SHOOT-TIP GRAFTING IN VITRO · 2021. 2. 8. · SHOOT-TIP GRAFTING IN VITRO TO OBTAIN CITRUS...

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

mailto:[email protected]

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)

http://ecoport.org/ep?SearchType=pdb&PdbID=35486

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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

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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.


20

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.


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.


7. Lay out the following supplies (spray them with 70% ethanol as they are


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


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.


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).


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



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.


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).



3. Lay out the following supplies (spraying them with 70% ethanol as they are


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.


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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ASBVd-infected Avocado (Persea americana) plants. Plant Cell, Tissue and Organ Culture 80:179-185.

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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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