Techniques for graft-transmission in citrus

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Of prime importance in grafting techniques is a good-quality budding knife. New knives must be sharpened using a medium-grit carborundum stone so that every trace of the original V-shaped tip on the cutting edge of the new blade is removed. When the cutting edge is observed with a magnifying glass, a smooth transition from the back of the blade to the very edge should be seen. The blade should be razor-sharp. The test for a well-sharpened blade is the ability to make a single, smooth slicing cut into the budstick and detach the bud or blind bud clear of the budstick without tearing the tissue as the cut is completed. The knife should be periodically resharpened with a fine-grit oilstone using oil, and honed on a leather strap. It is well to observe a number of professional or skilled budders to study their techniques and practice under their tutelage.

"Bud" graft inoculation

Figure 127 shows the three types of "bud" used for graft-transmission. The tissue illustrated at the top is a bud containing an eye. This is always used for propagation purposes but may be used for inoculation. The centre "bud" is called a blind bud i.e. a "bud" without an eye. It is cut from the portion of the stem between the buds and is used primarily for inoculation purposes.

Budsticks are collected from the field tree to be indexed and should be placed in a plastic bag, cooled in an ice chest and refrigerated at the laboratory until used. The bud or blind bud is sliced or cut from a stem or budstick. Using a well-sharpened knife, a slice is made into the stem using a continuous slicing motion. The bud should be cut free of the stem with one smooth slicing stroke. A T-cut is then made into the stem of the receptor or index plant to be inoculated. The upper portion of the bark at the top of the T-cut is opened and the bud or blind bud inserted (similar to the illustration in Figure 130). The blind bud is an excellent inoculum tissue and is preferred since it is easier to excise from a budstick and has the advantage of not having an eye, thus avoiding growth of the inoculum bud after the wrapping is removed. It is inserted in the same manner as the bud with an eye.

The third type of bud is the chip bud (Figure 127, lower). Chip buds are used when the bark of the receptor host does not slip or open up to accept a bud or blind bud. This bud is cut from the inoculum stem by first making a cut at right angles into the inoculum stem about 1-2 mm deep, and then making a slicing cut toward the initial cut to free the bud. Similar matching cuts are made into the receptor stem, and the two pieces are matched and fitted together and then wrapped. Chip buds may be cut with or without an eye.

After insertion, the inoculum "buds" are secured to the stem by wrapping with either rubber or plastic tape as shown in Figure 128. The tape should be kept stretched and the bud should be tightly wrapped. If a bud with an eye is used for propagation, avoid covering the eye with the wrapping tape so that the bud can grow. If, however, a bud with an eye is used only for inoculation, the eye should be covered and the bud wrapped completely with the wrapping tape.

Two bud-inoculations are generally sufficient for transmission. After two to three weeks, the wrapping tape is removed by cutting the tape with a knife or razor-blade and the bud is examined to see if it is alive (survival recording). If both grafts are found dead, the same plant should be reinoculated with buds or chip buds, or another test plant inoculated. If only one graft is dead, and there are sufficient replicated test plants, reinoculation is unnecessary.

When cutting tapes for removal, ensure that the knife blade is disinfected by dipping it into a 1 percent sodium hypochlorite solution.

Leaf-piece graft

• A rectangular piece approximately 3-4 mm wide and 2-3 cm long is cut from a young immature leaf as shown in Figure 129.
• The cut piece is inserted into a T-cut in the receptor stem exactly as is done for buds. The point of a knife is used to push the leaf into the T-cut as shown in Figure 130. It is important that the bark separate readily or be "slipping" to permit easy entry of the leaf piece into the T-cut. Two leaf-graft inoculations per plant are recommended.
• The inserted leaf piece is then wrapped in the same manner as with buds shown in Figure 128.
• The wrapping tapes may be cut two or three weeks after inoculation. The grafts are then examined for survival, and if both inoculum grafts are dead the plant can be reinoculated (if the bark is slipping) or a new plant inoculated.
After some time, the leaf piece of a successful graft can be seen to expand and grow inside the T-cut as illustrated in Figure 131.

Leaf disc graft

• A paper hole-punch is used to cut a disc from the midrib section of a leaf of the inoculum source, as shown in Figure 132. The leaf should be moderately mature or mature. Very young leaves should not be used since it is difficult to punch out discs and manipulate them, and the punched discs do not match up well with holes in the receptor leaf. The punched feat discs are placed on a piece of slightly moistened tissue paper during the inoculation procedure. A number of inoculum leaf discs may be cut at one time, and at least five discs are recommended for inoculation of each plant. The cut leaf discs should be placed on the moist paper with the top leaf surface facing up.
• A hole is punched in each of five leaves of the index or receptor test plant using the hole-punch as shown in Figure 132. Receptor leaves should also be moderately mature or mature, but not too young.
• A piece of adhesive tape, slightly longer than the width of the leaf, is placed on the bottom of each of the receptor leaves and pressed lightly into place.
• The cut discs, resting face upward on the moist tissue paper, are retrieved with a dissecting needle and carefully inserted into the hole previously made in the receptor leaf, as shown in Figure 133. The midribs should be as well aligned as possible. Similarly, the disc should be carefully matched to the hole. The adhesive tape at the bottom of the leaf will hold the leaf disc in place while necessary adjustments are made.
• Another piece of adhesive tape of similar length is then placed above the leaf and firmly pressed into place. The completed graft is shown in Figure 134.
• After one to two weeks, a determination can be made for graft survival. Dead leaf grafts will turn brown, and reinoculation is necessary if three or more of the five grafts are dead.

Caution. The brand of adhesive tape selected is extremely important. Some brands are toxic to the leaf. Scotch 600-brand is non-toxic. Whichever local brand is selected, it should be examined for toxicity by making some test grafts on a number of leaves. The tape should not be left in a warm greenhouse but kept refrigerated. Warm temperatures have been shown to induce chemical changes in the tape ingredients and induce toxicity.

Bark graft

• Rectangular pieces of bark are cut from the trunk of the tree to be tested or indexed. This should be done only during the months when the bark is readily slipping. The cut bark pieces are placed in a plastic vial, as illustrated in Figure 135. The vial should be designed to permit some breathing. The vials containing the bark samples should be placed in an ice chest immediately after sampling and later transferred to a refrigerator at the laboratory. Tests have shown that bark collected in this manner and refrigerated at +5-6°C remained viable for as long as one month.
• The bark pieces are removed from the vial, placed on a moist paper towel and cut into rectangular segments 3-4 mm by about 20 mm. The cambium face of the bark should always be kept in contact with the moist tissue paper (Figure 136).
• An "I" cut is made in the stem of the receptor or index test plant, and the bark is teased open slightly. The inoculum bark piece is then placed between the flaps of the open "I" cut and is held in place by the bark flaps (Figure 137, bottom).
• At least two bark grafts are made per index plant, and the inoculum is then tightly wrapped with rubber or polythene budding tape in the same manner as for bud grafts (Figure 128 and centre graft in Figure 137).
• After two to three weeks, the tape is removed by cutting it free with a disinfected knife or razor-blade, and the inoculum observed for survival. At this time, the survival or vitality of the bark tissue can be tested by gently slicing into the graft tissue just underneath the outer bark using a razor-blade or knife. Dead or dying tissue will be brown, shrivelled and sometimes loose; vital tissue will be green, yellow or white.

Side grafting

• Inoculum stem pieces approximately 8-15 cm long are cut from the test source tree or plant to be indexed and are matched, diameter for diameter, with the indicator test plant.
• Wedge cuts are sliced on both sides at the end of the stem piece giving the appearance of a long tapered "V". The cutting knife should be razor-sharp, and some skill and practice are needed to make the cuts very flat, smooth and straight.
• A slicing cut is then made into the receptor indicator plant using a knife or razor-blade. The depth of the cut should be about the same as the length of the wedge cut on the inoculum stem piece.
• Using a clipper, the inoculum piece is trimmed to about 3-4 cm long, as seen in Figure 138, and inserted into the stem. Some slicing and trimming may be necessary but, with practice, well-fitting grafts can be made repeatedly.
• The joined tissue is tightly wrapped with budding tape. At least two side grafts per test plant should be done (as illustrated in Figure 26 in the section on stubborn).
• The bottom end of a polythene bag is cut to convert the bag into a sleeve. This is then placed over the plant, and secured at the top and bottom with plant ties (Figure 138, left). It is important that some leaves be left on the stem near the graft to provide moisture within the bag. The cut end of the inoculum stem pieces need not be coated with protective tar. Research tests with thousands of grafts, with and without protective tar, indicate no benefit from this practice.
• After 10 days, the bottom of the polythene sleeve is opened to permit some air into the bag. At 15-20 days, the polythene sleeve is removed and grafts are examined for survival.
• The plant is then topped or cut back about 25-30 cm above the soil surface, but above the side grafts, and a single shoot is permitted to grow (as illustrated in Figure 26 in the section on stubborn).

Many thousands of side grafts have been made at the Riverside laboratory using this procedure, with a survival rate of over 95 percent. Whenever the graft inoculum died, the reason was found in a deficiency factor in the budwood rather than in human error. Certain mandarin budwood (i.e. Dancy) collected during the spring months gave a high rate of graft failure. However, most tissues of the major citrus cultivars were found to be highly graft-compatible.

A modified side graft can be made by inserting a small twig into a larger receptor stem and using a modified T-bud procedure, thereby reducing the importance of matching diameters of inoculum and receptor host. This is done by cutting the end of the twig at a sharp angle producing a long, elliptical surface area. This is then inserted into a T-cut made in the larger receptor plant.

REFERENCES

Baker, K.F., ed.1957. The UC system for producing healthy container-grown plants. Calif. Agric. Exp. Sta. Extension Service Manual 23. Reprinted1985 by Surrey Beatty and Sons, Chipping Norton, NSW 2170, Australia.

Klotz, L.J., deWolfe, T.A., Roistacher, C.N., Nauer, E.M. & Carpenter, J.B.1960. Heat treatment to destroy fungi in infected seeds and seedlings of Citrus.Plant Dis.Rep.,4(11): 858-861.

Nauer, E.M. & Carson, T.1985. Packaging citrus seed for long-term storage. Citrograph, 70(10): 229-230.

Nauer, E.M., Holmes, R.C. & Boswell, S.B.1980. Close spacing in the greenhouse inhibits lime seedling growth. HortSci.,15 (5): 591 -592. Nauer, E.M., Roistacher, C.N. & Labanauskas, C.K.1967. Effects of mix composition, fertilization, and pH on citrus grown in UC-type potting mixtures under greenhouse conditions. Hilgardia, 38(15): 557-567.

Nauer, E.M., Roistacher, C.N. & Labanauskas, C.K.1968. Growing citrus in modified UC potting mixtures. Calif. Citrogr., 53: 456, 458, 460-461.

Roistacher, C.N.1963. Effect of light on symptom expression of concave gum virus in certain mandarins. Plant Dis. Rep., 47(10): 914-915.

Roistacher, C.N. & Baker, K.F. 1954. Disinfesting action of wood preservatives on plant containers. Phytopathol., 44: 65-69.

Roistacher, C.N. & Nauer, E.M.1985. Effect of supplemental light on citrus seedlings in winter. Citrograph, 70(8): 181 - 182,196.

FIGURE.106 Three designs for the layout of plant laboratory greenhouses

a) A six-room design; each room an individual cubicle with a central walkway
b) A three-room design with a central walkway
c) Three separate small greenhouses with individual temperature control for cool, moderate and warm temperatures

FIGURE 107 Various greenhouses used worldwide for indexing a) An inexpensive wood-and-fibreglass greenhouse at Riverside, Calitornia, with two evaporator coolers

FIGURE 107 Various greenhouses used worldwide for indexing b) The interior of the greenhouse in (a) showing the woodandfibreglass structure. Excellent plants were grown in this inexpensive house using a UC system of soils, fertilizers and temperature control

FIGURE 107 Various greenhouses used worldwide for indexing c) The Rubidoux laboratory at Riverside, California, has a double-door entryway and three compartments, as in Figure 106b

FIGURE 107 Various greenhouses used worldwide for indexing d) A large fibreglass and aluminium-frame greenhouse. The screened portion at the tar end Is made of 32-mesh plastic screen to filter the outside air as it passes through cooling cells. Two large fans at the opposite end control air movement

FIGURE 107 Various greenhouses used worldwide for indexing e) The interior of the house shown in (d). This house was not divided Into compartments but was held at one temperature for large-scale testing for seedling-yellows tristeza

FIGURE 107 Various greenhouses used worldwide for indexing f) The interior of the fibreglass greenhouse at Moncada, Spain, used for extensive indexing of citrus. There are three such houses, maintained at different temperatures. Note the central walkway, metal benches with plywood top, and cooling cells at the opposite end of the house. This house is cooled in the same manner as the structure in (d) and (e)

FIGURE 107 Various greenhouses used worldwide for indexing g) A small aluminium-and-glass house at Riverside, Calitornia, with two internal heaters and evaporator coolers on the outside. This is an excellent type of greenhouse.

FIGURE 107 Various greenhouses used worldwide for indexing h) A small fibreglass house at Nelspruit, South Africa, used for indexing. This house is cooled by cooling cells at one end and a tan at the other

FIGURE 107 Various greenhouses used worldwide for indexing i) Glasshouse at Catania, Italy, with top and side vents for cooling and wire mesh over the structure to prevent damage to the glass from hail

FIGURE 107 Various greenhouses used worldwide for indexing j) A double-walled polythene greenhouse at Riverside, California. The double layer gives excellent insulation, and warm or cool air can be pumped into the space between the polythene layers for additional heating or cooling. This system is very energy-efficient

FIGURE 108 Examples of methods of heating greenhouses a) A small heating unit outside the structure at Riverside, California. Heating units placed outside the greenhouse are preferable since they minimize risk of ethylene damage to plants by leakage of exhaust fumes from faulty heaters

FIGURE 108 Examples of methods of heating greenhouses b) Two large heaters outside the Rubidoux glasshouse. The warm air is carried by ducts into the upper part of the house and distributed internally by tans and plastic tubing as shown in (c), (d) and (e)

FIGURE 108 Examples of methods of heating greenhouses c) A large internal heating unit with a perforated plastic tube attached. Note the circular hole in the plastic tube

FIGURE 108 Examples of methods of heating greenhouses d) Interior of greenhouse with large perforated plastic tube running the length of the greenhouse. The warm air is blown through the tubing and forced out through the many holes in the tubing

FIGURE 108 Examples of methods of heating greenhouses e) A separate tan attached near the heater. This tan can be activated independently of the heater tan by a separate thermostat. This permits the circulation of air within the greenhouse for the distribution of latent heat residing in the soil, floors and structure, and is energy-saving

FIGURE 109Different types of benches used to support plant containers a) Wooden benches and concrete block supports at Riverside, California. Note the gravel floor

FIGURE 109Different types of benches used to support plant containers b) Another view of wood-and-concrete block benches at Riverside, California. (Trees on benches are "virus" bank source plants)

FIGURE 109Different types of benches used to support plant containers c) Wooden runners to support large containers, keeping them off the ground. Note gravel floor (Riverside, California)

FIGURE 109Different types of benches used to support plant containers d) Benches made of concrete at Campinas, Brazil. Note the well-spaced plants

FIGURE 109Different types of benches used to support plant containers e) Steei and wire mesh benches, Mildura, Australia. Note concrete floor

FIGURE 109Different types of benches used to support plant containers f) A steel mesh bench on concrete blocks at the USDA greenhouse, Orlando, Florida

FIGURE 109Different types of benches used to support plant containers g) Plastic bench tops on a wooden frame set on concrete blocks in a screenhouse at Lake Alfred, Florida

FIGURE 109Different types of benches used to support plant containers h) Spraying wooden benches with copper naphthenate solution (an excellent wood preservative and disinfectant)

FIGURE 110.The ingredients and fertilizers used in the modified University of California mix for growing citrus

FIGURE 111Decives for steaming soil a) A trailer fitted with pipes perforated with holes drilled on the bottom of the pipes. Steam is conducted from the pipes through the soil. The top of the trailer is covered with a tarpaulin prior to steaming. AHer cooling, the soil can be taken directly from the trailer to fill containers

FIGURE 111Decives for steaming soil b) Flats and pots can be steamed directly in the trailer. Note steam pipes at the bottom

FIGURE 111Decives for steaming soil c) A fixed steam chamber used to steam containers. It can also be used to steam small quantities of soil

FIGURE 112 A metal tamper used to flatten and compress soil in flats prior to seed planting. The top of the soil should be level to prevent water accumulation at any low spots

FIGURE 113 A planting board used for the even distribution of seed. One seed is dropped into each hole and pressed gently into the soil. The seed is then covered with about 1 cm of soil and the soil is tamped lightly and levelled, using the tamper as shown in Figure 112

FIGURE 114a Seedling trays used for growing individual seedlings at the Citrus Research Center, Lake Alfred, Florida

FIGURE 114b Seedling trays with seedlings at the USDA greenhouse, Orlando, Florida. One seed is dropped into each cup and covered with soil medium or soil

FIGURE 115 A tray and pad with disinfectant placed in front of the entrance to the greenhouse (Nelspruit, South Atrica)

FIGURE 116 A recording thermograph should be kept in each room of a plant laboratory. It should be checked periodically with thermometers for accuracy, and maximum and minimum daily temperatures recorded in a separate book once a week when charts are changed

FIGURE 117 A very inexpensive device for siphoning fertilizer from a concentrated solution In a tub into the water supply. The flowing water creates suction in the Venturi restriction and draws the fertilizer through the watering hose. This device was used for many years at the University of Calitornia laboratory prior to installation of a Smith proportioning device (Figure 118)

FIGURE 118 A Smith Measuremix proportioner. This is a precision instrument. A plunger, activated by water pressure, injects a fixed amount of fertilizer Into the watering system. The amount of fertilizer injected is in proportion to water flow. This proportioner is expensive but has performed very well for many years with minimal maintenance

FIGURE 119 A large fan at one end of a greenhouse creates negative pressure in the house and brings in the outside air through cooling pads at the opposite end

FIGURE 120 A thermostatic control panel with four thermostats for four levels of control. Thermostats Independently control fans for the introduction of outside air or for pumping water over the cooling cells. They also control heating by circulating the inside air or by turning on heaters

FIGURE 121 A system used at the Riverside laboratory for filtering incoming air using spun-glass-andcharcoal filters

FIGURE 122 A standard commercial evaporator cooler. This cooler is effective it humidity is low. It is relatively inexpensive and, if properly maintained by replacing pads and by periodical cleaning and painting, it is effective in cooling greenhouses. Similar units were used for over 20 years at the Riverside laboratory and were very reliable

FIGURE 123 The same cooler as in Figure 122 but with the side panel removed to show the main squirrel-cage fan, the water pump, and water being pumped from the top and down over a pad of wood shavings. The outside air forced through the wet pads is cooled by evaporation

FIGURE 124 Cooling cells consisting of specially treated cardboard units placed together to make a continuous wall. Water is pumped from a reservoir tank below ground (bottom left) to the top, and drips freely down over the cells by gravity. Cooling is by evaporation

FIGURE 125 A battery of cooling cells at one end of a greenhouse. Cooling by evaporation is most efficient when humidity is low

FIGURE 126 Where humidity is too high, greenhouses may have to be cooled by electric refrigeration units. This method is expensive if power rates are high. Electric refrigeration units can be used to supplement evaporator coolers where humidity is moderate or high. They can be used in rooms designated for testing cool-temperature pathogens

FIGURE 127 Bud grafting. Showing three types of "bud" inserted into the stem of a citrus seedling and ready for wrapping. At the top is a bud containing an "eye" or the meristematic bud which can grow. In the centre is a blind "bud" containing no "eye". At the bottom is a chip bud fitted into the stem. The chip bud can be blind or can contain an eye. The one illustrated is a blind chip bud

FIGURE 128 During wrapping, the plastic tape should be kept stretched to secure a tight wrap

FIGURE 129 Leaf-piece grafting. Cutting a small rectangular segment from the central midrib of a young feat to be used for a feat graft

FIGURE 130 Insertion of the cut feat piece into a T-cut in the stem of a citrus seedling. "Buds" are inserted in the same manner. The point of a knife is used to slide the leaf piece or "bud" into the T-cut. Only seedlings in which the bark readily slips can be used. Seedlings with tight bark cannot be used for this technique

FIGURE 131 Showing a growing leaf piece which had been successfully grafted into the stem. The tapes are removed about three weeks after grafting. The photograph was taken about six weeks after grafting

FIGURE 132 Leaf punch graft technique. A hole is first punched into the leaf of the plant to be Inoculated, and a similar disc Is cut in a leaf of the Inwulum tissue

FIGURE 133 The inoculum disc is inserted into the receptor hole in a leaf of the Indlcator seedling using a dissecting needle. A piece of adhesive tape is first placed on the underside of the receptor leaf

FIGURE 134 Another piece of adhesive tape is then placed on top of the leaf and pressed firmly. After one or two weeks the feat disc will graft at the edges. Dead grafts wiii turn brown

FIGURE 135 Bark grafting. Showing the plastic tube used for collecting bark samples. A small piece of moist tissue is placed at the bottom of the tube and the bark pieces placed inside. The top cap should not be sealed but left open to allow aeration. Tubes are put into an ice chest immediately after field collection and refrigerated at the laboratory. Bark samples collected and stored in this manner will keep for over three weeks

FIGURE 136 The bark piece is trimmed on moist tissue paper with the cambium face downward

FIGURE 137 The bark piece is inserted into an l cut made in the stem of the indicator seedling. The side flaps of the l-cut will hold the bark in place as shown in the bottom graft. The bark is wrapped as shown in the centre graft

FIGURE 138 Side grafting. A side graft stem piece (top right) showing the tapered V-cut at the end ready for insertion. A cut is made into the receptor stem, and the inoculum piece is fitted and wrapped as shown on the bottom right. It is then covered with a polythene sleeve and tied above and below with plant ties. Some leaves should be left inside to provide moisture

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