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A) Cleft Grafting Protocol - GRAFTING AND BUDDING

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A) Cleft Grafting Protocol - GRAFTING AND BUDDING Powered By Docstoc
					                                  GRAFTING AND BUDDING

        Grafting and budding are similar processes. Both involve the physical joining of two or
more plants to form a single physiologically integrated unit. However, in budding, the grafting
partner that gives rise to all or most of the shoot system of the resultant plant, termed the scion,
consists of a single bud rather than a section of stem with multiple buds. Grafting, requires the
fusion, or intergrowth, of the vascular cambia of the grafting partners, such that the vascular
tissues (xylem, phloem) produced post-grafting are continuous between the scion and rootstock.
        Grafting, especially between tree roots, occurs naturally under field conditions, however,
in such cases both plants involved in the graft typically maintain separate root and shoot systems.
In the context of greenhouse and nursery propagation, separate plants are used to provide the root
system and shoot system, termed the „rootstock‟ (understock) and „scion‟, respectively. The
number and diversity of grafting techniques that have been developed is largely a reflection of
the divergent grafting preferences of plant taxa; with the optimal grafting method frequently
varying between plant species. In addition, several specialty grafting techniques have been for
use within the orchard industry; e.g., bridge grafting for the repair of damaged stems, top
working protocols for tree conversion between cultivars, etc.
        Although grafting is generally considered a form of cloning, since the aboveground
portion of the grafted plant is typically entirely derived from the scion the characteristics of
interest in the scion are typically conserved irrespective of the rootstock genotype, the resulting
plants are typically genetic chimeras. However, the lack of genetic uniformity between the root
and shoot systems of grafted plants is often advantageous since the rootstocks can be used to
contribute additional phenotypes; e.g., disease resistance, stress tolerance, growth control
(dwarfing), etc., that would not be possible if the scion had been propagated on its own root
system. In double-working, three genotypes are combined, a scion, a rootstock, and an
interstock. Interstocks, which are placed between the scion and the rootstock may also be
used for height control. In addition, interstocks are frequently used to prevent graft
incompatibility.
        Grafting is one of the oldest forms of plant propagation, dating back to 1000 b.c. and in
modern societies, it is also one of the most expensive, since it is intrinsically labor intensivene.
However, for some plants (e.g., red oak, black walnut) vegetative propagation is only possible
through grafting.
        Compared to other vegetative propagation techniques (stem cutting propagation, layering,
etc), grafting is technically challenging and it is more sensitive to differences in the physiology
and growth state of the stock plants. As a result of these difficulties, grafting is often referred to
as an art, more so, than as a science. However, there are several generalizations that can be made
about variables that contribute to grafting success. For starters, the stock plants used must be
healthy, disease-free, as is true of any vegetative propagation technique. In addition, the tools
used for grafting (knives, wedges etc.) should be well-maintained and properly sterilized before
each use. At a mechanistic level, the grafting cuts should be long, even, and well-matched and
the graft junctions should be well wrapped to ensure close contact between the graft partners and
prevent dessication. In addition, the vascular cambia of the scion and rootstock must be properly
aligned to facilitate their ultimate intergrowth. Proper aftercare is also important, especially for
grafts involve partners differing significantly in size, where the initial graft junctions are liable to
be structurally unstable.
        In this lab you will have the opportunity to practice several of the most common grafting
and budding techniques. We will initially work with herbaceous plants (tomato and potato) since
non-woody stems are easier to cut and there is less chance of injury. In addition, the amount of
woody scionwood that we have is limiting. However, we will ultimately also practice grafting
with aspen and apple.
Grafting Terminology:

Scion – a piece of detached twig or shoot (usually from last year‟s growth) that is used to
propagate the plant from which the twig was taken. The scion usually contains two to four buds.

Roostock (Understock) – the portion of the graft to which the scion is attached. It may be a piece
of root, a seedling, or a tree with part of the top removed in preparation for grafting.

Interstock – a piece of stem that is grafted between the rootstock and the scion

Budding – A form of grafting in which the scion consists of a single bud, that has been more or
less cut away from its parent stem

Budstick – a section of stem that is used as source of buds for budding. Budsticks are typically
collected from the most recent growth increment (1 year old branch regions)

Top Working – any grafting that involves the use of larger understocks, as in the case of grafting
onto the limbs of an established tree. Topworking can be used to combine multiple varieties on
the same tree or for orchard conversion; i.e., following severe pruning. Top working can also be
used to facilitate sexual reproduction. For example, for dioecious trees, male branches may be
grafted onto female trees to produce to create bisexual plants. In addition, for self-compatible
trees the branches of a pollinizer may be added through top working.

Double Working – any grafting the involves the use of an interstocks such that the shoot of the
grafted plant is derived from two separate plants, one which contributes to the basal regions of
the stem and a second partner which contributes to the crown.

Cambium - thin layer of cells located between the outer sapwood and the inner bark. The
cambium is the layer of cells that is responsible for the production of new wood and bark.

Callus – a mass of rapidly dividing, but largely unorganized cells. Callus tissues are generally
produced at wound sites, presumably as a result of hormonal imbalances In the case of grafting,
callus production is involved in formation of the graft union.

„Healing Over‟ – A term which used to refer to the process by which a functional connection is
formed between the grafting partners. Graft “healing” involves three stages: 1) proliferation and
interlocking of callus cells at the interface of the scion and rootstock to form a “callus bridge”, 2)
adhesion of the scion and rootstock, and 3) vascular tissue differentiation across the callus
bridge. Since cells associated with the vascular cambia contribute to callus bridge formation
disproportionately, alignment of the vascular cambia of the grafting partners facilitates callus
bridge formation. The formation of an interconnecting cambium is largely mediated by
hormones produced by the scion; however, for these hormones to be effective the vascular
cambia must also be aligned

Graft Incompatibility – a phenomenon whereby grafts fail to take not as a result of poor grafting
technique but rather as a result of a lack of stable physiological integration. In general the more
distantly related the plants being grafted the more likely they are to be incompatible. However,
the relationships are often complicated with graft compatibility depending upon which partner is
assigned as the rootstock or scion. Graft incompatibility may occur after months or years of
growth, in which case the incompatibility may be related to differences in growth rate.
I) Tomato-Potato Grafting:

In this exercise we will graft tomato scions onto potato rootstocks. Tomato and potato are in
different genera (Solanum vs. Lycopersicum), but are in the same family (Solonaceae) and are
easily grafted. The resulting plants, which have been affectionately termed „pomatoes‟, may
produce both tubers and tomato fruits. but it is a common urban legend that the grafting of
tomatoes and potatoes adversely affects the taste of both. In this exercise, you will prepare cleft
grafts and whip and tongue grafts. Each student should prepare two whip and tongue grafts and
four cleft grafts, two with scions and rootstocks of matching diameter and two with smaller
diameter scions.


Plant Material

Red Potato (Solanum tuberosum) - 3-4 wk old potato plant, that were produced vegetatively
from „seed potatoes‟. Seed potatoes are whole tubers of sections of tuber with one or more
axillary buds, or „eyes‟, that develop as new shoots upon sowing.

Tomato (Lycopersicum esculentum) - 4-5 wk old tomato seedlings. Several cultivars are
available. Most are determinate (bush) cultivars that should not require staking.


A) Cleft Grafting Protocol:

1) Obtain four pairs of potato and tomato stockplants for grafting. Two of the pairs should be of
equivalent diameter at the anticipated grafting sites; i.e., 10-15 cm below the growing tip on the
tomato seedlings and 3-10 cm above the base of the potatoes. For the other two pairs the scion
should be of distinctly smaller diameter

2) Working one graft at a time, use a sharp razor blade to decapitate the potato rootstock 3-10 cm
above soil level using a transverse cut and working at an internodal position.

3) Use the same razor to make a 2-3 cm long median longitudinal incision (cleft) in the top of the
rootstock.

4) Cut off the tomato scion approximately 10-15 cm below its growing point. Trim several of
the largest, lowest leaves on the scion to limit the amount of transpirational surface

5) Make opposite oblique longitudinal cuts at the base of the scion that met to create a wedge.
The cuts should be prepared at a low angle (sloping cuts), so that their length matches that of the
rootstock cleft (2-3 cm), to create a large surface area for contact with the rootstock.

6) Insert the base of the scion into the cleft at the apex of the rootstock and position the base of
the scion to ensure maximal contact between the cut surfaces of both partners. If the scion is
distinctly smaller in diameter than the rootstock, position it towards one side of the rootstock to
facilitate alignment of the vascular cambia (see diagram).

7) Wrap the graft union in parafilm starting on the rootstock below the point of grafting and
proceeding onto the scion. You should have enough parafilm to reverse direction and double
wrap the graft union. The parafilm supports the scion until adhesion and tissue intergrowth has
occurred. In addition, serves to maintain a high humidity at the graft union.

8) Obtain two bamboo stakes 15-20 cm taller than the height of the grafted plant and place in
opposite sides of the pot with the grafted plant.

9) Obtain a clear plastic bag and wet its inside by misting with a water sprizer. Place the bag
over top of the grafted plant using the bamboo stacks as a guide and “seal” the bag around the
base of the pot with a rubber band.

10) After you have finished grafting place the plants under indirect light, or on an open
greenhouse bench with 50% shadeclothe.

11) After 7 days remove the rubber band to start to lower the humidity level experienced by the
plant. Sprouts from the rootstock (which may appear water-soaked as a result of the high
humidity levels) should be pinched off at this time. Adventitious roots may be present at the
base of the scionwood, however, these do not need to be manually removed as they will
eventually air dry.

12) After an additional 3-4 days the bags can be completely removed. However, it is best,
remove the bags at the end of a day so that the plants will have several hours adjust to the
ambient humidity prior to addition of an illumination stress. The parafilm as it will rupture as
the plants increase in diameter


B) Whip-and-Tongue Grafting Protocol:

Obtain two pairs of tomato and potato stockplants for grafting with both members of each pair
being approximately the same size (diameter) at the anticipated grafting point (see above).
Using a single-edged razor blade prepare the grafting cuts as indicated in the whip-and-tongue
diagrams included with this handout. Aside from the differences in grafting technique, the
protocol followed should be equivalent as described for cleft grafting.


C) Aftercare and Evaluation

To produce both tomato and potato crops the plants will need to be transferred to larger pots (for
potato tuber development). Continue to remove rootstock suckers as they develop as they will
compete with the tomato scion for resources. Tubers will be set naturally after 2-3 months of
growth, irrespective of the photoperiod. Interested students may want evaluate the effect of fruit
thinning on tuber yield, since the processes of tomato fruit development and tuber growth would
be expected to compete for resources.
II) Whip-and-Tongue Grafting of Apple

         The whip and tongue graft is one of the most commonly used types of grafts in the U.S.
Whip and tongue grafts are primarily used with to join plant parts less than 1 cm (2.5 cm) in
diameter, with one year old seedlings being the most common rootstocks. Grafting take is
facilitated by the multiple wound surfaces on each grafting partner. In addition, since the
grafting partners are generally the same diameter,
         The grafting of woody plants is typically done early in the Spring using scionwood
collected from branches prior to bud break. Frequently the branches that will be used for the
scionwood are collected in the Fall and stored in a refrigerator for 2-3 months to break the
dormancy of the buds. The rootstocks may be dormant or non-dormant.
         We will be grafting onto containerized two year old grafted apple seedlings (Haralred
P4824) that have been maintained in an active state of growth in the IPFW greenhouse (25 C, 16
hr photoperiod). The scionwood (Sweet 16) consists of branches that were harvested from a two
year old graft plant on 8/11/04 and stored at 4 C until today‟s lab. Since we will be grafting onto
the limbs of the grafted apples rather than seedlings this exercise represents a form of „top
working‟.


Grafting Protocol:

See the diagrams for whip-and-tongue grafting provided with this handout. Each student will
prepare two grafts, each on a separate branch, with the grafts being prepared as close to the base
of the branch as possible. Scions will be cut from the stratified branch samples provided. The
scions contain two to four buds each. As with all grafting the cuts must be straight and smooth
to ensure good contact between the grafting partners; however, be sure to cut away from yourself
and others in the lab to avoid injury. After the scion and understock have been fitted together the
grafts should be wrapped tightly with budding rubbers, sealed with TreeCoat, and labeled.


III) Chip Budding of Aspen and Apple

         Whereas grafting utilizes a scion consisting of a short piece of stem tissue with two to
four buds, budding utilizes single buds. Budding offers several advantages to grafting. Budding
is more economical that grafting since a single bud is used per propagation, and is especially
important if propagation wood is scarce. Budding generally results in a stronger type of union
that is achieved with grafting especially for the first few years. Budding is quicker than grafting
(and more versatile in that budding can be done at more times during the year at least in terms of
nursery production). Techniques are simple and can be easily performed by an amateur. In
budding, the process of alignment is facilitated by the size differential of the grafting explants
         As with grafting several distinct budding techniques have been developed, the most
poplar of which include patch budding, T-budding, and chip budding. Patch budding and T-
budding require bark slippage and thus are most effective for spring grafting. In contrast, chip
budding does not require bark slippage and therefore can be performed at any time during the
growing season or in the Fall, however, chip budding is typically done in the early Spring or Fall.
When chip budding is done in the Spring the branch or seedling that was used for grafting is
typically cut off beyond the point of bud insertion after 10-15 days provided that the buds have
taken as assessed based on their color and degree of turgidity (swollen appearance). In the case
of fall budding, the branch or seedling is cut off the following spring before growth has resumed.
        In this exercise we will practice chip budding with apple and aspen. In both cases we
will be budding onto actively growing seedlings or grafts (in the case of apple). Although we are
working under greenhouse conditions, the grafting that we are attempting today is the equivalent
of summer grafting. For each seedling or branch selected for budding you can add two buds.
Add the buds close the base if possibleand


Review Questions:


1) Can you explain the production of adventitious roots at the base of the scion in terms of what
you have learned in class about activity and transport of auxins?




2) Can you think of any possible advantages to “taproot grafting”, where the scion is grafted into
the taproot of the rootstock?




3) Based on your knowledge of the differences between monocots and dicots and the mechanism
of graft healing would you expect many monocots to be amenable to graft propagation?

				
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