SHOOT-TIP GRAFTING IN VITRO TO OBTAIN CITRUS PLANTING MATERIAL FREE OF GRAFT-TRANSMISSIBLE PATHOGENS AND FOR THE SAFE MOVEMENT OF CITRUS BUDWOOD

Transcription

1 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

2 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) Prepared under the Project TCP-BZE Assistance to Manage Huanglongbing in Belize FAO, 2014 Food and Agriculture Organization of the United Nations Belize, 2014

3 Table of Contents INTRODUCTION MANDATORY CITRUS CERTIFICATION PROGRAMS Quarantine Program Clean Stock Program Certification Program... 4 II. WAYS OF OBTAINING PATHOGEN-FREE BUDWOOD SELECTION OF TREES ON THE FIELD (CLONAL SELECTION) PLANTS OF NUCELLAR ORIGIN THERMOTHERAPY SHOOT-TIP GRAFTING IN VITRO III. SHOOT-TIP GRAFTING IN VITRO (STG) SELECTION OF MOTHER TREES FROM THE LOCAL CULTIVARS (CLEAN STOCK PROGRAM) OBTAINING IN VITRO ROOTSTOCKS SOURCES OF FLUSHES FOR SHOOT TIPS ROOTSTOCK PREPARATION ISOLATING THE SCION AND PERFORMING THE GRAFT CARE OF IN VITRO STG PLANTS TRANSFER OF SHOOT-TIP GRAFTED PLANTS TO EXTERNAL ENVIRONMENTAL CONDITIONS Transfer to pots Re-grafting PLANT MAINTENANCE FACTORS INFLUENCING SHOOT-TIP GRAFTING RESULTS Rootstock chosen and variety used as scion Shoot tip size Pathogen Skill of the person performing STG IV. DIAGNOSTIC TESTS FOR PATHOGENS V. APPLICATIONS OF SHOOT-TIP GRAFTING IN VITRO RECOVERY OF PATHOGEN-FREE CITRUS PLANT MATERIAL RECOVERY OF PATHOGEN-FREE PLANTS OF OTHER WOODY SPECIES OTHER APPLICATIONS OF STG IN PLANT SPECIES SEPARATION OF PATHOGENS IN MIXED INFECTIONS STUDIES ON GRAFT INCOMPATIBILITY AND ON HISTOLOGICAL AND PHYSIOLOGICAL ASPECTS OF GRAFTING APPLICATIONS OF STG IN OTHER CITRUS RESEARCH IMPORTATION OF CITRUS GERMPLASM: QUARANTINE PROGRAMS VI. SAFE MOVEMENT OF CITRUS BUDWOOD VII. REQUIREMENTS, CULTURE MEDIA AND PROTOCOLS REQUIREMENTS Facilities Equipment Reagents Glassware, instruments and other STOCK SOLUTIONS AND CULTURE MEDIA Stock Solutions Culture Media PROTOCOLS In vitro sowing of seeds from the selected rootstock Collecting and surface sterilizing flushes Rootstock preparation Isolating the scion and performing the graft Removing rootstock sprouts Budstick culture in vitro VIII. BIBLIOGRAPHY... 66

4 IX. ACKNOWLEDGEMENTS

5 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 diseasecausing 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. 2

6 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 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 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: 3

7 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 humaninduced 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 grafttransmissible 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 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 4

8 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 5

9 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 6

10 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 Quarantine Program Importation of varieties Clean Stock Program Local selections Shoot-tip grafting Certification Program o Protected Germplasm Block o Protected Foundation Block o Protected Multiplication Block o Certified Nurseries o Lots for Horticultural Evaluation o Seed Source Trees 7

11 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. 8

12 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 9

13 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 ( 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 10

14 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. 11

15 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 12

16 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 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. 13

17 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 controlledtemperature 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 of this manual. Figure 2. Stripped plant producing flushes. (Photo: F. Arámburo) Figure 3. Flushes from a budstick. cultured in vitro. (Photo: L. Navarro, ECOPORT) 14

18 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 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). 15

19 The protocol for isolating the scion and performing the graft is provided in Chapter VII 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 µem -2 s -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) 16

20 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 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 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 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. 17

21 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 regrafting 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 cm. The STG plant is taken out of the test tube and a patchshaped 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 18

22 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 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. 19

23 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 regrafting 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 20

24 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 shoottip 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. 21

25 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 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: 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 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 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 mm from top to bottom, is recommended for routine STG application. This size gives a realistic frequency of successful grafts and healthy plants. 22

26 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 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. 23

27 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 Grafttransmissible 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). 24

28 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. 25

29 Shoot-tip grafting: 6-8 weeks in vitro Re-grafting: 3-4 months Indexing procedures: months Figure 14. Time required to obtain certified budwood. 26

30 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. 27

31 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. 28

32 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. 29

33 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 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 30

34 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 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. 31

35 VII. REQUIREMENTS, CULTURE MEDIA AND PROTOCOLS The materials and protocols related to shoot-tip grafting in vitro (STG) and regrafting 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 Requirements 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 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 32

36 7.1.3 Reagents Mineral Salts (for culture media) Magnesium sulphate heptahydrate (MgSO4.7H 2 O) Manganese sulphate monohydrate (MnSO 4. H 2 O) Zinc sulphate heptahydrate (ZnSO 4. 7H 2 O) Cupric sulphate pentahydrate (CuSO 4. 5H 2 O) Calcium chloride dihydrate (Cl 2 Ca.2H 2 O) Potassium iodide (KI) Cobalt chloride hexahydrate (CoCl 2.6H 2 O) Ammonium nitrate (NH 4 NO 3 ) Potassium nitrate (KNO 3 ) Potassium phosphate (KH 2 PO 4 ) Boric acid (H 3 BO 3 ) Sodium molybdate dihydrate (Na 2 MoO 4.2H 2 O) Ferrous sulphate heptahydrate (FeSO 4.7H 2 O) EDTA disodium salt (Na 2 EDTA.2H 2 O) 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 (KMnO 4 ) Formaldehyde 33

37 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) 34

38 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 mm 2 ) Fungicide Miticide Grafting knives Transparent polyethylene tape for grafting Labels to identify plants Plastic pots and/or polyethylene bags 35

39 7.2 Stock Solutions and Culture Media Stock Solutions 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 MgSO 4.7H 2 O g * MnSO 4. H 2 O g ZnSO 4. 7H 2 O g CuSO 4. 5H 2 O g Halides Cl 2 Ca.2H 2 O g KI g CoCl 2.6H 2 O g Nitrates NH 4 NO ,5 g * KNO ,0 g P B Mo KH 2 PO g H 3 BO g Na 2 MoO 4.2H 2 O g Na Fe EDTA FeSO 4.7H 2 O g Na 2 EDTA.2H 2 O g 36

40 Vitamins Dissolve separately in distilled water, adjust to 250 ml, and freeze in aliquots of 5 and 10 ml (Figures 16 and 17). Thiamine mg Pyridoxine mg Nicotinic acid mg Figure 16. Dispensing vitamin solution in tubes. Figure 17. Vitamin solutions distributed in aliquots in the freezer Culture Media 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. 37

41 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. 38

42 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 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 39

43 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. 40

44 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. 41

45 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). 42

46 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 seeds onto a square of cheesecloth. 4. Wrap the seeds in the square of cheesecloth (Figure 26). 5. Place seed package(s) in a beaker. Figure 26. Seeds ready for surface sterilization. 6. Spray the beaker with 70% ethanol; place it in the laminar air flow box. 43

47 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. 44

48 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 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. Figure 30. Carrizo citrange seedlings growing in vitro in the dark. Figure 31. Carrizo citrange seedlings ready to be used for STG. Important. The use of certified rootstock seeds should be guaranteed. 45

49 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. 46

50 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 of the flush. Figure 34. Flush terminals on a Petri dish. 10. Place group of 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. 47

51 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 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 48

52 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. 49

53 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. 50

54 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 ( mm long) (Figures 42 and 43). Keep the scion on the sliver razor blade. Figure 41. Isolating the scion. 51

55 Figure 42. Meristem, subjacent tissue And two primordial leaves (over the dotted line) ready to be excised for its use as scion in STG. Figure 43. Shoot tip excised ready to be grafted. 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. 52

56 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 -2 s -1 (about 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. 53

57 Figure 47. Clementine on Citrus macrophylla three weeks after STG 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 54

58 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. 55

59 7.3.6 Budstick culture in vitro As source of flushes for STG in a Clean Stock Program Budsticks of 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. 56

60 Figure 53. Budsticks after treatment with fungicide and miticide. Figure 54. Budsticks in plstic bags. 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) 57

61 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). 58

62 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. 59

63 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 -2 s -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. 60

64 17. The flushes obtained (generally 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. 61

65 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 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. 62

66 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). 63

67 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). 64

68 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. 65