
32
VII. REQUIREMENTS, CULTURE MEDIA AND PROTOCOLS
The materials and protocols related to shoot-tip grafting in vitro (STG) and re-
grafting STG plants onto vigorous rootstocks are described. Protocols related to the
essential diagnostic tests to which every STG plant obtained must be subjected, are
provided in the manual “Biological Indexing Procedures of Citrus Graft-transmissible
Pathogens (CGTPs)”, prepared as part of the activities of TCP-JAM-3302.
7.1 Requirements
7.1.1 Facilities
Laboratory for preparation of solutions and culture media
Culture room for maintenance of STG plants in vitro
Dark room or place for maintaining the rootstock seeds in vitro until the
etiolated seedlings are ready to be used for STG
Greenhouses for grafted plants as source of flushes for STG.
Greenhouses for re-grafted plants
7.1.2 Equipment
Laminar air flow box
Stereoscopic microscope
Water distillation machine
Autoclave
Technical balance
Analytical balance
Magnetic stirrer
pH-meter
Incubator
Refrigerator
Thermo-hygrograph
Air conditioning units
33
7.1.3 Reagents
Mineral Salts (for culture media) Magnesium sulphate heptahydrate (MgSO4.7H2O)
Manganese sulphate monohydrate (MnSO4.H2O)
Zinc sulphate heptahydrate (ZnSO4.7H2O)
Cupric sulphate pentahydrate (CuSO4.5H2O)
Calcium chloride dihydrate (Cl2Ca.2H2O)
Potassium iodide (KI)
Cobalt chloride hexahydrate (CoCl2.6H2O)
Ammonium nitrate (NH4NO3)
Potassium nitrate (KNO3)
Potassium phosphate (KH2PO4)
Boric acid (H3BO3)
Sodium molybdate dihydrate (Na2MoO4.2H2O)
Ferrous sulphate heptahydrate (FeSO4.7H2O)
EDTA disodium salt (Na2EDTA.2H2O)
Vitamins (for STG medium)
Thiamine.HCl
Pyridoxine.HCl
Nicotinic acid
Myo-inositol
Others
Sucrose
Agar-agar Oxoid No. 3
Sodium hydroxide (NaOH)
Hydrochloric acid (HCl)
Ethyl alcohol
Sodium hypochlorite (NaClO)
Tween 20
Potassium permanganate (KMnO4)
Formaldehyde
34
7.1.4 Glassware, instruments and other
Glassware
Test tubes 25 x 150 mm and polypropylene caps
Test tubes 38 x 200 mm and polypropylene caps, or glass jars appropriate for
budstick culture
Test tubes 15 mm diameter
Petri dishes (diameters 100 and 150 mm)
Graduated cylinders (10, 25, 50, 100, 250, 500, 1000, 2000 ml)
Beakers (25, 50, 100, 250, 500, 1000, 2000 ml)
Erlenmeyer flasks (250, 500, 1000, 2000 ml)
Volumetric flasks (250, 500, 1000 ml)
Amber bottles (25, 50, 100, 250, 500 ml)
Containers to freeze vitamin stock (5 and 10 ml)
Pipettes (5 and 10 ml)
Pipette pumps
Instruments
Straight forceps with grooves (13, 14.5, 16 and 20 cm long)
Curved pointed medium-size forceps (12-15 cm long)
Medium-size forceps with teeth
Optical pointed forceps (7 cm long)
Needle holders and needles, or handled needles or picks
Scalpels No. 7 or 3 and surgical blades No.11
Cuticle cutter
Razor blades
Beaver surgical handle (for a razor blade sliver) (Figure 15)
Curved pointed-tip scissors (Metzenbaum scissors 7” curved)
Pruning shears
Figure 15. Beaver surgical handle with razor blade sliver: important tool for STG. (Photo by: C. N. Roistacher, ECOPORT)
35
Others
Racks for test tubes
Sodium hypochlorite
Paper towel
Cheesecloth
Scissors
Aluminum foil
Parafilm or plastic wrap
Filter paper
Whatman hardened filter paper
Wash bottles
Autoclave tape
Permanent all-surface markers
Burners (alcohol / gas).
Lighters
Lab coats
Dust masks
Timer
Stir-bars
Brush
Bottle cleaners
Detergent
Wax
River sand or zeolite (sifted 1.6 mm2)
Fungicide
Miticide
Grafting knives
Transparent polyethylene tape for grafting
Labels to identify plants
Plastic pots and/or polyethylene bags
36
7.2 Stock Solutions and Culture Media
7.2.1 Stock Solutions
7.2.1.1 MS Mineral Salts (MS: Murashige and Skoog, 1962)
Dissolve salts separately in distilled water; if necessary, warm those that are
marked *. Combine salts of each group: sulphates, halides, nitrates, P B Mo
and Na Fe EDTA. Allow the mixture to cool. Adjust the final volume to 500
ml. Store the mixture in an amber container in a refrigerator. Store nitrates
in a dark place at room temperature. Check solutions and discard if
contamination is detected. Do not keep solutions longer than 3 months.
Sulphates
MgSO4.7H2O -----------------18.5 g *
MnSO4.H2O ------------------- 0.845 g
ZnSO4.7H2O ------------------ 0.430 g
CuSO4.5H2O ------------------ 0.00125 g
Halides
Cl2Ca.2H2O -------------------- 22.0 g
KI --------------------------------- 0.0415 g
CoCl2.6H2O ------------------- 0.00125 g
Nitrates
NH4NO3 ----------------------- 82,5 g *
KNO3 --------------------------- 95,0 g
P B Mo
KH2PO4 ----------------------- 8.5 g
H3BO3 ------------------------- 0.310 g
Na2MoO4.2H2O ------------- 0.0125 g
Na Fe EDTA
FeSO4.7H2O ---------------- 1.392 g
Na2EDTA.2H2O ------------ 1.862 g
37
7.2.1.2 Vitamins
Dissolve separately in distilled water, adjust to 250 ml, and freeze in aliquots of
5 and 10 ml (Figures 16 and 17).
Thiamine ---------------------- 5 mg
Pyridoxine ------------------- 25 mg
Nicotinic acid --------------- 25 mg
Figure 16. Dispensing vitamin Figure 17. Vitamin solutions distributed in solution in tubes. aliquots in the freezer.
7.2.2 Culture Media
7.2.2.1 Seed Germination (1 litre)
a) Weight 30 g of sucrose, pour it into a 1 litre beaker and dissolve in
distilled water.
b) Pour 10 ml of each MS salt stock solution.
c) Bring to 900 ml with distilled water.
d) Adjust to pH 5.7.
e) Pour into graduated cylinder and bring volume up to 1litre.
f) Add between 5 and 7 g of agar (depending on the quality of agar
being used).
g) Dissolve the agar by heating.
h) Distribute in aliquots of 25 ml into 25 x 150 mm test tubes.
i) Cap the test tubes.
j) Sterilize in autoclave 15 minutes at 121°C.
38
7.2.2.2 STG (liquid medium, 1 litre)
a) Weight 75 g of sucrose, pour it into a 1 litre beaker dissolve in distilled
water.
b) Weight 100 mg of myo-inositol, dissolve in distilled water and pour into
the beaker.
c) Pour 10 ml of each MS salt stock solution.
d) Add 10 ml of vitamins (frozen in aliquots) .
e) Bring to 900 ml with distilled water.
f) Adjust to pH 5.7.
g) Pour into graduated cylinder and bring volume up to 1 litre.
h) Distribute in aliquots of 20 ml into 25 x 150 mm test tubes (Figure 18).
Figure 18. Dispensing STG liquid medium in test tubes.
i) Insert the paper supportive platform (explained below). Lower the
support around 1 cm from the top of the tube (Figure 19).
Figure 19. inserting paper supportive platforms in the test tubes.
39
j) Cap the test tubes.
k) Sterilize in autoclave for 15 minutes at 121°C.
Paper supportive platform. This platform will provide support for the STG
plant in the liquid medium. For the platform, Whatman filter paper is used.
1. Cut circles of 5-7 cm diameter.
2. Fold flaps of the paper circle over the mouth of a 15 mm diameter
test tube, flat against the tube.
3. Punch a hole in the centre of the paper using a tooth-pick or a
needle (with handle) (Figure 20). The diameter of the hole should
be appropriate for insertion of the STG plant, in accordance with
the diameter of the root that will be placed into the liquid medium,
and to hold the STG plant with its epicotyl above the platform.
Figure 20. Preparing paper supporting platform for STG plants.
4. Punch another hole opposite to the previous hole.
5. Place paper supportive platform into 25 x 150 mm test tube with
liquid medium, pushing until around 1 cm deep inside the tube.
7.2.2.3 Budstick Culture
a) Pour 10 ml of each MS salt stock solution in a 1 litre beaker.
b) Bring to 900 ml with distilled water.
c) Adjust to pH 5.7.
d) Adjust the volume to 1 litre
40
A) If test tubes 38 x 200 mm will be used:
a) Add between 7 and 10 g of agar (depending on the quality of
agar being used).
b) Dissolve the agar by heating.
c) Distribute in aliquots of 50 ml into the test tubes.
d) Cap the test tubes.
e) Sterilize in autoclave for 15 minutes at 121°C.
B) If glass jars with zeolite or river sand are being used:
a) Distribute enough liquid solution into the jars to reach the top level
of the zeolite or sand (volume depending on the jar size).
b) Cover each jar with plastic caps or aluminium paper.
c) Sterilize in autoclave for 15 minutes at 121°C.
41
7.3 Protocols
While working under the laminar air flow box it is important to take necessary
measures to ensure the aseptic conditions that are essential for each in vitro culture
technique: wash hands and spray with 70% ethanol, wear laboratory coats and dust
mask, clean the laminar air flow box with 70% ethanol, appropriate manipulation of
materials and instruments.
Turn on the laminar air flow box at low speed, around 30 minutes before start of work.
When starting work under the laminar air flow box, turn off the ultraviolet light and
switch from low to high speed.
Light the burner. Make sure that a marker is at hand to make the notations on the
test tubes, and also strips of parafilm or plastic wrap to seal the caps.
When wrapping the top of each tube around the base of the cap with parafilm in the
laminar air flow box, ensure that the side of the parafilm touching the paper is what is
used against the glass.
It is necessary to be familiar with the operation of the stereomicroscope to make the
best use of its illumination and magnification, and to work comfortably through each
step that requires its use.
Between one STG and the next, the end of the Beaver handle that holds the sliver of
the razor blade must be dipped into the test tube with 1% sodium hypochlorite
solution and then rinsed by immersion in tubes with sterile distilled water. The
remaining instruments should be flamed in order to ensure their sterility.
Several sets of sterile instruments should be available in order to replace them after
every 2-3 STG being performed, or in case of a wrong manipulation.
After every four STG, it is recommended that the Petri dish, with double filter paper on
which work is being done, be replaced.
42
7.3.1 In vitro sowing of seeds from the selected rootstock
(two weeks before performing STG)
1. Wash hands and spray with 70% ethanol.
2. Clean the laminar air flow box with 70% ethanol.
3. Lay out the following supplies (spray them with 70% ethanol as they are
being placed inside the laminar air flow box):
a) bottles of sterile distilled water
b) sterile forceps
c) beaker to collect solution and washing water (waste beaker)
d) 0.7% sodium hypochlorite solution + 0.1% Tween 20 wetting agent
e) sterile Petri dishes
f) test tubes with culture medium for seed germination
g) spirit lamp or burner.
Working on the laboratory bench:
Extract the seeds from the fruits (Figures 21 and 22) and wash them under running
water until the mucilage is removed. Though the best choice is newly-extracted
seeds (from rootstock fruits), rootstock seeds that are adequately preserved in
accordance with established procedures, can also be successfully used.
Figure 21. ‘Carrizo’ fruit with seeds. Figure 22. Macrophylla fruit with seeds.
a) Remove manually the external and internal teguments of each seed. Work
carefully to avoid causing damage to the embryo(s) (Figure 23).
43
b) Peel away the outer seed coat, starting from the chalazal end (which is the
end opposite the embryo = micropylar end) (Figure 24).
c) Place seeds without outer coat on moist filter paper in a Petri dish.
d) Peel away the inner seed coat, starting from the chalazal end (Figure 25).
Figure 23. Newly extracted ‘Carrizo’ seeds. Figure 24. Peeling seed coats.
Figure 25. ‘Carrizo’ seeds: with both seed coats (left); with the inner seed coat (center); without seed coats (right).
1. Place peeled seeds on moist filter paper in a Petri dish.
2. Wash hands and spray with 70% ethanol.
3. Place group of 10-20 seeds onto a square of cheesecloth.
4. Wrap the seeds in the square of cheesecloth (Figure 26).
Figure 26. Seeds ready for surface sterilization.
5. Place seed package(s) in a beaker.
6. Spray the beaker with 70% ethanol; place it in the laminar air flow box.
44
Working under the laminar air flow box:
7. Pour the 0.7% sodium hypochlorite solution + 0.1% of Tween 20 into the
beaker with the packages of seeds (Figure 27): surface-sterilize the seeds for
10 minutes by immersion in this solution.
Figure 27. Surface sterilization of seeds with 0.7% NaClO.
8. After 10 minutes, discard the solution in the waste beaker.
9. Rinse several times with sterile distilled water.
10. Place the package(s) in a sterile Petri dish.
11. Using two forceps, open the seed package.
12. Using a large forceps, sow 1-2 seeds in each test tube, placing the micropylar
end inside the medium (Figures 28 (a and b) and Figure 29).
13. Cap the test tube.
Figures 28 a and b. Sowing seeds at the laminar air flow box.
45
Figure 29. ‘Carrizo’ seeds in test tubes with medium for seed germination.
14. Wrap the top of each tube around the base of the cap with parafilm or plastic
wrap.
15. Label each batch: rootstock and date.
16. Maintain cultures at 27-30°C and in constant darkness for around two weeks
(Figure 30) by which time the seedlings reach optimum development to be
used for STG, depending on the rootstock used (Figure 31).
17. Check twice a week for contamination. Discard immediately any tube with
contamination.
Important. The use of certified rootstock seeds should be guaranteed.
Figure 30. ‘Carrizo’ citrange seedlings growing in vitro in the dark.
Figure 31. ‘Carrizo’ citrange seedlings ready to be used for STG.
46
7.3.2 Collecting and surface sterilizing flushes
1. Wash hands.
2. Collect vegetative flushes of 1-3 cm long from the selected flush source
(actively-flushing trees, plants in bags or pots previously defoliated,
budsticks cultured in vitro).
3. Place flushes in Petri dishes with moist filter paper (Figure 32) or Ziplocs
bags.
Figure 32. Collected flushes on a Petri dish.
4. Label each Petri dish: selection or variety collected.
In the laboratory: 5. Wash hands and spray with 70% ethanol.
6. Clean the laminar air flow box with 70% ethanol.
7. Lay out the following supplies (spray them with 70% ethanol as they are
being placed inside the laminar air flow box):
a. bottles of sterile distilled water
b. sterile forceps
c. beaker to collect solution and washing water (waste beaker)
d. 0.25% sodium hypochlorite solution + 0.1% of Tween 20 wetting agent
e. sterile Petri dishes
f. spirit lamp or burner.
47
Working on the laboratory bench:
8. Remove the larger leaves of the flush with the help of a fine pointed
forceps and pinch off the terminal, approximately 1 cm long (Figure 33).
9. Place flush terminals on moist filter paper in a Petri dish (Figure 34).
Figure 33. Removing the larger leaves Figure 34. Flush terminals on a Petri dish. of the flush.
10. Place group of 10-15 flush terminals onto a square of cheesecloth. Flush
terminals may be positioned so that they are all facing the same way (to
make it easier to pick them up later).
11. Wrap the group of flush terminals in the square of cheesecloth (Figure
35).
12. Place flush terminal package(s) in a beaker.
13. Spray the beaker with 70% ethanol and place it in the laminar air flow box.
Figure 35. Flush terminals ready for surface sterilization.
48
Working under the laminar air flow box:
14. Pour the 0.25% sodium hypochlorite solution + 0.1% of Tween 20 into the
beaker with the flush terminal packages (Figure 36): surface-sterilize the
flush terminals for 10 minutes by immersion in this solution.
Figure 36. Surface sterilization of flush terminals with 0.25% NaClO.
18. After 10 minutes, discard the solution in a waste beaker.
19. Rinse several times with sterile distilled water.
20. Place the package(s) in a sterile Petri dish.
21. Using two forceps open the flush terminal package. Keep the Petri dish
covered except at the moment of taking one terminal flush to be used as
source of scion for STG.
7.3.3 Rootstock preparation
Following rootstock preparation, the next step: performing the graft is done
immediately after. Therefore, it is necessary to have all items to perform both
steps in the laminar air flow box.
1. Lay out the following supplies (spray them with 70% ethanol as they are being
placed inside the laminar air flow box):
a) sterile dissection instruments: forceps, scalpel with blade No. 11, Beaver
handle with sliver of razor blade
b) bottles of sterile distilled water
c) sterile Petri dishes with double filter paper
49
d) test tube with 1% sodium hypochlorite solution
e) test tubes (2) with sterile distilled water
f) test tube with ethanol
g) rack of test tubes with etiolated rootstock seedlings ready for STG
h) rack of test tubes with liquid culture medium for STG plants
i) stereomicroscope
j) spirit lamp or burner.
Working under the laminar air flow box:
2. Using sterile distilled water, moisten the double filter-paper of the Petri dish
(bottom) on which the work will be done (Figure 37).
3. Use a large forceps to remove the rootstock seedling from the test tube and
place it on the Petri dish (Figure 38).
Figure 37. Removing the rootstock Figure 38. Starting rootstock preparation seedling from the test tube. on a Petri dish with moistened double filter filter paper.
4. Using the forceps and scalpel with No. 11 blade:
a. decapitate the rootstock seedling leaving about 1.5 cm of the epicotyl
b. shorten the root to 4-6 cm and remove secondary roots (Figure 39)
c. remove the cotyledons and their axillary buds, under the
stereomicroscope
d. place cut-off pieces to one side on the Petri dish.
50
Figure 39. Etiolated ‘Troyer’ citrange seedling after growing
in the dark for 14 days (left); rootstock seedling ready for performing the graft (right).
5. Holding the seedling firmly with a forceps, perform the incision for the graft
under the stereomicroscope, using the scalpel with No. 11 blade: to make an
inverted-T incision, start by making a 1 mm-long vertical cut at the point of
decapitation, followed by a 1-2 mm-wide horizontal incision (Figures 40 a and
b). The cuts should be as “clean” as possible. The cuts are made through the
cortex and the flaps are lifted slightly to expose the cortical surface.
Figure 40 a and b. Performing the inverted-T incision under the stereomicroscope.
6. Place the seedling aside in the Petri dish, away from light. The rootstock is
ready to be grafted.
51
7.3.4 Isolating the scion and performing the graft
Isolating the shoot tip to be grafted and performing the graft must be conducted as
quickly and carefully as possible to avoid dehydration of the tissues or damaging the
apex.
1. Using a forceps take a single flush terminal from the Petri dish and place it on
the Petri dish (with double filter paper) where the rootstock is ready.
2. Holding the flush terminal as close as possible to the tip area, remove small
leaves and primordia with the help of a handled needle, a fine pointed forceps
or other appropriate instrument – based on the preference of the person
performing the STG – leaving the meristem and 2-3 primordial leaves (Figure
41).
3. By using the tool specially prepared for this technique (a sliver of razor blade
on a Beaver handle) excise the scion: shoot tip composed of the apical
meristem and subjacent tissue plus two or three primordial leaves (0.1-0.2
mm long) (Figures 42 and 43). Keep the scion on the sliver razor blade.
Figure 41. Isolating the scion.
52
Figure 42. Meristem, subjacent tissue Figure 43. Shoot tip excised And two primordial leaves (over the dotted ready to be grafted. line) ready to be excised for its use as scion in STG.
4. Using a forceps, place the rootstock at the centre of the Petri dish for proper
lighting (Figure 44).
5. Under the stereomicroscope, and holding the rootstock steady, place the
shoot tip with the basal cut surface in contact with the horizontal cortical
surface of the incision performed on the rootstock, sliding the tip off the sliver
razor blade onto the rootstock (Figure 45).
Figure 44. Performing the graft.
Figure 45. Shoot tip in the inverted-T incision.
53
Important. The cuts for the preparation of the rootstock and the excision of the shoot
tip should be as perfect as possible and the shoot-tip grafting steps should be done
as quickly as feasible to avoid drying-out of the tissues, particularly the sensitive tip.
6. Using long forceps, place the shoot-tip grafted plant on a test tube with liquid
medium, threading the root into the hole at the center of the paper supportive
platform; using the forceps, push the platform with the STG plant down until
the top of the support is level with the surface of the liquid medium (Figure 46
a and b).
7. Cap the test tube.
Figure 46 a and b. Inserting the root into the hole at the center
of the paper supportive platform.
7. Wrap the top of each tube around the base of the cap with parafilm or plastic
wrap in the laminar air flow box.
8. Label each tube: scion/rootstock and date.
9. Keep cultures at around 27°C, exposed daily to 16 hours of light at
45 µEm-2s-1 (about 1 000 lux) and 8 h darkness, or natural lighting.
10. Check weekly and record contamination, colour of scion (green or brown),
growth and appearance of rootstock sprouts (Figure 47). Discard any dead or
contaminated STG plant. Trim rootstock sprout as needed.
54
Figure 47. Clementine on Citrus macrophylla three weeks after STG.
7.3.5 Removing rootstock sprouts
1. Wash hands and spray with 70% ethanol.
2. Clean the laminar air flow box with 70% ethanol.
a. Lay out the following supplies (spray them with 70% ethanol as they
are being placed inside the laminar air flow box): sterile large forceps
b. sterile Metzenbaum scissors and sterile Petri dishes
c. test tubes in which rootstocks sprouts are growing.
3. Pick up culture tube and remove the cap (Figure 48).
Figure 48. Rootstock sprout at the top of the STG plant to be removed
55
4. With large forceps, pull the paper supportive platform with the STG plant to
the top of the tube.
5. Hold the plant with the forceps and remove any rootstock sprout using the
Metzenbaum scissors (Figure 49).
Figure 49. Removing the rootstock sprout with the Metzenbaum scissors. 6. Using large forceps push the support down until the top of the support is
level with the surface of the liquid medium.
7. Cap the test tube.
8. Wrap the top of the tube around the base of the cap with parafilm or plastic
wrap.
9. Return the STG plant to the rack in which STG plants are kept.
10. Record date and action.
56
7.3.6 Budstick culture in vitro
As source of flushes for STG in a Clean Stock Program
Budsticks of 15-20 cm long and 4-8 mm diameter are taken from plants to be
subjected to STG (Figures 50 and 51).
Figure 50. Collecting budsticks in the field to be used as source of flushes for STG. In the laboratory, a pruning shears is used to remove the leaves but the petioles are
retained (Figure 52) so that when the drop of the petioles occur, the wounds heal
naturally and in this way the possibility of damage that can be caused through the
sterilization process using varying sterilizing agents is reduced.
Figure 51. Budstick collected. Figure 52. Removing the leaves. The budsticks are carefully brushed with detergent, rinsed with running water and
treated with a fungicide and miticide, air dried (Figure 53) and saved in transparent
plastic bags (Figure 54). The bags are sealed and placed at room temperature.
57
Figure 53. Budsticks after treatment Figure 54. Budsticks in plstic bags. with fungicide and miticide. After petioles fall off inside the transparent plastic bags in which the budsticks were
kept (Figure 55), the following procedure should be carried out.
Figure 55. Budsticks with fallen petioles (ten days after introduction into plastic bags).
1. Wash hands and spray with 70% ethanol.
2. Clean the laminar air flow box with 70% ethanol.
3. Lay out the following supplies (spraying them with 70% ethanol as they are
being placed inside the laminar air flow box):
a. bottles of sterile distilled water
b. sterile forceps
c. sterile pruning shears
d. 70% ethanol for budstick surface sterilization
e. 2% sodium hypochlorite solution + 0.1% Tween 20 for budstick surface
sterilization
f. beaker to collect solution and wash water (waste beaker)
g. sterile Petri dishes (150 mm diameter)
58
h. test tubes 38 x 200 mm with solidified medium, or glass jars with zeolite or
river sand + liquid medium, for budstick culture
i. spirit lamp or burner.
4. Remove budsticks from the bags. Discard any budstick with damage or
contamination.
5. Brush the budsticks carefully with detergent and running water.
6. Seal the ends of the budsticks with melted wax (Figure 56).
Figure 56. Sealing the ends of a budstick with melted wax.
7. Place the budsticks in a graduated cylinder.
8. Spray the cylinder with 70% ethanol and place it in the laminar air flow box.
Working under the laminar air flow box:
9. Pour the 70% ethanol into the cylinder to cover the budsticks.
10. After 2 minutes, discard the ethanol in a waste beaker.
11. Pour the 2% sodium hypochlorite solution + 0.1% of Tween 20 into the
cylinder to cover the budsticks (Figure 57).
12. After 20 minutes, discard the solution in a waste beaker.
13. Rinse several times with sterile distilled water (Figure 58).
14. Hold a budstick with sterile forceps and remove its ends covered with wax
using a sterile pruning shears. Slant cuts are recommended (Figure 59).
59
Figure 57. Budstick surface sterilization by immersion in 2% NaClO.
Figure 58. Surface sterilization: rinsing budsticks for their culture in vitro.
Figure 59. Removing the waxed ends of budsticks.
60
15. Place the budstick vertically into the test tube or jar introducing the basal end
into the support (Figure 60).
Figure 60. Placing a budstick in a jar for its culture in vitro.
16. Maintain at 30 ± 2°C, exposed to 16 hours of light (minimum illumination of 45
µEm-2s-1) and 8 hours of darkness, or natural lighting, until the growth of
appropriate flushes for STG (Figures 61 a and b).
Figures 61 a and b. Budsticks cultured in vitro using agar (a) and zeolite (b) as support.
61
17. The flushes obtained (generally 10-15 days after the culture of the budsticks
and during several weeks, depending on the citrus species, varieties and
other factors) can be used as the source of scions for STG (Figure 62).
Figure 62. Flushes ready for STG produced by a budstick cultured in vitro. (Photo: L. Navarro) Citrus budwood importation/safe movement within a country (see Chapter VI) Preliminary inspection: budsticks should be visually inspected without opening the
bag.
1. If found abnormal or contaminated, or infested with living pests, the entire
package should be destroyed by autoclaving.
2. If after the visual inspection it is considered appropriate to move to the
procedure for budwood introduction, the procedure for budstick culture in vitro
described above can be followed.
Important: tissue destruction. It is important that all that remains in each step of the
procedure be destroyed in autoclave: the bags with the petioles, the waxed ends of
the budsticks, the budsticks after taking the flushes, what remains of the flushes after
taking the tips off, and wash water, so the only part introduced is the shoot tip grafted
of each STG plant cultured in vitro.
62
7.3.7 Re-grafting
1. Remove the STG plant from the test tube (Figure 63).
2. Make a wedge on the rootstock of the STG plant using a razor blade
(Figure 64).
3. Decapitate the vigorous rootstock growing in pot and perform a wedge cut.
4. Fit the rootstock of the STG plant on the wedge cut of the vigorous
rootstock, wrap and fasten with parafilm (Figure 65).
5. Place three wire sticks vertically into the pot introducing the end / extreme
into the substrate (Figures 66 and 67).
6. Cover with a transparent polyethylene bag to protect the re-graft from
dehydration.
7. Close with a rubber band.
8. Label: STG scion / rootstock, vigorous rootstock and dates.
9. Place in a shaded area of a temperature-controlled greenhouse at 18-
25°C.
10. After two weeks, open the bag for a short while. Over the next few days,
leave the bag open for longer period. After one week the bag is removed
and the plant allowed to grow under standard greenhouse conditions.
11. Irrigate with tap water two or three times a week, depending on the
necessities of the plant, during the first three weeks.
12. Remove adventitious shoots emerging from the rootstocks.
13. Based on the development of the re-grafted plants, follow cultural practices
established for nursery plants.
14. Plants can be moved from the temperature-controlled greenhouse to a
screenhouse protected with aphid-proof screens and strict measures to
prevent the entrance of vectors and appropriate management.
Important:
Cuts should be as perfect as feasible; a razor blade is preferred over of
a scalpel or grafting knife because the razor blade is thinner and tissues
are less damaged.
Handle with care during all steps, to avoid damaging the STG.
Avoid moving the re-grafted plants during at least the first four weeks.
63
Figure 63. STG plant ready for re-grafting.
Figure 64. Vigorous rootstock for re-grafting: 4-6 mm diameter; razor blade at the decapitation point (left); rootstock
decapitated and wedge cut performed (right).
64
Figure 65. Placing the STG on the vigorous rootstock: fitting the rootstock of the STG plant to the wedge cut made on the vigorous rootstock in pot (left); STG fastened with parafilm (right).
Figure 66. Placing three wire sticks for holding the plastic bag (left); covering with transparent plastic bag (right).
65
Figure 67. STG one week after re-grafting.
IMPORTANT. It is vital that every STG grafted plant obtained be subjected to
diagnostic tests for pathogens.
66
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Photos in this manual were taken by the authors – most of them during their
missions in Jamaica as part of TCP-JAM-3302 and of TCP-BZE-3402 in Belize –
except when another source is specified.
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IX. ACKNOWLEDGEMENTS The authors would like to express their pleasure for having had the opportunity to
contribute with their experience to the Belizean citrus industry throughout the Project
TCP/BZE/3402 “Assistance to Manage Huanglongbing in Belize”. They acknowledge
the FAO Representation in Jamaica (for Jamaica, Bahamas and Belize) and the FAO
Representation in Cuba, and to the staff of the Citrus Growers Association (CGA) and
its research arm, the Citrus Research and Education Institute (CREI) in Belize
involved in the Project. They also wish to acknowledge the Project TCP-JAM-3302
“Assistance to Manage Citrus Greening in Jamaica” under which they provided similar
assistance to several key personnel at the Bodles Research Station in Jamaica in
2012. They are particularly grateful to the trainees in both Caribbean countries for
their interest and concern on the establishment of STG for obtaining pathogen-free
citrus budwood. Special thanks to Dr. Vyjayanthi Lopez (Plant Production and
Protection Officer at the FAO Sub-regional Office for the Caribbean, and Lead
Technical Officer for both Projects) for her support and concern during the activities
related to the missions of the consultants, and for her careful revision of this manual.
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